Gravitational and Space Biology
Volume 18, Number 2 June 2005
Publication of the American Society for Gravitational and Space Biology ISSN 1089-988X
ASGSB EDITORIAL BOARD
Augusto Cogoli Luis Cubano Emily Holton Zero-G LifeTec GmbH Univ. Central del Caribe NASA Ames Research Center Zürich, Switzerland Camuy, Puerto Rico Moffett Field, CA John Kiss Patrick Masson Gloria Muday Miami University University of Wisconsin Wake Forest University Oxford, OH Madison, WI Winston Salem, CT Anna-Lisa Paul April Ronca Gerald Sonnenfeld University of Florida Wake Forest University SUNY Binghamton Gainesville, FL Winston Salem, CT Binghamton, NY Paul Todd Sarah Wyatt SHOT, Inc. Ohio University Greenville, IN Athens, OH PUBLISHING STAFF
Stan Roux Mary E. Musgrave Robert Blasiak Editor-in Chief Publishing Editor Assistant Editor University of Texas University of Connecticut University of Massachusetts Austin, TX Storrs, CT Amherst, MA
Nancy Searby Joan Vernikos Symposium Editor Symposium Editor NASA Ames Research Center Moffett Field, CA Alexandria, VA
GENERAL INFORMATION
Gravitational and Space Biology (ISSN 1089-988X) is a journal devoted to research in gravitational and space biology. It is published by the American Society for Gravitational and Space Biology, a non-profit organization whose members share a common goal of furthering the understanding of the biological effects of gravity and the use of the unique environment of spaceflight for biological research. Gravitational and Space Biology is overseen by a steering committee consisting of the Publications Committee, the Editor, the President, and the Secretary-Treasurer of the ASGSB.
The American Society for Gravitational and Space Biology was created in 1984 to provide an avenue for scientists interested in gravitational and space biology to share information and join together to speak with a united voice in support of this field of science. The biological effects of gravity have been acknowledged since Galileo’s time, but only since the 1970s has gravitational biology begun to attract attention. With the birth of the space age, the opportunity for experimentation over the full spectrum of gravity finally became a reality, and a new environment and research tool became available to probe biological phenomena and expand scientific knowledge. Space and spaceflight introduced new questions about space radiation and the physiological and psychological effects of the artificial environment of spacecraft.
The objectives of ASGSB are:
• To promote research, education, training, and development in the areas of gravitational and space biology and to apply the knowledge gained to a better understanding of the effect of gravity and space environmental factors on the flora and fauna of Earth. • To disseminate information on gravitational and space biology research and the application of this research to the solution of terrestrial and space biological problems. • To provide a forum for communication among professionals in academia, government, business, and other segments of society involved in gravitational and space biological research and application. • To promote the study of concepts and the implementation of programs that can achieve these ends and further the advancement and welfare of humankind.
A Collaborative Production: This issue of the Gravitational and Space Biology was produced through collaboration with the Professional Writing and Technical Communication Program at the University of Massachusetts Amherst. Under the direction of Dr. John Nelson, the PWTC Program has trained students for a variety of professions that demand excellent technical writing and editing skills. Since its inception in 1990, the program has placed nearly 100% of its graduates. The American Society for Gravitational and Space Biology is pleased to sponsor an editorial fellowship for students in the PWTC program at the University of Massachusetts, and gratefully acknowledges the contributions of its students and directors to the production of our journal.
MEMBERSHIP: The American Society for Gravitational and Space Biology welcomes individual, organizational, and corporate members in all of the basic and applied fields of the space and gravitational life sciences. Members are active in the fields of space medicine, plant and animal gravitational physiology, cell and developmental biology, biophysics, and space hardware and life support system development. Membership is open to nationals of all countries. Members must have education or research or applied experience in areas related to the Society’s purposes: i.e., Doctorate, Masters with 2 years experience, Bachelors with 4 years experience (student members must be actively enrolled in an academic curriculum leading toward a career related to the Society’s purposes), or special appointment by the Board of Directors. Membership applications may be obtained by writing the American Society for Gravitational and Space Biology, P.O. Box 2581, Chapel Hill, NC 27515, or at the society website (http://www.asgsb.org).
Gravitational and Space Biology is sent to all members of the American Society for Gravitational and Space Biology. Requests for copies, information about subscriptions and membership, changes of address, questions on permission to reproduce parts of this volume, and other correspondence should be sent to the American Society for Gravitational and Space Biology P.O. Box 2581, Chapel Hill, NC 27515.
Copyright © 2005 by the American Society for Gravitational and Space Biology
ii Gravitational and Space Biology 18(2) June 2005
American Society for
Gravitational and Space Biology
Proceedings of the 20th Annual Meeting of the American Society for Gravitational and Space Biology
Featuring:
Symposium I: Model Organisms for Exploration Biology
Symposium II: Pharmacological Countermeasures to Physiological Changes Induced in Space Flight
Additional Short Papers
Gravitational and Space Biology 18(2) June 2005 iii
Table of Contents
Symposium I: Model Organisms for Exploration Biology………………………………...…………..1
THE YEASTS SACCHAROMYCES CEREVISIAE AND SCHIZOSACCHAROMYCES POMBE: MODELS FOR CELL BIOLOGY RESEARCH – S.L. Forsburg………………………………………………………………………………………………….3
WORMS IN SPACE? A MODEL BIOLOGICAL DOSIMETER – Y. Zhao, R. Johnsen, D. Baillie and A. Rose.……………11
DROSOPHILA MELANOGASTER - THE MODEL ORGANISM OF CHOICE FOR THE COMPLEX BIOLOGY OF
MULTI-CELLULAR ORGANISMS – K.M. Beckingham, J.D. Armstrong, M.J. Texada, R. Munjaal, D.A. Baker………..…17
USE OF ANIMAL MODELS FOR SPACE FLIGHT PHYSIOLOGY STUDIES, WITH SPECIAL FOCUS ON THE IMMUNE SYSTEM – G. Sonnenfeld…………………………………………………………………………………………………….31
Symposium II: Pharmacological Countermeasures to Physiological Changes…………………….37
EXERCISE AND PHARMACOLOGICAL COUNTERMEASURES FOR BONE LOSS DURING LONG-DURATION SPACE FLIGHT – P.R. Cavanagh, A.A. Licata, and A.J. Rice………………………………………………………………………..39
CONSEQUENCES OF CARDIOVASCULAR ADAPTATION TO SPACEFLIGHT: IMPLICATIONS FOR THE USE OF PHARMACOLOGICAL COUNTERMEASURES – V.A. Convertino………………………………………………………...…….59
DIET AS A FACTOR IN BEHAVIORAL RADIATION PROTECTION FOLLOWING EXPOSURE TO HEAVY PARTICLES – B.M. Rabin, B. Shukitt-Hale, J. Joseph and P. Todd…………………………………………………………………71
Short Papers……………………………………………………………………………………….…...79
Advanced Life Support and Biotechnology:
EVALUATION OF A SILANE QUATERNARY AMMONIUM SALT AS AN ANTIMICROBIAL SURFACE TREATMENT – D. Blustein, N. Hinkle and A. Smith…………………………………………………………………………………..81
SCREENING AND IDENTIFICATION OF CRYOPRESERVATIVE AGENTS FOR HUMAN CELLULAR BIOTECHNOLOGY EXPERIMENTS IN MICROGRAVITY – T.F. Elliott, G.C. Das, D.K. Hammond, R.J. Schwarzkopf, L.B. Jones, T.L. Baker and J.E. Love……………………………………………………………………………………………….……..83
PSEUDOMONAS AERUGINOSA GROWTH AND PRODUCTION OF EXOTOXIN A IN STATIC AND MODELED MICROGRAVITY ENVIRONMENTS – S. Guadarrama, E. deL. Pulcini, S.C. Broadaway and B.H. Pyle………………...85
DEVELOPMENT AND TESTING OF AN EFFICIENT LED INTRACANOPY LIGHTING DESIGN FOR MINIMIZING EQUIVALENT SYSTEM MASS IN AN ADVANCED LIFE-SUPPORT SYSTEM. – G. D. Massa, J. C. Emmerich, M. E. Mick, R. J. Kennedy, R. C. Morrow and C. A. Mitchell…………………………………………………………………………………87
CELL BEHAVIOR IN SIMULATED MICROGRAVITY: A COMPARISON OF RESULTS OBTAINED WITH RWV AND RPM – A.Villa, S. Versari, J.A.M. Maier and S. Bradamante…………………………………………………………….…….89
Animal Development, Physiology and Gravity Response:
METHODS FOR THE CULTURE OF C. ELEGANS AND S. CEREVISIAE IN MICROGRAVITY – T. Fahlen, J. Sunga, J. Rask, A. Herrera, K. Lam, L. Sing, K. Sato, R.A. Ramos, M. Kirven-Brooks, and D. Reiss-Bubenheim………………………....91
DEVELOPMENT OF THE EMCS HARDWARE FOR MULTIGENERATIONAL GROWTH OF DROSOPHILA MELANOGASTER IN SPACE – M.E. Sanchez, M. Shenasa, A. Kakavand, R.S. Stowers, D. Leskovsky and S. Bhattacharya…………………………………………………………………………………………….…………………………………93
iv Gravitational and Space Biology 18(2) June 2005
NEUROPHYSIOLOGICAL LONG-TERM RECORDINGS IN SPACE: EXPERIMENTS SCORPI AND SCORPI-T – M. Schmäh and E. Horn…………………………………………………………………………………………………….95
SPACE SHUTTLE FLIGHT ENVIRONMENT INDUCES DEGENERATION IN THE RETINA OF RAT NEONATES – J. Tombran-Tink and C.J. Barnstable………………………………………………………………………………………………………..97
Cell Biology:
ANTIGENIC PROTEIN IN MICROGRAVITY-GROWN HUMAN MIXED MÜLLERIAN OVARIAN TUMOR (LN1) CELLS PRESERVED IN RNA STABILIZING AGENT – D.K. Hammond, J. Becker, T.F. Elliott, K. Holubec, T.L. Baker
and J.E. Love………………………………………………………………………………………………………………………………….99
COUNTERMEASURE FOR SPACE FLIGHT EFFECTS ON IMMUNE SYSTEM: NUTRITIONAL NUCLEOTIDES – A.D. Kulkarni, K. Yamauchi, A. Sundaresan, G.T. Ramesh and N. R. Pellis………………………………………………….……101
CELL SURVIVAL AND PRESERVATION OF SIRNA-MEDIATED PROTEIN KNOCK-DOWN UPON SERUM-FREE CRYOPRESERVATION (-80°C) – C. A. Lambert, C. Deroanne, S. Servotte, P. Mineur, C. M. Lapière, B. Nusgens…...…103
TESTING OF SACCHAROMYCES CEREVISIAE MORPHOLOGICAL FIXATIVES AND FIXED SAMPLES STORED AT AMBIENT TEMPERATURE – D. Ly, D. Yu, B. Girten and J. Cohen……………………………………………………………..105
POSSIBLE INVOLVEMENT OF FLOW DETECTION IN THE ACTIVATON OF OSTEOBLASTS – M. Takaoki, H. Park,
N. Murakami, D. Shiba, and J-I Gyotoku………………………………………………………………………………………………..107
POLYAMINES PROTECT AGAINST RADIATION-INDUCED OXIDATIVE STRESS – A.W. von Deutsch, C.D. Mitchell, C.E. Williams, K. Dutt, N.A. Silvestrov, B.J. Klement, I.K. Abukhalaf and D.A. von Deutsch………………………………...…109
ERYTHROPOIETIN AND IL-3 RECEPTOR CELL SURFACE EXPRESSION IS DECREASED UNDER CONDITIONS THAT MODEL SOME ASPECTS OF MICROGRAVITY – K. Xu, L. Feldman, K.L. Davis and A.J. Sytkowski…………...111
Plant Development and Gravity Response:
SECRETION AS A KEY COMPONENT OF GRAVITROPIC GROWTH: IMPLICATIONS FOR ANNEXIN INVOLVEMENT IN DIFFERENTIAL GROWTH – G. Clark, A. Cantero-Garcia, T. Butterfield, M. Dauwalder and S.J. Roux…………………………………………………………………………………………………………………………………………..113
PHOTOTAXIS AND AEROTAXIS IN A CALCIFYING ALGA – P. Jackie Duke, W. Callejas, L. Doan and M. Marsh...115
CO-EXPRESSION AND HORMONAL REGULATION OF GENES IN RESPONSE TO GRAVITY AND MECHANICAL STIMULATION IN THE ARABIDOPSIS ROOT APEX – J.M. Kimbrough, C.S. Brown and H. Winter Sederoff…………..117
ALTERATION OF GROWTH AND GRAVITROPIC RESPONSE OF MAIZE ROOTS BY LITHIUM – T.J. Mulkey..…119
GRAVITY AND LIGHT: INTEGRATING TRANSCRIPTIONAL REGULATION IN ROOTS – R. Salinas-Mondragón, A.
Brogan, N. Ward, I. Perera, W. Boss, C.S. Brown and H. Winter Sederoff…………………………………………………………121
Author Index…………………………………………………………………………………………A - 1
Gravitational and Space Biology 18(2) June 2005 v
vi Gravitational and Space Biology 18(2) June 2005
Symposium I: Model Organisms for
Exploration Biology
Nancy Searby, Editor
Gravitational and Space Biology 18(2) June 2005 1
2 Gravitational and Space Biology 18(2) June 2005 THE YEASTS SACCHAROMYCES CEREVISIAE AND SCHIZOSACCHAROMYCES POMBE: MODELS FOR CELL BIOLOGY RESEARCH Susan L Forsburg University of Southern California, Los Angeles, CA
ABSTRACT organization, budding yeast became a favorite system to Yeast species provide excellent models for fundamental tackle cell biology questions. It was the first eukaryote to biological research. In this review, I will describe be sequenced (Goffeau et al., 1996), which has sparked a characteristics of the two most common laboratory systems: the whole new era developing genomics tools. fission yeast Schizosaccharomyces pombe, and the budding yeast Saccharomyces cerevisiae. They have substantial S. pombe lagged behind as an experimental system. It similarities that make them powerful as research tools, and also was isolated as late as the 1890s from East African millet striking biological differences that make them complementary beer (pombe means beer in Swahili), but limited genetic experimental models. Each provides unique tools for understanding environmental effects on cellular systems. studies only began in the late 40s. It was picked up further in the 60s for studies of growth control, which were facilitated by its regular rod shaped morphology INTRODUCTION (Leupold, 1993; Mitchison, 1990). For some time, fission yeast was a model primarily for cell division and Yeast is a general term, covering a wide range of very sexual differentiation (meiosis). At first, the molecular different single-celled fungi. In the molecular biology tools developed for S. cerevisiae were adapted to S. laboratory, two species are commonly employed as pombe, but were subsequently replaced with pombe- models for biomedical research: the budding or brewer’s specific methods. In recent years, more investigators yeast Saccharomyces cerevisiae, and the fission yeast have chosen to work on S. pombe, and additional areas of Schizosaccharomyces pombe. Although they share the research have opened up, although the community is still designation “yeast”, these two species are evolutionarily smaller than that working on budding yeast. The very distinct from one another, with estimates of up to complete genome sequence was published in 2002 1000 million years (MY) since they diverged from a (Wood et al., 2002). common ancestor (Heckman et al., 2001; Hedges, 2002). Although single cells, they are true eukaryotes, and share In the laboratory, both species are typically maintained as fundamental cell processes with metazoan systems. Each haploid cells. Cells are tolerant of cold and can be stored offers unique tools to the cell biologist, providing frozen at –70°C. Generation time varies with media and complementary approaches and insights into functions of temperature, but is generally in the 2-4 hour range. In larger eukaryotes (e.g., (Forsburg, 1999; Forsburg and response to nutrient limitations, yeast cells exit the cell Nurse, 1991; Lew et al., 1997; MacNeill and Nurse, 1997; cycle and enter stationary phase; this a period of Russell and Nurse, 1986)). Both yeasts are harmless, dormancy, more severe than the G0 phase in mammalian tractable genetic systems, easily manipulated in the cells. A particular strength of both systems is that they laboratory using superb molecular tools (Forsburg, 2001). also have a diploid sexual cycle: haploid cells of opposite mating types can mate, resulting in cell and nuclear fusion. Diploids can be maintained in the laboratory or HISTORY AND CHARACTERISTICS induced to enter meiosis and sporulate. The four spores packaged in the yeast ascus are the fungal equivalent of S. cerevisiae was adopted as a model system for human gametes. Thus, the entire life cycle of yeast cells laboratory study in the 1930s, as investigators developed provides a simple model for events occurring in human genetic tools to understand its life cycle and cells. differentiation (Hall and Linder, 1993). It provided an important tool to understand recombination and the Both cell types have highly organized internal structures transmission of genetic material, and launched into with the membrane-delimited compartments typical of greater prominence with the molecular era in the 70s, eukaryotic cells, including a nucleus, mitochondria, Golgi when it became identified as a sort of eukaryotic E. coli. and other structures. S. cerevisiae grows by budding, With potent genetic tools and a typical eukaryotic cell which requires highly targeted cell growth and mechanisms for spatial coordination with nuclear ______division (Pruyne et al., 2004). S. pombe is a linear, rod * Correspondence to: Susan L Forsburg shaped cell that grows at its ends and divides by medial Molecular & Computational Biology Section fission. This likewise requires significant positional University of Southern California awareness (Hayles and Nurse, 2001). The maintenance of 8335 W 37th St SHS172 cellular organization can be perturbed with drugs or Los Angeles CA 90089-1340 mutants, and provides an interesting problem for effects Email: [email protected] of microgravity. Phone: 213-740-7342; Fax: 213-740-8631
Gravitational and Space Biology 18(2) June 2005 3 S. Forsburg — Models for Cell Biology Research GENOME STUDIES AND SPECIES which have large, degenerate, and diffuse structures. For CONSERVATION example, while the budding yeast origin of replication is a 100bp element with a short, highly conserved consensus Comparison of the genomes reveals significant sequence, the fission yeast origin is 10 times larger (rev. differences between the species (Goffeau et al., 1996; in (Clyne and Kelly, 1995; Newlon and Theis, 1993; Zhu Wood et al., 2002; Wood et al., 2001). Although both et al., 1994)). There is no consensus sequence in the have a haploid genome with over 12 megabases (Mb) of fission yeast origin, and no single mutation abolishes its DNA, haploid S. cerevisaie has 16 chromosomes while S. function (Clyne and Kelly, 1995; Dubey et al., 1996; Kim pombe has only 3. There is no synteny (conserved gene and Huberman, 1998). Importantly, a large, diffuse order), not surprising given their long period of structure appears to be characteristic of metazoan divergence. S. cerevisiae has about 5800 likely protein- replicators as well (rev. in Gilbert, 2001), suggesting the encoding genes. A significant fraction of these are fission yeast origin is particularly appropriate as a model paralogues (genes of related sequence that are not exact for function of higher eukaryotic origins. Similarly, the functional homologues). Evidence suggests that the centromeres of the two species are very different in size budding yeast genome has undergone several large scale and structure (reviewed in Clarke, 1998; Hegemann and duplications through evolution , and following gene loss, Fleig, 1993; Sullivan et al., 2001). In budding yeast, the the remaining duplicates may have diverged in centromere is a small element (about 100 bp) with a expression or function (Dietrich et al., 2004; Kellis et al., discrete consensus sequence. In contrast, the fission yeast 2004; Wolfe and Shields, 1997). Thus, it has proven a centromere is a large, degenerate element of between 40- good model for genome evolution. In contrast, the 100 kilobases (kb) with numerous simple repeats, very fission yeast, with a similar total genome size has about similar to the centromere structure reported for 4800 genes, with no evidence for large scale duplication Drosophila (reviewed in Clarke, 1998; Hegemann and (Wood et al., 2002). Genome organization also differs: Fleig, 1993; Sullivan et al., 2001). For this reason, S. over 40% of fission yeast genes have introns (non-coding pombe has become a particularly powerful system for sequences that interrupt the gene), while a scant 5% of understanding chromosome dynamics. budding yeast genes are interrupted. S. cerevisiae proves to be a better model for other cellular S. pombe has proportionally more genes conserved in functions. For example, S. cerevisiae has been used to metazoans than does S. cerevisiae, although each yeast study the peroxisome, a metabolic organelle that functions species shares genes with metazoans that the other yeast in oxidation of fatty acids and oxidative stress (Brown lacks (Aravind et al., 2000; Wood et al., 2002; Wood et and Baker, 2003; Lazarow, 2003); in contrast, S. pombe al., 2001). Interestingly, these often fall into functional lacks many of the conserved proteins involved in groups (Aravind et al., 2000). For example, budding peroxisomal biogenesis and function ((Wood et al., 2002), yeast has lost many of the genes associated with the and V. Wood, pers. comm.). signalosome, a proteosome-related complex required for diverse signaling pathways that is present in fission yeast Even where functions are superficially similar, study in (Aravind et al., 2000). S. cerevisiae lacks a number of the two yeasts provides unique and complementary genes required for the spliceosome, that is responsible for information. For example, both species were studied removing introns: again, these are genes that are intensely to determine mechanisms of cell division preserved in S. pombe (Aravind et al., 2000). The control (rev. in Forsburg and Nurse, 1991). These studies proteins required for RNA interference (Dcr1, Ago1, allowed identification and characterization of many genes Rdp1; (Hall et al., 2002; Schramke and Allshire, 2003; that are required to regulate normal events in the cell Volpe et al., 2002) are absent from budding yeast, but cycle progression and respond to any defects. However, present in fission yeast. at first it appeared that they used a different regulatory logic: S. cerevisiae regulates its cell cycle at the G1/S Finally, a significant number of chromosome associated phase transition, when DNA replication occurs; in proteins are absent in budding yeast but shared between contrast, data suggested that S. pombe primarily regulates fission yeast and metazoa, including the conserved the decision to initiate mitosis (chromosome segregation). Clr4/SuVar3-9 histone methyltransferase (Bannister et al., Only by combining the data using a “compare and 2001; Nakayama et al., 2001), and the Swi6 and Chp2 contrast” approach was it apparent that both species used HP1-heterochromatin proteins (Eissenberg and Elgin, the same regulatory molecules, and in fact, both control 2000; Ekwall et al., 1995; Halverson et al., 2000; Thon points were present in both species, with the balance and Verhein-Hansen, 2000), telomere proteins shifting between them due to growth conditions. This Taz1/TRF2 (Cooper et al., 1997; Nimmo et al., 1998)and resulted in a powerful experimental synergy that Pot1 (Baumann and Cech, 2001) and the centromere significantly expanded our understanding of cell cycle associated CENP-B proteins (Baum and Clarke, 2000; regulation and provided unique insights into mammalian Irelan et al., 2001; Nakagawa et al., 2002). This cell function. Neither yeast was sufficient alone to explain difference extends beyond gene sequences to cell cycle dynamics in all larger eukaryotes: both yeasts chromosomal elements. Typically, chromosome contributed essential information. structures in budding yeast are very small and streamlined, in contrast to fission yeast and metazoan 4 Gravitational and Space Biology 18(2) June 2005 S. Forsburg — Models for Cell Biology Research These cell cycle genes are conserved throughout metazoa, and Elledge, 2000). Recent data suggest that exposed showing that they define fundamental pathways common single strand DNA (ssDNA) coated by replication protein to eukaryotes. Defects in their function are implicated in A (RPA) as damaged regions are exposed or resected many forms of human cancer (reviewed in (Evan and may be an activating signal for Rad3-family kinases (Zou Vousden, 2001; Fodde and Smits, 2002; Ford and Pardee, and Elledge, 2003). 1999; Maser and DePinho, 2002; Vessey et al., 2000; Wassmann and Benezra, 2001; Willers et al., 2002)). Although the checkpoint proteins are largely in common The significance of these studies in both yeasts was between S. pombe and S. cerevisiae, the details of their recognized by the 2001 Nobel prize that S. cerevisiae responses differ. Unusually, in S. cerevisiae the G2, or geneticist Lee Hartwell and S. pombe geneticist Paul damage checkpoint operates by controlling the metaphase Nurse shared with biochemist Tim Hunt. to anaphase transition, while in fission yeast and metazoans, it operates by controlling the Cdc2 kinase DAMAGE RESPONSE (reviewed in Nyberg et al., 2002). In addition, several of the checkpoint proteins are essential for viability in Further studies have examined the response of normal and normal vegetative S. cerevisiae, due to an additional role mutant cells to specific genome insults caused by ionizing in regulating nucleotide synthesis; while they are not or ultraviolet radiation, alkylating agents, nucleotide essential in S. pombe (Liu et al., 2003; Zhao et al., 1998). starvation, or replication defects, thus providing a These differences provide a further example of the fingerprint of cellular responses that can be used to complementary data obtained by studying both yeasts. identify specific forms of genetic damage. These studies are well-developed and have provided significant insights Many repair and replication genes interact with these into the maintenance of genome integrity. This wealth of checkpoint kinases providing the cell with multiple information provides important tools to study the effects mechanisms to respond to genotoxic insults (rev in of space radiation on living systems, and moreover, (Caspari and Carr, 2002; O'Connell et al., 2000; Rhind provides a clear context to relate results to known effects and Russell, 2000)). There are also differences in the on Earth. repair of DNA damage. Fission yeast is significantly more resistant to UV or ionizing radiation than budding When DNA is damaged or DNA synthesis is blocked, yeast, which suggests additional repair pathways are cells activate checkpoint pathways (rev. in Boddy and operating (discussed in (McCready et al., 2000; Murray et Russell, 2001; Carr, 2002; Caspari and Carr, 2002; al., 1994)). It is known that fission yeast has an Osborn et al., 2002). These pathways recognize a damage additional UV-repair system (the Uve1 endonuclease) not signal, transmit the signal and respond appropriately. found in budding yeast (Yasui and McCready, 1998). They ensure that cells arrest S phase progression, protect Both species, however, show significantly more radio- replication structures, repair any defects, and finally resistance than metazoan cells. Mutants defective in restart the replication process and recover. Checkpoints various repair pathways can be used to categorize the also ensure that the cell chooses the correct mechanism of damage that the cells have suffered. repair for the lesion it encounters. The response to DNA damage in the yeasts is mediated by kinase response Probably the most threatening form of damage that a cell pathways (reviewed in (Carr, 1998; Foiani et al., 1998; can suffer is double strand breaks (DSBs) in the genome. Huberman, 1999; McGowan, 2002; Murakami and Nurse, Therefore, checkpoint and repair pathways operate to 2000; Nyberg et al., 2002; O'Connell et al., 2000; Rhind minimize the conditions where DSBs can occur and and Russell, 1998; Rhind and Russell, 2000; Walworth, maximize efficient repair. Ionizing radiation induces 2000)). The type of damage determines the response: DSBs which also occurs following treatment with the replication fork stalling and exposed single stranded DNA alkylating agent MMS, radiomimetic drugs such as activates one arm of the pathway, and reparable lesions bleomycin, or collapse of unprotected replication forks such as double strand breaks activates the other. A (Kostrub et al., 1997; Memisoglu and Samson, 2000; common group of proteins activates the response. Rhinds and Russell, 2001). DSBs are repaired by two broad pathways (reviewed in Haber, 2000; Krejci et al., Damage is recognized by a ring-shaped structure related 2003). The first is recombination, either error-free to the replication protein PCNA, called the 9-1-1 complex homologous recombination (HR) or error-generating (Sp Rad9-Hus1-Rad1, or ScDdc1-Mec3-Rad17). Just as single strand annealing (SSA). The second is non- PCNA is loaded onto the DNA by the clamp loader RFC, homologous end joining (NHEJ), which is likely to the 9-1-1 complex is loaded by an RFC variant in which generate errors, rearrangements and translocations. HR the RFC1 subunit is replaced by a related, checkpoint and NHEJ may be distinguished by the enzymes they specific protein called SpRad17 (ScRad24). This employ. Mutants lacking these functions are not complex contributes to activation of the ATR homologue surprisingly very sensitive to ionizing radiation and other SpRad3/ScMec1 and its associated activator ATRIP DSB related challenges. (SpRad26/ScDdc2) which in turn activates the appropriate downstream pathway (Caspari and Carr, UV radiation produces a variety of lesions including 2002; Cortez et al., 2001; Murakami and Nurse, 2000; pyrimidine dimers and other photoproducts. These are O'Connell et al., 2000; Rhind and Russell, 2000; Zhou generally repaired by photolyases (light-activated repair Gravitational and Space Biology 18(2) June 2005 5 S. Forsburg — Models for Cell Biology Research enzymes), or by recombination and in some species, particularly in the cell cycle field, has shown that there is including fission yeast, there is an additional excision a terrific synergy to examining phenomena in both repair pathway termed UVDE (rev. in Yasui and species. If the goal is insight into the responses of McCready, 1998). Interestingly, recombination mutants metazoa, particularly human cells, long experience shows in S. pombe are generally also sensitive to UV radiation, that these yeasts provide complementary information, suggested a role for HR proteins in processing UV which has led to important advances in our understanding damage in this species (McCready et al., 2000). of mechanisms of cell growth and regulation. Alkylating agents such as MMS have a range of effects. They typically generate abnormal bases, which are REFERENCES substrates for repair by base or nucleotide excision repair (BER and NER). In addition, MMS damage ultimately Aravind, L., Watanabe, H., Lipman, D. J., and Koonin, E. results in DSBs (discussed in (Memisoglu and Samson, V. (2000). Lineage-specific loss and divergence of 2000)). DNA damage associated with UV and MMS functionally linked genes in eukaryotes. 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Gravitational and Space Biology 18(2) June 2005 9 S. Forsburg — Models for Cell Biology Research
10 Gravitational and Space Biology 18(2) June 2005 WORMS IN SPACE? A MODEL BIOLOGICAL DOSIMETER Yang Zhao1, Robert Johnsen2, David Baillie2 and Ann Rose1. 1 University of British Columbia, Vancouver, Canada V6T 1Z3 2 Simon Fraser University, Burnaby, Canada V5A 1S6
INTRODUCTION
Although it is well known that radiation causes mutational system, muscle, intestine and gonad. Many of the damage, little is known about the biological effects of developmental and biochemical pathways are conserved long-term exposure to radiation in space. Exposure to with human. There is a large community of C. elegans radiation can result in serious heritable defects in researchers taking genetics-based approaches to experimental animals, and in humans, susceptibility to understanding fully the biology of this organism. An cancer, radiation-sickness, and death at high dosages. It is introduction to the C. elegans system and literature has possible to do ground controlled studies of different types been reviewed in Riddle et al.(1997) and is available at a of radiation on experimental animals and to physically web site maintained by Leon Avery at measure radiation on the space station or on space probes. http://elegans.swmed.edu/. At this site there is also However, the actual biological affects of long-term information about up-coming meetings, abstracts, in- exposure to the full range of space radiation have not been house publications, researchers’ contact information, and studied, and little information is available about the current methodologies. biological consequences of solar flares. Biological systems are not simply passive recording instruments. EXPERIMENTAL RESOURCES They respond differently under different conditions, and thus it is important to be able to collect data from a living The genome has been completely sequenced and consists animal. There are technical difficulties that restrict the of approximately 20,000 predicted protein-encoding placement of an experimental organism in a space genes (Sequencing Consortium 1998) and at least 50% of environment for long periods of time, in a manner that C. elegans’ genes have human homologs (McKay, et al. allows for the recovery of genetic data. Use of the self- 2004). A powerful approach for studying loss of gene fertilizing hermaphroditic nematode, Caenorhabditis function technology has recently been developed. elegans offers potential for the design of a biological Double-stranded RNA can be introduced be either dosimeter. In this paper, we describe the advantages of injection into the worm or be feeding. The most widely this model system and review the literature of C. elegans used approach, developed by A. Fire’s laboratory, in space. involves feeding worms bacteria, which are producing double-stranded RNA for the gene of interest. The THE C. ELEGANS SYSTEM ingestion of the dsRNA causes inhibition of gene expression (RNAi) (reviewed in Fire et al. 1999). C. elegans is a well-established animal model, which is Libraries containing bacterial strains for most of the easy to culture in a laboratory. Normally it is maintained predicted protein-encoding genes have been constructed, on agar culture plates and fed a non-pathogenic bacteria. and the phenotype for each of the genes observed (Fraser C. elegans is a self-fertilizing, effectively isogenic et al. 2000; Kamath, et al. 2003). In addition, the targeted hermaphrodite which produces approximately 300 deletion of specific genes has been undertaken by theC. progeny from a single individual. Hermaphrodites have elegans Reverse Genetics Consortium (a collaboration of two X chromosomes while males, which are XO, are the R. Barstead laboratory in the USA, the D. Moerman produced spontaneously as a result of X-chromosome loss laboratory in Canada and the Y. Mitani laboratory in or nondisjunction. Once mated by a male, a Japan), and requests and information are available at hermaphrodite produces out-cross progeny. This is a very http://www.celeganskoconsortium.omrf.org/. An useful feature for doing genetic crosses. The alternate approach to obtaining gene deletions is the use hermaphroditic life style is especially useful for of transposable element insertion and excision by L. maintaining animals over several generations, on for Segalat’s laboratory in France. Mutant strains of the gene example, sustained missions. The complete cell lineage is knockouts are made available by the Caenorhabditis known, including those cells genetically programmed to Genetics Centre (CGC) (http://www.cbs.umn.edu/CGC/). die (apoptosis). The worms are transparent at all The CGC maintains and provides thousands of strains developmental stages, making it possible to examine cell generated by these and classical genetic approaches used division and development in real time. C. elegans is a by the hundreds of researchers that make up the C. metazoan with a number of tissue types, such as nervous elegans research community. In addition there are ______numerous rearrangements, duplications and deletion strains generated by the Genetic Toolkit Project, the * Correspondence to: Ann Rose cosmid transgenetic rescue project, and other researcher University of British Columbia projects. A further aid to mapping mutants is the database Vancouver, Canada V6T 1Z3 descriptions of single nucleotide polymorphisms (SNP) Email: [email protected] between strains. Many of these SNP markers can be Phone: 604-822-5467; Fax: 604-822-5348 assayed by polymerase chain reactions (PCR) followed by Gravitational and Space Biology 18(2) June 2005 11 Y. Zhao, R. Johnsen, D. Baillie and A. Rose — A Model Biological Dosimeter restriction enzyme digestion, making this a fast efficient REPAIR SYSTEMS way to find the molecular basis of a desired phenotype (Wicks, et al. 2001; Swan et al. 2002). A large scale In the case of assaying the response to radiation, reporter project to examine the expression profile of genes with analysis of repair genes would be of interest. C. elegans human homologs (the “C. elegans II” project funded by has a large number of repair genes which function in GenomeBC, D. Baillie, p. comm.) has resulted in the highly conserved pathways, that is the protein sequences construction of more than 2000 Promoter::GFP strains are conserved with both yeast and higher organisms, (McKay, et al. 2004). Large scale identification of including man. A review of the repair pathways (Rose, protein interactions, the ‘interactome’ (Walhout et al. unpublished), and many other aspects of C. elegans 2002; Reboul et al. 2003 Li et al., 2004), and stage- biology, will be accessible in the upcoming worm book, specific serial analysis of gene expression (SAGE) (Jones C. elegans III which will be available on-line in 2005 at et al. 2001; McKay et al. 2004) have been developed. www.wormbase.org. Genes involved in responding to Access to these data and more can be obtained from radiation damage were first identified by Hartman and WormBase http://www.wormbase.org/ ). Herman (1982). The review presents a summary of the genes for which mutant phenotypes have been described, ANALYSIS OF GENE EXPRESSION including components of the pathways for nucleotide excision repair, mismatch repair, DNA damage There are a number of resources for studying global gene checkpoint, non-homologous end joining, homologous expression in C. elegans. Microarray analysis was first recombination repair, and chromosomal structure used by Reinke et al. (2000) to produce a profile of surveillance. A broad perspective on genes involved in germline expression. It has been used subsequently for a DNA repair has been gained using high throughput, number of descriptions of differential gene expression, genome-wide analysis of RNAi phenotypes (Piano et al., including a profile of the genes differentially expressed in 2002; Kamath et al. 2003; Pothof et al., 2003; Lettre et al., the chemically defined CeMM media compared to the 2004; vanHaaften et al., ) and protein interactions (B) and commonly used nematode growth media (NGM) (C. protein interactions (Boulton et al. 2002; Li et al.). As Conley & N. Szewczyk, NASA, p. comm.). In addition to part of the interactome analysis, known proteins microarray analysis, SAGE profiles of each of the implicated in replication, nucleotide excision repair, developmental stages of C. elegans, including the dauer mismatch repair, base excision repair, nonhomologous larvae stage have been done ((Jones et al. 2001; end joining, homologous recombination and checkpoint www.wormbase.org ). Expression of individual genes pathways were used in yeast 2-hybrid experiments to which are turned on in specific developmental stages has identify physical interactors in the predicted proteome been characterized by GFP promoter analysis (McKay, et (Boulton et al. 2002; Li et al. 2004). Components of the al. 2004). In this case, a fluorescent reporter lights up checkpoint signaling networks assemble into more when the gene is transcriptionally active (expressed). For complicated networks. Sensors, transducers and example, a muscle-specific promoter expresses only in mediators are shared when generating different responses muscle (Fig. 1). The potential exists to adapt this including chromatin remodeling, altered gene expression approach to a reporter detection system in space, which and DNA replication. The data demonstrate that many of would record which genes are turned on under particular the pathways are interrelated, and that pathway conditions, for example, responses to lift-off and solar components exhibit previously unrecognized links flares. between repair mechanisms and checkpoints.
A CHEMICALLY-DEFINED MEDIA
C. elegans also has many characteristics that make it an excellent model system for use in space. C. elegans reproduces as a self-fertilizing hermaphrodite, is small (adults are approximately 1 mm long), and thus easily grown in a small space. The life-cycle is short, approximately one-week under conditions at the International Space Station (ISS), and the progeny numbers high, a few hundred per hermaphrodite. The normal food source for C. elegans is bacteria; however for space experimentation it can be fed a chemically defined, axenic media (CeMM) adapted for space travel by C. Conley’s laboratory at NASA, Ames (Lu and Goetsch (1993; Szewczyk N J et al. 2003). The worms will survive for several months in CeMM media, and can be transferred to fresh media and maintained apparently
Figure 1. Promoter expression of the muscle gene, B0228.4 indefinitely. In addition, under conditions of using green fluorescent protein (GFP) as the reporter signal. overcrowding and limited food larvae can enter a dormant “dauer larva” stage. These dauers can survive for several 12 Gravitational and Space Biology 18(2) June 2005 Y. Zhao, R. Johnsen, D. Baillie and A. Rose — A Model Biological Dosimeter months at ambient temperature and will resume their THE ICE-FIRST PROJECT normal life-cycle when introduced to fresh axenic media. In this context, we took part in a recent International For experimental purposes, samples can be prepared collaboration to use C. elegans as a model system for either on the ISS or on the ground for subsequent data biological studies in space, ICE-First (Fig. 2). The project analysis. At the re-entry site the samples can be was coordinated by Michel Viso of the Centre Nationale preserved alive in a frozen state in liquid nitrogen and be d’Etudes Spatiales (CNES) with the help of the European recovered later for biological analysis. Space Agency (ESA) and the Space Research Organization of the Netherlands (SRON) and was flown on the Dutch Science Mission (DSM) to the International Space Station during April 19-30 2004. Researchers from France, Japan, USA and Canada participated. The scientific projects included validation of liquid culturing in the space flight environment; studies on muscle protein growth and maintenance; whole genome microarraay responses to spaceflight; and morphology of larval development during space flight (Table 1). The effects on nematodes being grown for three to four generations in CeMM media and of being in space for the 10 days of the mission is being analyzed by A. Rose’s laboratory for eT1-balanced mutations (Zhao et al., ‘Spaced-out Worms’ abstract available at http://elegans.swmed.edu/) and by D. Baillie’s laboratory for changes in RNA expression (unpublished).
Figure 2. Logo of the ICE-First mission.
C. ELEGANS IN SPACE
Johnson and Nelson (1991) first proposed using C. elegans as a model system for space biology studies. Since then C. elegans has flown on several missions to Earth orbit, and was shown to develop and reproduce normally, making it an excellent model system for biological research in space. Nelson et al. (1989; 1994a; 1994b) investigated mutations induced by cosmic rays in C. elegans on Spacelab in low Earth orbit. Their analysis was for short-term (8 days) radiation exposure. Currently nothing is known about longer term exposure to the different types of radiation in space, nor about the effects of exposure to the range of radiation in the space environment. In his review, Nelson (2003) states that “The unique feature of the space radiation environment is the dominance of high-energy charged particles (HZE or high LET radiation) emitted by the Sun and galactic sources, or trapped in the Van Allen radiation belts. These Figure 3. Dose curve of gamma radiation. Data from charged particles present a significant hazard to space Rosenbluth et al., 1983. flight crews, and accelerator-based experiments are underway to quantify the health risks due to unavoidable ACCUMULATED MUTATION RATE radiation exposure”. Recently, Nelson et al. (2002) MEASUREMENT examined the effect of different types of radiation, gamma rays, accelerated protons, and iron ions at the same The most common type of easily identified mutation is physical dose. Using RT-PCR differential display and that affecting expression of an essential gene (lethals). whole genome microarray hybridization experiments, Lethals will not accumulate if normal animals are used. they described unique transcriptional profiles for the We have developed a system to measure the accumulated different radiation treatments. The genes affected by each mutation rate, the eT1-system (Rosenbluth and Baillie, radiation species were associated with unique regulatory 1981; Rosenbluth et al., 1983) was used. eT1(III;V) clusters, highlighting our limited knowledge of the (eT1) is a reciprocal translocation that recombinationally biological responses to radiation exposure. balances the left half of Linkage Group V [LGV(left)] and the right half of Linkage Group III [LGIII(right)] (Rosenbluth and Baillie 1981) which is nearly 20% of the Gravitational and Space Biology 18(2) June 2005 13 Y. Zhao, R. Johnsen, D. Baillie and A. Rose — A Model Biological Dosimeter nematode’s genome. eT1(III) breaks in unc-36 thus the effects of traveling and living in space. A high giving eT1 a visible phenotype. LGV(left) contains priority could be the development of an accumulating approximately 7% (23 m.u.) of the recombinational dosimeter. NASA, for example, has called for protocols distance in the genome and approximately 10% of its whose objective is to “determine the effects that long- DNA. It is relatively straightforward to calculate forward term exposure to the space environment has over multiple mutation rates using this system, and that has been done generations in space”. for mutagens routinely used in the laboratory, such as EMS and gamma radiation (Rosenbluth et al., 1983; In summary, the resources and knowledge of the C. 1985); formaldehyde (Johnsen and Baillie 1988) and UV elegans system make it an excellent biological model for radiation (Stewart et al., 1991). In the analysis of both studies of gravitational effects on muscle gene exposure to gamma radiation Rosenbluth et al., (1983) expression and mutational consequences of radiation observed that at low doses, the curve was non-linear (Fig. exposure. 3). The data show that low doses of radiation are non- damaging, apparently due to repair mechanisms, which ACKNOWLEDGEMENTS may be very good news for those spending long periods of time in space, if the radiation exposure is low level. We acknowledge funding from the Canadian Space Agency (CSA) for the Canadian participation in the ICE- The eT1 methodology can also be used to determine what FIRST project and GenomeBC/Genome Canada for the types of mutations were generated. That is, are they GFP promoter project. Special thanks to Michel Viso, putative point mutations or are they predominately small CNES for organizing the international participation. or large rearrangements? The mapping can determine if the new mutations occurred randomly or whether there REFERENCES were some mutational “hotspots”. Any putative point mutations can also be identified as new alleles of known Boulton S.J., Gartner A., Reboul J., Vaglio P., Dyson N., genes or as newly identified genes. 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16 Gravitational and Space Biology 18(2) June 2005 DROSOPHILA MELANOGASTER - THE MODEL ORGANISM OF CHOICE FOR THE COMPLEX BIOLOGY OF MULTI-CELLULAR ORGANISMS Kathleen M. Beckingham 1*, J. Douglas Armstrong2, Michael J. Texada1, Ravi Munjaal1, Dean A. Baker2 1 Department of Biochemistry and Cell Biology, MS-140, Rice University, Houston, Texas 77251. 2 School of Informatics, Institute for Adaptive and Neural Computation Edinburgh, EH1 2QL, UK.
ABSTRACT Although the route from mutation to sequenced gene may Drosophila melanogaster has been intensely studied for almost be long and hard, genetics is an extremely powerful 100 years. The sophisticated array of genetic and molecular approach because it can be applied to much more tools that have evolved for analysis of gene function in this complex biological processes than biochemistry. Clearly organism are unique. Further, Drosophila is a complex multi- for fundamental, universal processes that can be studied cellular organism in which many aspects of development and in cell-free extracts, the biochemical approach is highly behavior parallel those in human beings. These combined advantages have permitted research in Drosophila to make effective. But for complex processes, such as seminal contributions to the understanding of fundamental developmental events or behavioral responses, in which biological processes and ensure that Drosophila will continue to many components of the whole organism are in play, provide unique insights in the genomic era. An overview of the genetics offers perhaps the only viable route to dissecting genetic methodologies available in Drosophila is given here, out the protein components. together with examples of outstanding recent contributions of Drosophila to our understanding of cell and organismal biology. The genetic approach has been used to probe gene/protein The growing contribution of Drosophila to our knowledge of function in many organisms. However, the extent to gravity-related responses is addressed. which genetic methods and tools have been developed for
Drosophila melanogaster far exceeds that for any other complex multi-cellular organism. As a result, Drosophila INTRODUCTION stands alone in terms of its ability to provide insight into complex biological processes. Many of the dramatic All biological processes are based on the use of a selected advances in understanding the genetic basis of set of proteins to perform the many steps that underlie the development and behavior have come, and will continue final coherent set of events. It is an implicit assumption to come, from Drosophila. of modern biology that every activity of every organism can be viewed in this way. Given this recognition, two In this review we will first delineate the historical and fundamentally different approaches to identifying these practical reasons that have led to Drosophila becoming proteins can be applied. The biochemical approach takes such a pre-eminent model organism and then discuss the route of actually isolating the proteins themselves and some of the unique advantages and molecular characterizing them, first in vitro and more recently, with methodologies that it now offers for studying gene/protein the advent of recombinant DNA technology, by function. We will then provide examples of biological reintroducing versions of the gene encoding a given processes where genetic work in Drosophila has opened protein back into the cell to test the roles hypothesized for up molecular understanding with relevance across all of the protein. In contrast, the genetic approach begins at the evolution and has permitted insights into our own level of the organism with the proposition that the molecular makeup. In particular, recent work that uses proteins involved in a particular process can be identified Drosophila to address gravity-related phenomena will be by first identifying their genes. The genes are identified discussed. by their ability to disrupt the process in question when mutated. In other words, if a mutation to a particular gene disrupts a particular process, then the protein encoded by THE DEVELOPMENT OF DROSOPHILA AS A that gene must play a role in that process. With this GENETIC SYSTEM approach, much activity is spent in determining what type of protein is encoded by each mutated gene. Mendel's genetic studies of the garden pea were the serendipitous consequence of his duties in the monastery ______garden. In contrast, Drosophila came to be a central * Correspondence to: Kathleen M. Beckingham organism in genetics as a result of careful consideration Dept. Biochemistry and Cell Biology by T. H. Morgan, who, in the early 1900's, was looking Houston, PO Box 1892, TX 77251 for a suitable species in which to perform studies of Email: [email protected] heredity. Drosophila met all his criteria; a small species Phone: 713-348-4016; Fax: 713-348-5154 with a short life cycle that could be easily reared to produce large numbers of progeny. The intense studies of Drosophila that Morgan and his highly talented students (C. B. Bridges, H. J. Muller and A. H. Sturtevant) went on to perform gave rise to many of the concepts that are
Gravitational and Space Biology 18(2) June 2005 17 K. Beckingham, J. Armstrong, M, Texada, R. Munjaal, D. Baker - DROSOPHILA MELANOGASTER fundamental to genetics today. The recognition of genes 1). The maps created for these chromosomes by Bridges as linear arrays on the chromosomes grew directly from (1935) are still in use and the positions of many of the their research, as did the demonstration of recombination deficiencies (and duplications) in the chromosomes used between homologous chromosomes and the chromosomal today in genetic mapping were identified by the changed basis of sex determination (Sturtevant, 1965). banding patterns seen in the polytene chromosomes. A method for hybridizing DNA fragments encoding But beyond these theoretical contributions, the genetic particular genes to the polytene chromosomes was tools evolved from these studies have placed Drosophila developed by M.-L. Pardue (Pardue and Gall, 1969: in its unparalleled position in terms of understanding Pardue et al., 1970) and with the advent of recombinant gene/protein function. In particular, two types of special DNA this provided an even more rapid route to chromosomes were developed as result of their work. positioning a gene within the genome - by hybridizing its Balancer chromosomes were first created by Muller (18). DNA to the polytene chromosome set. These chromosomes with multiple re-arrangements cannot recombine with a homologous chromosome partner, and therefore their use allows particular arrangements of mutations on a given chromosome to be maintained through many generations. Balancer chromosomes are available for all three of the major Drosophila chromosomes and their use is an essential component of most genetic manipulations in the organism. Deficiency chromosomes, each lacking a defined chromosome region, were another offshoot of their work. These chromosomes are invaluable in genetic mapping since a recessive mutation will show a phenotype when paired with a deficiency chromosome that is lacking the region containing the mutated gene. At this point in time, sets of deficiency chromosomes are available from the Drosophila stock centers that cover the entire genome and allow straight forward mapping of a mutation to a unique small chromosome region. These
sophisticated tools are not available for any other Figure 1. The polytene chromosome set of the Drosophila organism. larval salivary glands. The Drosophila chromosome complement consists of two large metacentric chromosomes In addition to these "man-made" genetic tools, the (chromosomes 2 and 3) and the single telocentric X (first) organism itself has conferred further advantages for chromosome. Although seen here, the tiny fourth chromosome genetic studies. The rich array of external appendages is rarely visible. The tips of the left and right arms of (multiple bristle types, wings, eyes, antennae and so on) chromosomes 2 and 3 are labeled, as is the single arm of the X. that can be mutated without producing lethality has The chromosomes are fused at their centromeres at a structure allowed for the isolation of many marker mutations that which is called the chromocenter. The banding patterns seen here are highly reproducible. Genes can be mapped to are the work-horse tools of most genetic schemes. In individual bands by hybridization of their cloned DNA to addition, several unusual aspects of Drosophila biology preparations of the chromosomes. have proved extremely valuable. These biological eccentricities were not known, or anticipated, at the time of Morgan's decision to work on Drosophila. Along with THE TRANSPOSON BASED TOOL KIT FOR IN- insects from several other evolutionary groups, members DEPTH MOLECULAR GENETIC ANALYSIS IN of the Genus Drosophila do not show meiotic DROSOPHILA recombination in the male. This provides another useful mechanism in genetic schemes for avoiding The revolution initiated by the invention of DNA cloning rearrangements in arrays of mutations assembled on technology has enhanced gene function analysis not just particular chromosomes. In addition, Drosophila belongs in Drosophila but in all organisms under study. One to a group of organisms that makes extensive use of an special consequence in Drosophila, however, was that this unusual growth mechanism in certain tissues: DNA methodology permitted the molecular basis of an unusual replication proceeds without nuclear division or genetic phenomenon termed hybrid dysgenesis, identified cytokinesis such that polyploid nuclei, often with and studied by M. Kidwell, to be explained. Kidwell thousands of copies of the chromosomes, are produced in (Kidwell et al., 1977) discovered that high levels of huge cells. In some tissues the chromosome copies are mutagenesis occurred when certain strains (termed P and retained together in perfect register such that giant, so- M strains) of Drosophila were crossed to one another, but called polytene, chromosomes with intricate, reproducible only if the P strain was used to provide the male parent, patterns of bands are produced. The polytene not the female. O'Hare and Rubin (1983) established that chromosomes of the Drosophila larval salivary glands are the behavior of a class of transposable DNA elements, particularly fine and have been intensely studied (Figure 18 Gravitational and Space Biology 18(2) June 2005 K. Beckingham, J. Armstrong, M, Texada, R. Munjaal, D. Baker - DROSOPHILA MELANOGASTER present only in P strains and hence termed P elements, is introducing different modified versions of a gene into its the underlying basis of hybrid dysgenesis. Movement of null genetic background through P element P elements is repressed in the P strains but in crosses to M transformation, the ways in which the modified gene can strains, which lack the elements, repression is released. support the complete set of functions of the normal gene Transposition of the elements to new locations in the can be addressed. For example, by this approach an genome is thus allowed, and as a result, new mutations investigator could address the function of particular are generated. regions of the protein coding sequence in vivo, by determining the ability of appropriately modified gene Since their discovery, the biology of P element constructs to restore the mutant phenotype to normalcy. transposons has been exploited to generate an impressive variety of molecular tools for in vivo analysis of gene But P elements have been put to even more sophisticated function in Drosophila. In initial ground-breaking work, genetic uses. Three powerful P-based methodologies Spradling and Rubin (1982; Rubin and Spradling, 1982) deserve special mention. One technique addresses the made modified versions of P elements and demonstrated central quest in genetics - that of determining, once a that these could be used to reintroduce cloned genes back mutant phenotype of interest has been identified, which into the organism. The benefits of this so-called gene is affected by the mutation. Most of Drosophila transformation technique are multiple. First, it permits genetics to date has relied upon chemically induced genetic rescue of mutations - that is, reversion of a mutant mutations. Such mutagens typically produce single base phenotype to the normal wild type state by re-introduction changes in DNA. Given that the genome of Drosophila is of a "good" version of the gene. But more importantly, ~ 1.65 x 108 bases in total, identifying the location of that when combined with the extensive mutant collections single base change has some of the quality of finding a available in Drosophila, it permits a detailed analysis of needle in a hay stack. Without the sophisticated gene function not possible in other organisms. Thus, for genetic/molecular methods developed in Drosophila, this many genes, a completely non-functional mutation (null task would be impossible, but even with these tools, it can mutation) of the gene has been isolated previously. By take years of effort to identify the genes affected by such
Figure 2. The Gal4 system for directed gene expression. The upper diagram shows a Gal4 P element transposon (pGawB - see Brand and Perrimon, 1993) inserted into the Drosophila genome in a typical position close to an enhancer element that would normally regulate an adjacent gene (shown here to the right of the transposon). The Gal4 coding sequence within the transposon will be expressed in the pattern dictated by the enhancer element. In addition to the Gal4 gene, pGawB contains the white +gene (a marker used to identify successful integration of the transposon into the genome) and bacterial plasmid sequences (amp ori) to allow cloning of the DNA adjacent to the insertion site. In order to identify the Gal4 expression pattern produced by the enhancer, flies carrying the pGawB insertion are crossed to flies carrying a second P-based insertion that has the bacterial β galactosidase (lacZ) gene linked to a promoter region carrying five Gal4 binding sites (UAS sequences) (see A in figure). The progeny of this cross will carry both constructs in their genomes and thus will express lacZ in the pattern of the Gal4. Given that lacZ is very easily detected histochemically, this allows simple determination of the Gal4 expression pattern. Once the pattern is known, it can be used to express any gene of interest (see B in figure) by a similar procedure.
Gravitational and Space Biology 18(2) June 2005 19 K. Beckingham, J. Armstrong, M, Texada, R. Munjaal, D. Baker - DROSOPHILA MELANOGASTER mutations. P elements, by inserting themselves into the An example from the Beckingham lab. will illustrate this genomic DNA, naturally act as mutagens. In order to be point (Wang et al., 2002). We identified a mutation to the able to determine very rapidly which gene is affected by a universally expressed calcium signaling protein particular P element insertion, versions of P elements that calmodulin that affected muscle behavior in the permit the rapid and simple cloning of the DNA adjacent larval/pupal stages. We wished to determine whether to their insertion point have been generated (Cooley et al., these defects reflected problems in the musculature itself, 1988). Given that the Drosophila genome is completely in the nervous system innervating the muscles, or perhaps sequenced and at an advanced level of annotation, in most elsewhere in the animal. By expressing wild type cases, sequencing of the P-element adjacent DNA permits calmodulin in either the muscles or the nervous system immediate and unambiguous positioning of the P using this technique, we were able to show convincingly insertion within the genome. This ability to make that the defects originated entirely within the muscles "tagged" mutations that readily lead to the affected gene is themselves. a powerful advantage that is now being used throughout the Drosophila community. Although the Gal4-UAS system allows directed gene expression in particular tissues it does not permit temporal A second P element based technique allows the control over when, in the development of that tissue, the identification of the regulatory elements associated with gene will be expressed. However, systems are now particular genes. As a result of the molecular analysis of evolving to address this need to control gene activation in the insertion points of thousands of P elements, it has terms of not just tissue but also time (reviewed in become clear that they preferentially insert into regions of McGuire et al., 2004). Modification of the Gal4-UAS genes that contain sequences regulating the expression of system so that Gal4 is activated only in response to the genes. This is an intriguing finding and presumably feeding the hormone analog RU486 is one approach; use reveals some aspect of chromatin function that has yet to of a temperature sensitive version of the Gal4 repressor be understood. Many of these regulatory sequences, protein Gal80, which represses Gal4 at permissive termed enhancers, lie in the 5' flank of the transcribed temperatures but allows Gal4 expression upon shift to region. Modified versions of P elements have been higher temperatures, is another. generated that will report the expression pattern dictated by an enhancer flanking a P insertion site (O'Kane and Many proteins have vital roles in different tissues at Gehring, 1987). These modified versions contain the different stages of development. A null mutation in such coding sequence for a bacterial protein β galactosidase a gene will produce death at the first point in the life cycle (lacZ) but with no associated transcription regulatory of the organism at which the protein is essential. Thus, a elements. As a result there is no expression of the lacZ in classic null mutation cannot be used to address the roles Drosophila unless its host P element is inserted so as to of the protein beyond this point of death. For any allow an adjacent enhancer to confer an expression recessive mutation, this problem can be overcome by so- pattern on lacZ. The pattern of lacZ expression has been called mosaic analysis. In this approach, the whole examined in hundreds of these so-called "enhancer trap" organism is heterozygous for the mutation and therefore lines and thus regulatory sequences that give gene phenotypically wild type. Then, some method is used to expression in many different tissues have been identified. render the mutation homozygous in particular cells of the body so that the effects of the mutation can be addressed Building on this approach, a further powerful P element in those cells. Although classical Drosophila genetics based system (the GAL4-UAS system -see Figure 2) has provided some techniques for generating mosaic animals been developed that allows patterns of enhancer-driven they were cumbersome and not generally applicable to expression to be converted into the expression of any any gene. Another advance that has grown out of the gene of interest (Brand and Perrimon, 1993; Duffy, 2002). ability to readily transfer gene constructs into the genome This is achieved by using the gene for a yeast with P element vectors is a generally applicable system transcription factor, Gal4, in place of lacZ, in the for generating mosaic animals (Xu and Rubin, 1993). "enhancer trap" P construct and then introducing into the This system, termed the FLP-FRT system (Figure 3) uses genome, in a second P element, the gene of interest under a recombinase enzyme (FLP) from yeast that will induce the control of a promoter that will be activated by Gal4 sequence-specific recombination at a site termed FRT. binding. Thus the gene of interest will be expressed FRT sequences are appropriately positioned on two wherever Gal4 is expressed. The resulting ability to homologous chromosomes one of which carries a express particular genes in pre-selected tissues has many mutation of interest. FLP activity is then used to induce valuable applications. For example, although a gene of an illegal recombination event that will result in a cell in interest may be expressed in many tissues, the phenotype which both homologous chromosomes carry the mutation. of a particular mutation to the gene may result from By using versions of the FLP enzyme that are under the defects in only one of these tissues. This hypothesis can control of either tissue-specific enhancers or a promoter be tested by expressing the wild type, non-mutant, version induced by heat-shock, cell lineages homozygous for the of the gene in particular tissues of the mutant animal and mutation can be induced in either particular tissues, or determining in which tissues the mutant phenotype is throughout the animal at particular times. rescued.
20 Gravitational and Space Biology 18(2) June 2005 K. Beckingham, J. Armstrong, M, Texada, R. Munjaal, D. Baker - DROSOPHILA MELANOGASTER It is important to recognize that, effective as these methods for re-introducing genes into the genome target transposon-based methods are for gene analysis, any gene their integration by homologous recombination so that reintroduced into the organism in this way is not inserted they are substituted for the endogenous gene at the correct so as to replace the version of the gene already present in chromosomal locus (Thomas and Capecchi, 1987; Petes et the genome. Hence as alluded to above, to examine the al., 1991). Recently methods for this type of targeting of function of the transgenic construct without interference genes have also been developed in Drosophila and will be from the endogenous chromosomal version of the gene valuable for those genes for which a null mutation is not requires the pre-existence of a null mutation of the gene in available (Rong and Golic, 2000). question. In contrast, in the mouse and in yeast, the
Figure 3. The FLP-FRT system for generation of mosaic tissues. The upper panel shows a pair of homologous chromosomes in a single cell of an animal that has been generated to allow mosaic analysis of a particular mutation (represented here by a star symbol on one of the chromosomes). The cell is heterozygous for the mutation and therefore phenotypically normal. The chromosomes are shown in G2 of the cell cycle, when sister chromatids for both chromosomes are present. FRT sequences are present at identical positions on the two chromosomes. Flp recombinase, expressed from another chromosome in the cell, causes an illegal recombination event between the FRT sequences on chromatids of the homologous pair. As a result, the two chromatids containing the mutation are now attached to different centromeres (see middle panel). After mitosis, when chromatids derived from each chromosome pair have segregated to the daughter cells (lower panel), one cell will be homozygous for the mutation and the other wild type. Reproduced with permission from Nature Reviews in Genetics 3, 176-188, 2002 (www.nature.com/reviews) copyright 2002, Macmillan Magazines Ltd.
Gravitational and Space Biology 18(2) June 2005 21 K. Beckingham, J. Armstrong, M, Texada, R. Munjaal, D. Baker - DROSOPHILA MELANOGASTER THE INITIAL UNLEASHING OF THE POWER OF the biological phenomenon that interested him most - that DROSOPHILA GENETIC SCREENS of behavior. Although Benzer received considerable criticism for attempting to unravel complex phenomena The mutations initially studied by Morgan's group were one gene at a time, the genetic screening initiated in his naturally occurring ones. Indeed the discovery of the first lab. identified mutants that have played ground-breaking white-eyed mutant fly by Morgan was a defining moment roles in understanding such universal phenomena as in Drosophila genetics. The critical advance of directly circadian rhythms, courtship and mating activity, and generating mutations was pioneered by Muller, when he learning and memory. Mutants of the gene period were initiated the use of ionizing radiation as a mutagen isolated by Konopka in Benzer's lab (Konopka and (Muller, 1927). Subsequently, more easily useable Benzer, 1971) and subsequent cloning of the period gene chemical mutagens - in particular DNA alkylating agents by the labs of Hall (a Benzer post-doc) and Rosbash such as ethylmethane sulfonate - have been identified and (Reddy et al., 1984) has led to recognition of its pivotal used. The ability to generate mutants in a scattershot role in regulating diurnal activity across all evolutionary manner throughout the genome, led to the concept of the orders. The first learning mutant, dunce, isolated in the genetic screen; that is, a two-stage experimental protocol Benzer lab by Quinn (Dudai et al., 1976) proved to whereby mutants are generated and then those that affect encode an enzyme involved in cyclic nucleotide a particular process of interest are isolated and studied. metabolism. Along with rutabaga, another Quinn Although genetic screening of this type was established in mutation that affects adenylyl cyclase (Livingstone et al., microbial systems relatively early, it was not pursued in 1984) these learning mutants have provided part of the complex multi-cellular animals initially for two now large body of evidence demonstrating the role of compelling reasons: first, in most species existing genetic cyclic AMP in learning and memory in all biological analysis was too primitive to permit in-depth analysis of systems. mutants, and second, the resources and time necessary for multi-generational analysis of hundreds or thousands of mutant lines made such screening completely impractical c) Mutants in embryonic patterning in most species. Nusslein-Volhard and Wieschaus as young new In essence, Drosophila was the only complex animal independent investigators at the EMBL labs in species for which these two criteria could be met and in Heidelberg, undertook a large scale screen to identify the 1970's directed screening efforts to identify mutants in genes that affect formation of the Drosophila embryonic particular processes were initiated. Three of these early body plan. A large number of mutations were produced, screening efforts deserve particular mention for the far- some with remarkable effects on the final form of the reaching impact of their findings. animal (Nusslein-Volhard and Wieschaus, 1980; Nusslein-Volhard et al., 1984; Jurgens et al., 1984). For a) Mutants in visual processes example, the even-skipped and odd-skipped mutations generate larvae in which every other body segment is Pak (1979; 1995) used an electrophysiological assay to missing. The impact of this screen cannot be over- identify mutants which altered the electroretinogram of emphasized. Determining what kinds of genes were the fly and thus, by implication, to find genes with roles behind such unusual phenotypes was of great interest. in Drosophila phototransduction and other vision-related Further, this work appeared just at the moment when events. Several of these mutations have proved to affect cloning the gene affected by a given mutation was genes with general roles in intracellular signaling becoming feasible. The cloning and further study of processes and thus they provide insights with broad genes identified by this screen is one of the seminal significance into intracellular regulation. The trp contributions of Drosophila to modern biology. Many of mutations in particular have played an enormously these genes are conserved in higher species and over the important role in advancing understanding in neural past 20 years we have seen the mammalian homologs signaling in mammalian systems. Originally named for identified and their fundamental roles in vertebrate cell the electrophysiological effect of the mutants (a transient function established. Indeed whole conserved pathways receptor potential,) the trp gene proved to encode a novel used across evolution to execute similar functions have kind of ion channel. Trp is now recognized as the been identified. The Hedgehog signaling pathway (Lum founding member of a vast and varied family of related and Beachy, 2004) is a striking example of the impact of channels used throughout the nervous system across this screen: besides roles in embryonic development in all evolution and with roles in many sensory processes such species, this pathway plays a critical part in regulation of as taste, touch, pain and temperature perception tissue growth in adult vertebrates and is implicated in the (Clapham, 2003; Huang, 2004). transition to tumor growth in several cancers (Scott, 2003). Three key members of this pathway (Hedgehog, b) Mutants in behavioral processes Patched and Smoothened) were identified by the Nusslein-Volhard/Wieschaus screen. Benzer, after his successful exploitation of bacteriophage genetics to address the nature of the genetic code, turned his attention to Drosophila genetics as a route to exploring 22 Gravitational and Space Biology 18(2) June 2005 K. Beckingham, J. Armstrong, M, Texada, R. Munjaal, D. Baker - DROSOPHILA MELANOGASTER FURTHER DEVELOPMENT OF GENETIC expressed a fluorescent marker protein (GFP -for cell SCREENING IN DROSOPHILA identification) and a gene that causes the cells to transition to non-invasive tumorous growth. In addition, Numerous Drosophila screens have been carried out since this recombination event rendered the cells homozygous this period and genes with roles in processes as diverse as for one of a collection of random mutations being tested olfaction (Woodward et al., 1989), hearing (Eberl et al., for their effects in a non-invasive tumor cell. Those 1997), sensitivity to ethanol (Guarnieri and Heberlein, mutations that caused a transition to metastatic growth 2003) and development of various organ systems have could then be easily identified since fluorescent cells were been identified. As proved true for the embryonic no longer confined to the eye, but rather were spread patterning screens, the extensive screening in the Rubin throughout the organism. No other organism allows this lab. focused on eye development (e.g. Therrien et al., kind of sophisticated, directed approach to identifying 2000; Greenwald and Rubin, 1992) has uncovered genes genes with roles in metastasis. The implications of this and pathways that are universally conserved. The so- particular screen in terms of understanding human cancer called Pax-Six-Eya-Dach signaling network is one such are obvious. conserved pathway to emerge from this work (Ozaki et al., 2004). In addition, a "second order" class of screens has been developed that allows further proteins in a given DROSOPHILA IN THE GENOMIC ERA pathway to be identified once one pathway member has been isolated through mutation. These so-called genetic With the sequencing of the genomes of several model interaction (or enhancer/suppressor) screens involve organisms, a new so-called genomics era has arrived. searching for second site mutations that suppress or Interestingly, the genomes of the vertebrate model enhance the phenotype of a given mutation in a starting organisms have proved to be "quasi polyploid" in that gene of interest. The underlying assumption here is that three or four highly related copies of many genes are only mutations that affect protein products that interact present. Pleasingly this is not true of Drosophila; most with one another, or at least have roles in the same Drosophila genes are unique and the complication of process, could modify one another's phenotype in this genetic redundancy is thus greatly diminished. way. The new central approach of the genomics era is whole As discussed above, a major advance for genetic genome gene expression analysis using probes for all the screening has involved the development of modified P genes in the organism displayed by micro-array transposons that can be used as insertional mutagens that technology. This approach compares expression patterns then allowed rapid cloning of the adjacent DNA (Cooley for two different experimental situations, such as a et al., 1988). This approach is now widely in use, and our particular tissue before and after some experimental own studies of gravitaxis took advantage of this approach treatment, or at two different stages of development. (see below). But in addition, genetic screening methods Again, Drosophila brings particular advantages to this have been developed that take advantage of some of the type of approach. Most notably, the ability to pursue more sophisticated P element based tools discussed array-based expression data by subsequent study of above. One such technique allows the identification of mutants and altered gene constructs adds a depth of genes with roles in particular processes by examining the analysis not possible in most organisms. effects of their over-expression or mis-expression in particular tissues (Rorth, 1996). The technique uses Gal4 A wealth of Drosophila community resources has expressed in a specific pattern to activate randomly developed over the years including bioinformatic tools to inserted UAS-type promoter elements and thus to initiate handle the large data sets now accumulating from expression of any endogenous gene adjacent to the genomic studies. Several stock centers around the world insertion in the pattern determined by the Gal4 expression maintain both classical and transgenic mutants. A variety characteristics. For a fraction of the genes in Drosophila, of international consortia make datasets and molecular even null mutations do not produce a phenotype, perhaps reagents available to the community at large. The as a result of functional redundancy. This new method is FlyBase database ([email protected]) maintains a list valuable in that it permits functional analysis of such of Drosophila researchers, references, genome and mutant genes. information, and many user-contributed articles and tutorials. Of particular note is the Berkeley Drosophila By combining aspects of the Gal4-UAS system, the FLP- Genome Project (BDGP: http://www.fruitfly.org/) which FRT system and mutagenesis extremely sophisticated maintains developing genomic resources such as banks of screens are now being developed and executed in cDNAs and stocks of newly generated gene disruption Drosophila. One example will suffice. In order to mutants. There is now also an initiative to maintain identify genes that might contribute to the transition from extensive raw datasets in a central, searchable database; a non-invasive to a metastatic tumor, Pagliarini and Xu FlyMine (http://www.flymine.org). FlyBase is a central (2003) devised a scheme in which FLP-FRT was used to repository for links to developing bioinformatics tools. generate mosaic cell lines in developing eye tissue. As a result of a single FLP-induced recombination event they With the genome completely sequenced, the Drosophila could generate a clone of cells that simultaneously community has begun development of tools for the Gravitational and Space Biology 18(2) June 2005 23 K. Beckingham, J. Armstrong, M, Texada, R. Munjaal, D. Baker - DROSOPHILA MELANOGASTER ultimate phase of genomic research - a full understanding phenotype. By this route, completely novel genes of the function of each individual gene. Two useful tools involved in disease progression will be identified. In are already available for this work. First, a large fraction time, this will provide new potential targets for of the proteins of the Drosophila proteome have been development of therapeutic drugs. tagged with GFP by genomic insertion of an appropriate reporter construct (Kelso et al., 2004; http://flytrap.med.yale.edu). The GFP tag permits the DROSOPHILA'S CONTRIBUTION TO RESEARCH expression pattern and subcellular location of the protein INTO GRAVITY- BASED RESPONSES from a given gene to be easily investigated. Second, as a complement to standard mutations, libraries of DNA a) Earth-based studies constructs that can be used to create mutant phenotypes by RNAi-mediated mRNA destruction have been Interestingly, geotaxis (now more correctly termed generated (http://www.hgmp.mrc.ac.uk/geneservice). gravitaxis) was one of the first behaviors investigated in Currently, constructs for ~90% of the Drosophila genome Drosophila at the beginning of the last century (Carpenter, are available. In addition, a large collection of new 1905). However, further work was not undertaken again gene/small genomic deletions has been made by the until the late 1950's/ early 1960's when Hirsch began company Exelixis, using more recently generated extensive studies of the phenomenon. At that time, the transposons such as piggybac, razorbac and warthog. issue of inheritance of behavioral traits was of intense These will aid studies of uncharacterized genes interest because of its far-reaching social implications in (http://drosophila.med.harvard.edu/index.php?option=con terms of human behavior. Hirsch's primary goal was thus tent&task=view&id=5&Itemid=27). to identify defined behavioral responses and then to address their heritability. He designed vertical multi- choice mazes to examine gravitaxic behavior in walking, DROSOPHILA AND HUMAN DISEASES as opposed to flying, flies (Hirsch, 1959). By selecting flies from a wild type population that emerged either at The promise of using the capacity for deep genetic the high or the low maze exits he was able to demonstrate analysis in Drosophila to advance molecular stable heritance of the maze behavioral traits over many understanding of specific human diseases has recently generations with maximal behavioral separation between begun to be realized. The major approach to date in high and low lines occurring after 48 generations (Hirsch modeling a human disease in Drosophila has been to and Erlenmeyer-Kimling, 1961). Today, after more that express the protein product associated with a particular 700 generations, these lines still breed true in terms of disease state in the fly and then to establish whether the their maze behavior. changes associated with the disease are mimicked in this organism. For three major classes of neurodegenerative Erlenmeyer-Kimling and Hirsch (1961; Hirsch and diseases, Alzheimer's, Parkinson's disease and the Erlenmeyer-Kimling, 1962) established that the polyglutamine class of diseases (exemplified by determinants for these traits were dispersed throughout Huntington's disease) this approach has been successful the chromosome set, but prevailing methodology and the model disease state generated in the fly is being precluded actually identifying any of the loci involved. used for further analysis (Bonini and Fortini, 2003; Iijima More recently however, Toma et al. (2002) have used the et al., 2004). Candidate genes that might modify the genomics approach described above to identify these loci. severity of the disease-related phenotype (see suppressor They compared patterns of gene expression in the high and enhancer screens above) are being investigated. For and low strains by DNA microarray technology and both Parkinson's and the polyglutamine diseases, this identified several genes for which expression levels approach has already established that chaperone proteins differed significantly between the two strains. For three such as hsp70, which regulate protein folding, can genes, they were able to use previously identified mutants ameliorate the disease phenotype (Bonini and Fortini, of the loci to demonstrate that the genes had roles in 2003). Current indications also suggest that general gravitaxis. Thus mutants at these genes showed altered insights into the biological function of the disease protein behavior in the maze. (or its interaction partners) will come from this strategy of expressing in Drosophila. For example, over-expression We have taken an alternative approach to identify genes in Drosophila of APP, the protein associated with with roles in gravitaxis (Armstrong, Texada, Munjaal, Alzheimer's disease, produces a blistered wing phenotype Baker and Beckingham, manuscript submitted). We have (Fossgreen et al., 1998). This defect is associated with used the gravitaxic maze as the central assay of a genetic failed cell-cell adhesion in the wing epithelia and suggests screen to identify mutants with altered behavior in this that the processing enzyme that modifies APP (which is gravitaxic response. By examining mutations caused by a already present in Drosophila) might also play a role in Gal4 expressing "enhancer trap" type P transposon (see processing critical cell adhesion molecules. above), we have been able to quickly move from the mutants we have identified to the affected genes. The However, the great power of this Drosophila disease actual sense organs that detect gravitational force are not modeling approach lies in large scale genetic screening known in Drosophila. However, recognizing that Gal4 for mutations that enhance or suppress the disease will be expressed in those tissues that are the likely 24 Gravitational and Space Biology 18(2) June 2005 K. Beckingham, J. Armstrong, M, Texada, R. Munjaal, D. Baker - DROSOPHILA MELANOGASTER origins of the mutant phenotype, we have been able to by both Toma et al. and Bhattacharya and colleagues identify candidate peripheral sense organs that might (Table 1). The Bhattacharya lab has also identified a role mediate graviperception. In particular, a set of sensory for conserved genes of the cAMP pathway (dunce, structures on the Drosophila head show strong expression rutabaga) known to be involved in learning and memory of Gal4 in many of our mutant lines (Figure 4). in addition to genes affecting longevity and stress (methuselah and I’m not dead yet). The roles of these Bhattacharya and collaborators are addressing another various genes are thus much broader than the original behavioral response to gravity in Drosophila (Sanchez, studies of them might have indicated. Further, it is hard Stowers, Shenasa, Fahlen and Bhattacharya, manuscript to imagine any route by which the roles of these genes in in preparation). They have established that upon gravity-based responses could have been identified in any subjecting flies to hypergravitational force (1.8g-8g) a mammalian system, given the limited methodology highly stereotypic locomotor response is initiated, the currently available in these organisms. But with the features of which suggest that increased g force stimulates characterization of these genes in Drosophila, the an adaptive program of gene expression. Whole genome possibility for mammalian studies is now open. micro-array studies and candidate gene approaches are Hopefully future studies will address the roles of these being used to identify genes important for this locomotor proteins in gravitaxic responses in mammals. response. The mushroom body in the Drosophila brain, which is important for olfactory learning and memory and b) Micro-gravity experiments for locomotion, has been shown to be involved in regulating this behavioral response to hypergravity. Our The features of Drosophila that make it ideal for genetic de novo forward genetic screen studies have identified studies of a multi-cellular organism also make it ideal for several novel genes with roles in gravitaxis. But a experiments in the micro-gravity environment. On the striking aspect of all three of these approaches is that International Space Station (ISS), where mass and volume many of the gravity-related genes identified have clear are critical issues, the small size and fecundity of homologs in human beings. As shown in Table 1, several Drosophila make it possible to use large numbers of of these genes have predicted roles in neural signaling or individuals for experiments - at least two orders of morphogenesis. Interestingly, gravity-related genes with magnitude more than could be used with mammals. roles in regulating circadian activity have been identified Further, the ease with which Drosophila can be reared in
Figure 4. Gal4 expression patterns in facial sense organs of two Drosophila gravitaxic mutants. Heads from two different mutant fly lines with altered gravitaxic behavior in vertical mazes (see text) are shown. Each mutation is caused by a P{GawB}transposon insertion and shows Gal4 expression in the pattern of an adjacent enhancer sequence associated with the affected gene (see Figure 2). A. Head of a fly from the yuri gagarin mutant line. B. Head of a fly from the neil armstrong mutant line. The expression patterns for Gal4 were visualized by crossing each Gal4 transposon line to a UAS-lacZ line (see Figure 2) and detecting lacZ by a histochemical stain in the progeny. In each line, three pairs of sense organs in the head stain prominently for lacZ - these are the antennae (uppermost), the vibrissae (middle) and the maxillary palps (lower). Each of these structures contains mechano-sensory receptors that are therefore candidates for sensing gravity.
Gravitational and Space Biology 18(2) June 2005 25 K. Beckingham, J. Armstrong, M, Texada, R. Munjaal, D. Baker - DROSOPHILA MELANOGASTER
TABLE 1 Genes with Roles in Gravitaxic Behavior in Drosophila that Have Conserved Homologs in Humans
Gene Protein Function
aPendulin (Pen) Nuclear importin acryptochrome (cry) Photopigment protein with role in circadian rhythms aPigment-dispersing Factor (Pdf) Neuropeptide that mediates circadian locomotor activity bbroad (br) Zinc-finger class transcription factor boff-track (otk) Receptor tyrosine kinase with role in neural pathfinding (Human homologs are Trk neurotrophin receptors) bdiscslarge (dlg1) MAGUK class protein with guanylate kinase, PDZ, SH3 and P-loop domains with a role in synapse structure (Human homolog is chapsyn-110) bescargot (esg) Zinc-finger class transcription factor with role in peripheral nervous system development (Human homolog is SLUG) bConnector of kinase to AP-1 (Cka) WD-40 domain protein, part of JNK signaling cascade (Human homologs are striatin, zinedin and cell cycle autoantigen SG2NA) cperiod (per) Transcription factor that regulates circadian rhythm (Human homolog is PER3) ctimeless (tim) Transcription factor that regulates circadian rhythm (Human homolog is hTimeless) cdunce (dnc) Phosphodiesterase that regulates cAMP levels (Human homolog is cAMP-specific 3', 5'-cyclic phosphodiesterase 4D) crutabaga (rut) Adenylate cyclase responsible for cAMP synthesis (Human homolog is brain adenylate cyclase 1) cI’m not dead yet (indy) Sodium dicarboxylate cotransporter implicated in longevity (Human homolog is NADC3) cG-salpha60A Component of transmembrane receptor signal transduction cascade involved in associative learning in mushroom body (Human homolog Guanine nucleotide-binding protein G-s-al 3)
a = Toma et al. (2002); b = Armstrong et al., submitted (see text); c = Sanchez et al, in preparation (see text).
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Kelso, R.J., Buszczak, M., Quinones, A.T., Castiblanco, Pagliarini, R.A. and Xu, T. 2003. A genetic screen in C., Mazalupo, S. and Cooley, L. 2004. Flytrap, a database Drosophila for metastatic behavior. Science 302: 1227- documenting a GFP protein-trap insertion screen in 1234. Drosophila melanogaster. Nucleic Acids Research 32: D418-D420. Pardue, M.L. and Gall, J.G. 1969. Molecular hybridization of radioactive DNA to the DNA of Kidwell, M.G., Kidwell, J.F.and Sved, J.A. 1977. Hybrid cytological preparations. Proceedings of the National dysgenesis in Drosophila melanogaster: a syndrome of Academy of Sciences USA 64: 600-604. aberrant traits including mutation, sterility and male recombination. Genetics 86: 813-833. Pak, W.L. 1979. Study of photoreceptor function using Drosophila mutants. In: Neurogenetics: Genetic Konopka, R.J. and Benzer, S. 1971. Clock mutants of approaches to the nervous system. (Breakfield , X. Ed.) Drosophila melanogaster. Proceedings of the National New York: Elsevier-North Holland, pp. 67-99. Academy of Sciences USA 68: 2112- 2116. Pak, W.L. 1995. Drosophila in vision research. The Livingstone, M.S., Sziber, P.P. and Quinn, W.G. 1984. Friedenwald Lecture. Investigative Opthalmology and Loss of calcium/calmodulin responsiveness in adenylate Visual Science 36: 2340-2357. cyclase of rutabaga, a Drosophila learning mutant. Cell 37: 205-215. Pardue, M.L., Gerbi, S., Eckardt, R.A. and Gall, J.G. 1970. Cytological localization of DNA complementary to Lum, L. and Beachy, P.A. 2004. The Hedgehog response ribosomal RNA in polytene chromosomes of Diptera. network: sensors, switches, and routers. Science 304: Chromosoma 29: 268-290. 1755-1759. Petes, T.D., Malone, R.E. and Symington, L.S. 1991. In: McGuire, S.E., Roman, G. and Davis, R.L. 2004. Gene Molecular and cellular biology of the yeast expression systems in Drosophila: a synthesis of time and Saccharomyces. (Broach, J.R., Pringle, J.R. and Jones E. space. Trends in Genetics 20: 384-391. W., Eds.) Plainview, New York: Cold Spring Harbor Laboratory Press, pp. 407-521. Muller, H.J. 1918. Genetic variability, twin hybrids and constant hybrids, in a case of balanced lethal factors. Reddy P, Zehring, W.A., Wheeler, D.A., Pirrotta, V., Genetics 3: 422-499. Hadfield, C., Hall, J.C. and Rosbash, M. 1984. Molecular analysis of the period locus in Drosophila melanogaster 28 Gravitational and Space Biology 18(2) June 2005 K. Beckingham, J. Armstrong, M, Texada, R. Munjaal, D. Baker - DROSOPHILA MELANOGASTER and identification of a transcript involved in biological Therrien, M., Morrison, D.K., Wong, A.M. and Rubin , rhythms. Cell 38: 701-10. G.M. 2000. A genetic screen for modifiers of a kinase suppressor of Ras-dependent rough eye phenotype in Rong, Y.S. and Golic, K.G. 2001. Gene targeting by Drosophila. Genetics 156: 1231-1242. homologous recombination in Drosophila. Science 288: 2013-2018. Thomas. A.R. and Capecchi, M.R. 1987. Site-directed mutagenesis by gene targeting in mouse embryo-derived Rorth, P. 1996. A modular misexpression screen in stem cells. Cell 51: 503-512. Drosophila detecting tissue-specific phenotypes. Proceedings of the National Academy of Sciences USA Toma, D.P., White, K.P., Hirsch, J. and Greenspan, R.J. 93: 12418-12422. 2002. Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait. Nature Rubin, G.M. and Spradling, A.C. 1982. Genetic Genetics 31: 349-353. transformation of Drosophila with transposable element vectors. Science 218: 348-353. Wang, B., Bolduc, C. and Beckingham, K. 2002. Calmodulin UAS-constructs and the in vivo roles of Scott, M.P. 2003. Cancer: a twist in a hedgehog's tale. calmodulin: analysis of a muscle-specific phenotype. Nature: 425: 780-782. Genesis 34: 86-90.
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Gravitational and Space Biology 18(2) June 2005 29 K. Beckingham, J. Armstrong, M, Texada, R. Munjaal, D. Baker - DROSOPHILA MELANOGASTER
30 Gravitational and Space Biology 18(2) June 2005 USE OF ANIMAL MODELS FOR SPACE FLIGHT PHYSIOLOGY STUDIES, WITH SPECIAL FOCUS ON THE IMMUNE SYSTEM Gerald Sonnenfeld Department of Biological Sciences, Binghamton University, State University of New York, Binghamton, NY
ABSTRACT Animal models have been most useful when carrying out Animal models have been used to study the effects of space experiments that could not be carried out on human flight on physiological systems. The animal models have been subjects (Sonnenfeld, 2005). These experiments involve used because of the limited availability of human subjects for exposure to conditions that would be dangerous for studies to be carried out in space as well as because of the need humans. Also, developmental biology studies have been to carry out experiments requiring samples and experimental carried out in animals flown in space that could not have conditions that cannot be performed using humans. Experiments have been carried out in space using a variety of been accomplished using humans. Additionally, since the species, and included developmental biology studies. These number of crewmembers is small and their time is species included rats, mice, non-human primates, fish, extremely limited, animal models have been utilized to invertebrates, amphibians and insects. The species were chosen provided preliminary data on which to base future human because they best fit the experimental conditions required for the studies. Therefore, animal models have proven to be experiments. Experiments with animals have also been carried extremely useful in providing information on detrimental out utilizing ground-based models that simulate some of the effects of space flight conditions on physiological effects of exposure to space flight conditions. Most of the function as well as for the development of animal studies have generated results that parallel the effects of space flight on human physiological systems. Systems studied countermeasures to ameliorate of prevent such have included the neurovestibular system, the musculoskeletal detrimental effects. No doubt, animal models will play a system, the immune system, the neurological system, the crucial role in the realization of the initiative that has been hematological system, and the cardiovascular system. Hindlimb announced to develop exploration class missions for the unloading, a ground-based model of some of the effects of space exploration of space. flight on the immune system, has been used to study the effects of space flight conditions on physiological parameters. For the The nature of the changes induced by space flight in immune system, exposure to hindlimb unloading has been animal physiological system has been included in a shown to results in alterations of the immune system similar to definitive review volume, and they will not be replicated those observed after space flight. This has permitted the development of experiments that demonstrated compromised here (Sonnenfeld, 2005). Instead, recent work using resistance to infection in rodents maintained in the hindlimb animal to study the effects of space flight conditions on unloading model as well as the beginning of studies to develop the immune system will be highlighted in this review. countermeasures to ameliorate or prevent such occurrences. Additional background information on the overall use of Although there are limitations to the use of animal models for animals to study the effects of space flight on physiology the effects of space flight on physiological systems, the animal can be found in the review volume (Sonnenfeld, 2005). models should prove very valuable in designing countermeasures for exploration class missions of the future. Additionally, since the opportunities to carry out space flight animal studies are rare and expensive, ground-based systems have been developed that model some of the INTRODUCTION AND OVERVIEW effects of space flight on animal physiology (Giron et al., 1967; Il'in and Novikov, 1980; Morey, 1979; Musacchia Animal models have been used extensively to study the et al., 1980; Space Studies Board, 1998). The need for effects of spaceflight on physiological conditions. The rat these models has been heightened as we have embarked has been the animal used most extensively, but some upon the era of planning for exploration class space studies have also been carried out utilizing mice and missions (Sonnenfeld, 2005). The limited ability to carry rhesus monkeys (Sonnenfeld, 2005). Invertebrates have out space flight animal experiments during the also been extensively used, as have fish and amphibians. construction phase of the international Space Station Studies have been carried out on just about every compounded by the difficulties in carrying out flight physiological parameter that could be studied, including experiments when the activities of the Space Shuttle fleet the musculoskeletal system, the cardiovascular system, are limited or suspended, has enhanced our need for the neurovestibular system, the immune system, and overall ground-based models. Without them, it seems unlikely developmental biology. that sufficient information will be available to allow for ______planning of human exploration class space flight missions. * Correspondence to: Gerald Sonnenfeld, Ph.D. Binghamton University State University of New York There are several ground based animal models available, P.O. Box 6000 including low pressure chambers and centrifugation Binghamton, NY 13902-6000, USA hypergravity models (Giron et al., 1967; Oyama and Platt, Email: [email protected] 1965) for studying the continuum of the force of gravity, Phone: 607-777-4818; Fax: 607-777-2501 but the most commonly utilized ground based model is hindlimb unloading of rodents (Il'in and Novikov, 1980; Gravitational and Space Biology 18(2) June 2005 31 G. Sonnenfeld — Animal Models in Space Morey, 1979; Musacchia et al., 1980; Space Studies those observed during space flight (Sonnenfeld and Board, 1998). This model is also known as hypokinetic, Shearer, 2002; Sonnenfeld, Butel and Shearer, 2003). hypodynamic antiorthostatic suspension or tail Again, these have been recently reviewed (Sonnenfeld suspension. In the model, rats or mice are suspended so and Shearer, 2002; Sonnenfeld, Butel and Shearer, 2003), that their hindlimbs are not weight bearing and with a and details will not be given here. Functional immune head down tilt so that there is a fluid shift to the head. responses have been shown to be altered after rodents The forelimbs remain load-bearing. This results in have been placed in the hindlimb unloading model, conditions similar to some of those observed during space including the same functional immune response that have flight, and effects on physiological systems are most often been shown to be affected by space flight such as similar to those observed during space flight (Il'in and cytokine production, leukocyte blastogenesis, response of Novikov, 1980; Morey, 1979; Musacchia et al., 1980; bone marrow cells to colony stimulating factors, Space Studies Board, 1998). Chapes et al., (1993) neutrophil activity, and resistance to infection. Leukocyte reviewed the effects of hindlimb unloading on the subset distribution was not affected in the same way by immune system and showed that hindlimb unloading was hindlimb unloading as it was by space flight, suggesting an acceptable, but not perfect, model for the effects of that the model is not useful for lymphocyte cell space flight on the immune system. Most of the effects of distribution studies (Sonnenfeld and Shearer, 2002; space flight on the immune system were replicated using Sonnenfeld, Butel and Shearer, 2003). the hindlimb unloading model, with the exception of leukocyte subset distribution in rats. SPACE FLIGHT CONDITIONS, GROUND-BASED MODELS AND RESISTANCE TO INFECTION SPACE FLIGHT CONDITIONS, GROUND-BASED MODELS AND THE IMMUNE SYSTEM Although it is clear that exposure of animals to space flight conditions results in alterations of immunological Exposure to space flight has been shown to modify many parameters, the question can still be asked, does this immunological parameters (Sonnenfeld and Shearer, affect the actual health and well-being of the host? This 2002; Sonnenfeld, Butel and Shearer, 2003). These question could only be answered by carrying out studies parameters have also been recently extensively reviewed, using infectious disease or tumor models. For obvious and have been demonstrated using rats, mice and rhesus reasons, such studies could not be carried out using monkeys. In brief, the immunological factors shown to humans. Animal models have proved invaluable for be affected by space flight in animals include: leukocyte carrying out infectious disease studies and have increased subset distribution, leukocyte blastogenesis, the response our understanding of the biological significance of the of bone marrow cells to colony stimulating factors, effects of space flight conditions on the immune system. cytokine production, natural killer cell activity, and neutrophil activity. The development of these Since space flight experimental opportunities for animal immunological responses in offspring of pregnant rats studies have been so limited, infection studies have not flown in the Space Shuttle were not affected (Sonnenfeld been carried out in space. Therefore, the hindlimb and Shearer, 2002; Sonnenfeld, Butel and Shearer, 2003). unloading model has proven to be invaluable for carrying out experiments on the effects of space flight conditions Exposure of rodents to the hindlimb unloading model has on resistance to infection (Table 1). also resulted in changes in the immune system similar to
32 Gravitational and Space Biology 18(2) June 2005 G. Sonnenfeld — Animal Models in Space In an early study, female Swiss/Webster mice were possible pathogen form space travelers who might inoculated with the D variant of encephalomyocarditis develop a compromised immune system. Pseudomonas virus (EMC-D virus). Females of the Swiss/Webster aeruginosa is an opportunistic gram negative pathogen strain normally are totally resistant to infection with that has already caused infectious difficulties during a EMC-D virus (Gould et al., 1987) with the resistance space flight mission (Hawkins and Ziegelschmid, 1975; mediated, at least in part, by interferon (Gould et al., Taylor, 1974). One of the astronauts in the Apollo 13 1987). Hindlimb-unloaded mice became susceptible to mission developed a urinary tract infection with P. infection, whereas mice that were restrained without head aeruginosa during the mission (Hawkins and down tilt and carrying full load on their hind limbs were Ziegelschmid, 1975; Taylor, 1974). For these reasons, still resistant to infection (Gould et al., 1987). The ability these two organisms were chosen for additional studies on of mice to produce interferon-α/β correlated with the the effects of hindlimb unloading on resistance to alteration in resistance to EMC-D virus. infection.
An additional experiment using mice subjected to A study was undertaken to determine the effects of hindlimb unloading was carried out by Fleming et al., hindlimb unloading on resistance of mice to infection (1990). The results of the study showed that hindlimb- with K. pneumoniae (Belay et al., 2002). Mice were unloaded mice had impaired ability to produce infected with K. pneumoniae. Mortality, as expected, was superoxide, decreased ability to kill phagocytosed 50% for control mice that were normally caged and bacteria (Propionibacterium acnes), and altered housed and received 1 LD50 (lethal dose for 50% of the corticosterone levels. This suggested a compromise of animals) of K. pneumoniae. Mortality was 43% for innate immune defenses, particularly neutrophils, by control mice that were restrained and received 1 LD50 of hindlimb unloading. K. pneumoniae., which was not significantly different from control mice. Mortality was 86% for experimental Mice that were placed in the hindlimb unloading model mice that were hindlimb-unloaded and received 1 LD50 of showed enhanced resistance to primary infection with K. pneumoniae, which was a statistically significant Listeria monocytogenes, an intracellular pathogen (Miller different from the other groups of mice (Belay et al., and Sonnenfeld, 1993, 1994). This was probably due to 2002). Mean time to death was also significantly reduced enhanced macrophage function and production of in the hindlimb-unloaded mice. Additionally, the cytokines produced by macrophages that resulted in hindlimb-unloaded mice could not readily clear the enhanced clearance of the pathogen (Miller and bacteria from the blood and other organs, suggesting that Sonnenfeld, 1993, 1994). At the same time that the the innate immune system was compromised in this resistance to the primary L. monocytogenes infection was infection (Belay et al., 2002). Therefore, it appears that strengthened by hindlimb unloading, the ability to exposure of mice to hindlimb unloading decreased generate long-term T cell mediated immunologic memory resistance K. pneumoniae. to the L. monocytogenes was inhibited (Miller and Sonnenfeld, 1993, 1994). Therefore, although initial Additional studies were also completed to determine the resistance to an obligate intracellular organism was effects of hindlimb unloading on resistance of mice to P. enhanced by hindlimb unloading, the ability to develop aeruginosa (Aviles et al., 2003a). Hindlimb-unloaded long-term T cell-mediated resistance to challenge with the mice had significantly enhanced mortality when infected organism was inhibited (Miller and Sonnenfeld, 1993, with P. aeruginosa in a fashion similar to that obtained 1994). Therefore, it is possible that in space flight with K. pneumoniae (Aviles et al., 2003a). The conditions, although initial resistance to some organisms hindlimb-unloaded mice that were infected had a might be enhanced, over the long-term, that resistance prolonged elevation of corticosterone, indicating that a would be likely to be compromised. stress response might play some role in decreasing resistance to infection (Aviles et al., 2003a). Although the previous studies generated evidence that resistance to infection could be compromised by hindlimb If resistance to infection is compromised in mice by unloading, the organisms studied were not likely to be hindlimb unloading, could a countermeasure be pathogens during a space flight mission. EMC-D virus developed that could minimize the deleterious effects of and L. monocytogenes are excellent organisms for hindlimb unloading on resistance to infection? This is a studying resistance to infections in models, but they are question of some importance, because it is possible that not pathogens for humans that would be found in space such a countermeasure could be further developed to flight conditions. Therefore, additional studies were protect humans from infections during space flight carried out using potential pathogens. missions.
Klebsiella pneumoniae is a gram negative enteric bacteria Studies were begun with active hexose correlated that is a major problem in immunocompromised surgical compound (AHCC). AHCC is an extract prepared from patients (Polk et al., 1992). It is part of the normal co-cultured mycelia of several species of Basidiomycete microbiota in the intestine, and when the host is mushrooms (Kidd, 2000). It contains polysaccharides, immunocompromised, can spread from the gut to cause amino acids, and minerals and is orally bioavailable. It sepsis and death (Polk et al., 1992). Therefore, it is a has been shown to have a beneficial effect on the immune Gravitational and Space Biology 18(2) June 2005 33 G. Sonnenfeld — Animal Models in Space system of normal humans (Matsui et al., 2002) and both ground-based and flight animal models to enhance rodents, including enhancement of natural killer cell our ability and enable u to protect future space travelers activity (Burikhanov et al., 2000; Matsushita et al., 1998; from any detrimental effects of the space flight Wakame et al., 1999; Yagita et al., 1998, 2002). AHCC environment. is available “over-the-counter” as a nutritional supplement). ACKNOWLEDEGMENTS
In the study carried out with AHCC, mice pre-treated for The US National Aeronautics and Space Administration one week by gavage with AHCC and then hindlimb- (NASA) supported some of the studies in the author’s unloaded with continued AHCC treatment were laboratory described above by direct grant support and significantly protected from infection with K. pneumoniae through NASA Cooperative Agreement NCC 9-58 with (Aviles et al., 2003b). In fact, protection against infection the National Space Biomedical Research Institute. was greater in hindlimb-unloaded mice than in normally Additional funding was provided for AHCC studies by caged mice, suggesting that more beneficial effects would the Amino Up Chemical Company of Japan. be observed in immunocompromised hosts (Aviles et al., 2003b). This surprising result stimulated additional mechanistic studies with AHCC. Hindlimb-unloaded mice REFERENCES infected with K. pneumoniae could not clear the infection from the blood, but hindlimb-unloaded mice pre-treated Aviles, H., Belay, T., Fountain, K., Vance, M., with AHCC and infected with K. pneumoniae were able Sonnenfeld, G. (2003a). Increased susceptibility to to clear the bacteria from the blood (Aviles et al., 2003b). Pseudomonas aeruginosa infection hindlimb unloading These results suggested that the immune system was conditions. J. Appl. Physiol. 95:73-80. stimulated by pre-treatment with AHCC, resulting in clearing of the infection. Aviles, H., Belay, T., Fountain, K., Vance, M., Sun, B., Sonnenfeld, G. (2003b). Active Hexose Correlated Additional mechanistic studies have been carried out to Compound Enhances Resistance to Klebsiella determine the role of AHCC in enhancing resistance to pneumoniae Infection in Mice in the Hindlimb-Unloading infection. Hindlimb–unloaded mice infected with K. Model of Space Flight Conditions. J. Appl. Physiol. pneumoniae and pre-treated with AHCC showed 95491-496. enhanced cytokine production, spleen cell blastogenesis and peritoneal cell nitric oxide production (Aviles et al., Aviles, H., Belay, T., Vance, M., Sun, B., Sonnenfeld, G. 2004). These results again supported that AHCC (2004). Active Hexose Correlated Compound Enhances treatment enhanced innate immunity in the immune function of mice in the hindlimb-unloading immunocompromised hosts, but also showed a possibility model of space flight conditions. J Appl Physiol. that AHCC treatment could enhance acquired immunity 97:1437-1444. (Aviles et al., 2004). Belay, T., Aviles, H., Vance, M., Fountain, K., CONCLUSIONS Sonnenfeld, G. (2002). Effects of the hinlimb-unloading model of spaceflight conditions on resistance of mice to The results of the infectious disease studies show that infection with Klebsiella pneumomniae. J. Allergy Clin. exposure of mice to hindlimb unloading conditions Immunol. 110262-268. resulted in compromised immune responses and compromised resistance to infection. AHCC may be a Burikhanov, R.B., Wakame, K., Igarashi, Y., Wang, S., useful countermeasure to protect mice from the Matsuzaki, S. (2000). Suppressive effect of Active compromising effects of hindlimb unloading on resistance Hexose Correlated Compound (AHCC) on thymic to infection. This certainly raises an issue that will apoptosis induced by dexamethasone in the rat. Endocr. require further study in actual space flight experiments. Regul. 34:181-188.
The above results could not have been obtained without Chapes, S.K., Mastro, A.M., Sonnenfeld, G., Berry, W.D. the use of the hindlimb unloading animal model. The (1993). Antiorthostatic suspension as a model for the space flight studies that should be carried out will not be effects of spaceflight on the immune system. able to be completed without the use of animals. The J Leukoc Biol. 54:227-235. countermeasures that must be developed must have their efficacy and safety tested in animal models. Fleming, S.D., Rosenkrans, C.F., Jr., Chapes, S.K. (1990) Test of the antiorthostatic suspension model on the The need for these models is not limited to the immune inflammatory cell responses. Aviat. Space Environ. Med. system. Similar problems as well as the need for 61: 327-332. development of countermeasures exist in all disciplines (Sonnenfeld, 2005). Therefore, it is extremely important, Giron, D.J., Pindak, F.F., Schmidt, J.P. (1967). Effect of as we go forward with planning for exploration class a space cabin environment on viral infection. Aerosp human space flight missions, that we include the use of Med. 38:832-834. 34 Gravitational and Space Biology 18(2) June 2005 G. Sonnenfeld — Animal Models in Space
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36 Gravitational and Space Biology 18(2) June 2005
Symposium II: Pharmacological
Countermeasures to Physiological Changes Induced by Space Flight
Joan Vernikos, Editor
Gravitational and Space Biology 18(2) June 2005 37
38 Gravitational and Space Biology 18(2) June 2005
EXERCISE AND PHARMACOLOGICAL COUNTERMEASURES FOR BONE LOSS DURING LONG- DURATION SPACE FLIGHT Peter R. Cavanagh1,2,3,5, Angelo A. Licata1,3,4, and Andrea J. Rice1,2 1Department of Biomedical Engineering, Lerner Research Institute, 2Center for Space Medicine, 3Department of Orthopaedic Surgery, 4Department of Endocrinology, Diabetes & Metabolism 5Orthopaedic Research Center, The Cleveland Clinic Foundation, Cleveland, OH
ABSTRACT 2003). This adaptation to microgravity renders the skeleton “at risk” for fracture, increases the risk of renal Bone loss in the lower extremities and lumbar spine is an stones (Whitson et al., 1999), and poses potential long- established consequence of long-duration human space flight. term health risks for astronauts on their return to Earth Astronauts typically lose as much bone mass in the proximal with reduced bone mass. femur in 1 month as postmenopausal women on Earth lose in 1 year. Pharmacological interventions have not been routinely In this article, we will examine the evidence for loss of used in space, and countermeasure programs have depended solely upon exercise. However, it is clear that the osteogenic bone mass during long-duration space flight, discuss the stimulus from exercise has been inadequate to maintain bone mechanisms for such loss, review countermeasures that mass, due to insufficient load or duration. Attention has have been attempted to date, and examine the potential of therefore been focused on several pharmacological interventions pharmaceutical countermeasures in the future. The that have been successful in preventing or attenuating implications of recent findings regarding the genetic osteoporosis on Earth. Anti-resorptives are the class of drugs determinants of bone mass will also be discussed. most commonly used to treat osteoporosis in postmenopausal women, notably alendronate sodium, risedronate sodium, BONE LOSS IN SPACE: THE EVIDENCE zoledronic acid, and selective estrogen receptor modulators, such as raloxifene. There has also been considerable recent interest in anabolic agents such as parathyroid hormone (PTH) Bone loss during space flight has been a concern since the and teriparatide (rhPTH [1-34]). Vitamin D and calcium Gemini flights (1-14 day missions, 1962-1966). Mack supplementation have also been used. Recent studies of and colleagues (Mack et al., 1967; Mack and LaChance, kindreds with abnormally high bone mineral density have 1967), reported what they called “small but significant” provided insight into the genetic regulation of bone mass. This bone loss. If one extrapolated their results to long- has led to potential therapeutic interventions based on the LRP5, duration flights, these changes would have been Wnt and BMP2 pathways. Another target is the RANK- alarming—ranging from 5.3% per month in the calcaneus L/osteoprotegerin signaling pathway, which influences bone during Gemini 7 to 89% per month in the finger turnover by regulating osteoclast formation and maturation. phalanges during Gemini 4. The observations were based Trials using such therapies in space are being planned. Among the factors to be considered are dose-response relationships, on the use of densitometry of plain X-rays, which is now bone quality, post-use recovery, and combination therapies—all regarded as an inaccurate methodology (Rambaut et al., of which may have unique characteristics when the drugs are 1975). It is, however, interesting to note that these used in space. authors also measured urinary and fecal loss of calcium in a group of bed rest subjects and reported a correlation of -0.7 between loss of bone mass and mean calcium intake. INTRODUCTION Soviet researchers (Biriukov and Krasnykh, 1970; The human skeleton has evolved in an environment where Krasnykh, 1969—reported by Rambaut et al., 1975) the force of Earth’s gravity has been a continual presence. found average losses of 4.7% per month in the os calcis in It is, therefore, not surprising that removal of gravity subjects in a bed rest study, and they reported that net during long-duration space flight results in a loss of losses during 8-10 weeks of bed rest were equivalent to homeostasis in the skeleton, which adapts to the new those seen in the 18-day Soyuz 9 flight. environment by shedding calcium (Lang et al., 2004) at a rate that is almost 10 times greater than that in a In the Apollo flights (6-14 day missions, 1968-1972), postmenopausal woman (Iki et al., 1996; Sirola et al., neutron activation analysis of fecal specimens (Brodzinski ______et al., 1971) (Apollo VII-XI; 6-11 days), densitometry from plain radiographs (Mack and Vogt, 1971) (Apollo * Correspondence to: Peter R. Cavanagh, Ph.D., D.Sc. The Cleveland Clinic Foundation VII and VIII; 11 and 6 days, respectively), and single- 9500 Euclid Avenue / ND-20 photon absorptiometry of calcaneus and forearm Cleveland, OH 44195 (Rambaut et al., 1975) (Apollo XIV-XVI; 9, 12, and 11 Email: [email protected] days, respectively) were used to assess changes in bone Phone: 216-445-6980; Fax: 216-445-6083 status.
Brodzinski et al. (1971) called the Gemini findings of Mack and LaChance (1967) “dubious,” and their own Gravitational and Space Biology 18(2) June 2005 39 P.R. Cavanagh - Preventing Bone Loss in Space measurements of calcium loss in Apollo VII-XI changes was not correlated with flight time, presumably crewmembers suggested less substantial changes. They due to individual differences in rates of bone loss. estimated that loss of total body calcium could be as little as 7.5% per year of space flight, but they suggested that a McCarthy et al. (2000) used three techniques (dual energy calcium balance experiment should be conducted on X-ray absorptiometry [DXA], ultrasonic measurements of Skylab, and this was in fact accomplished (see below). velocity [SOS], and broadband attenuation [BUA] of the calcaneus) to evaluate changes in bone during two Using similar methodology to their earlier studies, Mack missions, of 180 and 20 days, to the Mir space station, and Vogt (1971) reported average losses of 11.6% per involving three subjects. DXA measurements resulted in month in the lower extremity and 22.6% losses in the significant variation between different sites in the body upper extremity of six Apollo VII and VIII crew for changes in BMD, with the greatest losses occurring in members. As discussed above, in retrospect, these the lumbar spine and proximal femur. changes in “bone density” measured from plain radiographs were clearly erroneous; control subjects on Earth did not show such large changes. 0
The single gamma photon absorptiometry of Rambaut et -0.5 al. (1975) found a loss of 5.1% per month in the lower extremity, a gain of 0.6% per month in the radius, and a loss of 4.6% per month in the ulnae of nine Apollo XIV- -1 XVI crew members.
One of the most complete series of calcium balance -1.5 studies in space was conducted during the Skylab Delta BMD/Month (%) Space Flight missions (Skylab 2, 28 days, 1973; Skylab 3, 59 days, -2 Bedrest 1973; Skylab 4, 84 days, 1973-1974—Rambaut and Johnston, 1979; Smith et al., 1977; Smith et al., 1998; Smith et al., 1999; Tilton et al., 1980; Whedon et al., -2.5 Spine Neck Trochanter Pelvis Arm 1977). Commencing 21 days prior to flight, during flight, and for 18 days post flight, the intake of 30 nutrients were Figure 1a: Change in bone mineral density in different monitored, 24-hour pooled urine collections were made, anatomical regions (in percent change per month; negative and fecal samples were vacuum dried for analysis. values represent loss) during Mir missions and bed rest. Data Weekly plasma samples were also taken. A 56-day adapted from LeBlanc et al. (2000). ground-based control experiment was also conducted. During the Skylab 4 mission, average negative calcium The application of modern imaging techniques to bone balances of -100 (+25), -180 (+36), -229 (+60), -223 changes during space flight was first accomplished by (+42), -88 (+52) were reported for the pre-flight, flight LeBlanc and colleagues (LeBlanc et al., 1996; LeBlanc et days 1-24, 25-56, 57-84, and post-flight measurements, al., 1998; LeBlanc et al., 2000). In 1989, they installed a respectively. In addition, using single-photon absorp- Hologic 1000W dual X-ray absorptiometry (DXA) tiometry, Smith et al. (1977) indicated that a mean loss of scanner at the cosmonaut training center in Star City, 0.4% per month occurred in the calcanei of all nine Moscow, in the former USSR. Between 1990 and 1995, Skylab 2-4 crew members, while the investigators they studied 18 cosmonauts who had flown for between detected negligible losses in the radii (0.06% per month) 126 and 438 days (LeBlanc et al., 1996; LeBlanc et al., and a gain in the ulnae (0.4% per month). 2000). These measurements showed regional losses during flight of between 1.06% and 1.56% per month in Rambaut et al. (1979) pointed out that “the chain of the spine, pelvis and proximal femur, but no significant events leading ultimately to bone loss inflight remains changes in the upper extremities (Figure 1a). Losses were elusive.” In an article almost 20 years later, in which the parallel, but smaller, during bed rest, except in the arms, urine of Skylab crew members was re-analyzed, Smith et where losses were greater than during flight. These data al. (1998) shed light on this mechanism by demonstrating showed, for the first time, a pattern of lower-extremity that urinary excretion of collagen breakdown products loss and upper-extremity preservation during flight. The during the Skylab 4 mission was 40-45% higher than pre- authors concluded that the in-flight exercise programs flight values, indicating that space flight is associated were not sufficient to completely ameliorate bone loss with increased bone resorption. during flight (no countermeasures were used during bed rest). Using computer tomography (CT), Oganov et al. (1990) measured mineral density of lumbar vertebrae in four Lang et al. (2004) provided data from DXA, volumetric Salyut-7 crew members before and after extended flights quantitative computer tomography (vQCT), and (5-7 months’ duration). These authors reported that bone quantitative ultrasound (QUS) on crew members from the mineral density (BMD) diminished only in some of the Expeditions 2-6 to the International Space Station (ISS; test subjects and emphasized that the magnitude of 2001-2003, 130-197 days). The authors’ data confirmed 40 Gravitational and Space Biology 18(2) June 2005 P.R. Cavanagh - Preventing Bone Loss in Space
that little progress had been made in preventing loss of PRIOR COUNTERMEASURES bone mineral in the 30 years since Skylab. Notably, vQCT allowed an examination of the loss in both The only countermeasure that has so far been used in trabecular and cortical fractions of bone and also space for bone loss, albeit unsuccessfully, is exercise. estimates of the volumetric BMD (vBMD) as well as the Astronaut-physician William E. Thornton was a tireless conventional areal BMD (aBMD). These data confirmed proponent of exercise countermeasures, and his accounts the large losses in the spine and proximal femur (Figure of exercise countermeasures and devices (Thornton, 1b), and indicated that the rate of loss of bone mineral 1989a; Thornton, 1989b; Thornton, 1989c) are required content (BMC) in trabecular bone in the proximal femur reading in order to understand the history of use of this was approximately twice that of the cortical loss. Since modality. There is also a good description of exercise and trabecular bone cannot be replaced after loss of trabecular other countermeasures in Nicogossian et al. (1994). The continuity (Langton et al., 2000), this later finding is of countermeasure tradition began in the confined space of particular concern. The authors also found that calcaneal the Gemini capsule, where a bungee cord held by a loop estimates are not good surrogates for central or upper to the feet was pulled to exercise the arms and legs extremity skeletal measures and concluded that there was (Dietlein, 1965). There is no record of its efficacy, a continuing need to improve countermeasures to bone although measurements of heart rate, blood pressure, and loss, as it has become clear that current efforts are respiration rate were taken during exercise to record inadequate. cardiac response to exercise in space (Dietlein and Rapp, 1966). 0 The Soyuz 9 flight (18 days, 1970), on which bungee and -0.5 expanders were also used for exercise (Nicogossian et al., 1994), highlighted the need for more effective -1 countermeasures to combat the general loss of
-1.5 conditioning (Yegorov et al., 1972). Subsequently, some of the Salyut Space orbital stations (Salut 1, 1971 to
Delta BMD/month (%) BMD/month Delta -2 Salyut 6, 1985) were equipped with a passive treadmill, a bicycle ergometer, and a gravity simulation suit for long -2.5 wear (Gazenko et al., 1976). The efficacy of this Vertebral Vertebral Lumbar Femoral Total femur Trabecular Cortical trabecular posterior spine neck proximal proximal “Penguin Suit” (Nicogossian et al., 1994) has not been element femur femur confirmed. Figure 1b: Data showing change in regional bone (in percent change per month; negative values represent loss) from 13 crew The Skylab astronauts used several on-board exercise members on the International Space Station. Data adapted from devices, including a bicycle ergometer and a Teflon® Lang et al. (2004). plate, not available until Skylab 4, on which they performed an unusual form of tethered locomotion It is interesting to note that long-duration space flight (Figure 2a; Thornton and Rummel, 1977; Thornton, continues to be a male bastion, and thus we do not have 1989a). They also had a Mini Gym exercise device, adequate data on gender differences in bone loss in space. which allowed concentric muscular exercise to be For the 32 subjects for whom DXA data are available, performed, primarily benefiting the arms and trunk. there are only two women: one in the LeBlanc et al. Although this device probably transmitted higher forces (2000) series, who was reported to have similar responses to the legs than those from the bicycle ergometer, the to the mean of the group, and one in the Lang et al. (2004) force levels were still considered inadequate (Thornton series, whose data were not uniquely identifiable. and Rummel, 1977). No systematic record of the use of Presumably, privacy issues prevented this disclosure, but these devices by Skylab crew members is available in the one would hope that all crew members would make such literature, although it is likely that such records were kept. data available in the future in the interest of science.
Gravitational and Space Biology 18(2) June 2005 41 P.R. Cavanagh - Preventing Bone Loss in Space
Cosmonauts on Mir have been said to perform exercise “up to 3 hours per day” (Nicogossian et al., 1994), while others believe that the exercise was 2-3 hours on 3 of 4 days (LeBlanc et al., 2000). The passive treadmill was considered the “stadium” from which exercise was performed. While the subject was tethered in place using bungees, he not only walked and ran, but also performed calisthenics and upper-body exercises using additional bungee cords for resistance (Figure 3). The data from LeBlanc et al. (2000) showed clearly that this protocol, even if faithfully performed, is not an effective countermeasure for bone loss.
The exercise facilities available on the ISS through Expedition 12 consist of a Treadmill Vibration Isolation and Stabilization System (TVIS; Figure 2c; McCrory et
Figure 2a: A Teflon® plate on which Skylab astronauts al., 1999), a cycle ergometer with vibration isolation exercised in an unusual form of locomotion (Thornton and (CEVIS; Figure 2d), and the Interim Resistive Exercise Rummel, 1977; Thornton, 1989a). Artwork courtesy of NASA. Device (iRED; Figure 2e; Schneider et al., 2003). There is also a bicycle ergometer available in the Russian Thornton is thought to be the first man to run around the segment. None of these devices has a force measurement world in low Earth orbit. This feat was performed during capability, and there is very little published information one complete orbit of STS-8 (1983) on a treadmill that about their performance characteristics. When running on Thornton helped to design. Because the mid-deck of the the treadmill, a subject must be tethered using a subject Space Shuttle was not particularly spacious, the passive load device (SLD) to restrain him on the treadmill treadmill had to be stowable in a locker, a fact that surface, and, optionally, a subject position device (SPD) severely limited its belt length (Figure 2b). The subject is used to keep the subject in an area of the treadmill was tethered by bungee cords, which applied an unknown where a pitch oscillation of the treadmill will not be tension to return the crew member to the treadmill initiated. Each crew member is assigned a period of 2.25- surface. Kinematic analysis of on-orbit film taken during 2.5 hours every day for exercise—including set-up and running on the treadmill (Thornton et al., 1998) indicated break-down time, which can consume more than 50% of that there was restricted range of motion at the lower- the assigned period. The work of Lang et al. (2004) extremity joints and a plantar-flexed “tip-toe” gait. No showed that these devices as they are presently used are measurements of the foot forces were made. not effective as a countermeasure for bone loss during long-duration flights.
As we shall discuss below, prolonged bed rest is considered to be a viable analog of space flight. Shackelford et al. (2004) conducted a program of vigorous resistance training (averaging 74% of one repetition maximum) in nine individuals during a 17- week confinement. The exercise was found to have a beneficial effect on BMD during bed rest compared to controls, specifically in the lumbar spine (+3% vs. -1%), total hip (+1% vs. -3%), heel (+1% vs. -3%), total body (0% vs. -1%), and pelvis (-0.5% vs. -3%). However, the high levels of load imposed on the muscle groups studied have never been achieved in space, and it is unlikely that in-flight exercise devices currently in use will permit such loads to be achieved.
Figure 2b: The passive Shuttle treadmill designed by Astronaut-Physician William Thornton (Thornton, 1989c). Artwork courtesy of NASA.
42 Gravitational and Space Biology 18(2) June 2005 P.R. Cavanagh - Preventing Bone Loss in Space
Figure 2c: The International Space Station Treadmill with a Figure 2d: The Cycle Ergometer with Vibration Isolation and Vibration Isolation and Stabilization System (TVIS). (NASA Stabilization System (CEVIS) in use on the International Space photography) Station. (NASA photography)
Figure 2e: The Interim Resistive Exercise Device (iRED) in use on the International Space Station (ISS). (NASA photography)
Gravitational and Space Biology 18(2) June 2005 43 P.R. Cavanagh - Preventing Bone Loss in Space
Figure 3: A page from the Mir cosmonaut exercise instruction manual showing a 24-stage exercise session performed on the treadmill.
44 Gravitational and Space Biology 18(2) June 2005 P.R. Cavanagh - Preventing Bone Loss in Space
WHY HAVE EXERCISE COUNTERMEASURES IN BONE REMODELING SPACE NOT BEEN EFFECTIVE? Bone is an active tissue that is constantly being Since exercise has been the only countermeasure to bone remodeled, principally by the action of two cell types: loss so far attempted in space, and since considerable osteoclasts, which resorb bone, and osteoblasts, which bone loss has occurred on all flights to date, it would be build new bone (Figure 4). It is estimated that all of the tempting to conclude that exercise is not an appropriate bone in the adult skeleton is replaced every 10 years countermeasure. There are, however, several reasons why (Marx, 2004). Homeostasis of bone is only maintained if such a conclusion may be premature: (1) There has never the opposing—or perhaps complementary—actions of been a controlled study of exercise, either in space or osteoblasts and osteoclasts are balanced. A defect in during bed rest. The lack of such a study in the more than either process can result in accumulation of bone (as in 40 years that this problem has been recognized is highly osteopetrosis) or in a net loss of bone (as in osteoporosis) perplexing to the current authors and perhaps reflects the (Helfrich, 2003; Phan et al., 2004). The mineral phase of fact that NASA has traditionally been an engineering bone is of primary importance to density, and therefore rather than a science agency; (2) The loads applied to the BMD has been used in the past as a main indicator of body by any piece of exercise equipment were not bone status (Kanis, 2002). However, there is now measured prior to 2003, so it is not known whether or not increasing interest in measures of bone “quality” that equipment exerted 1-g-like loads; (3) Exercise adherence include structural as well as compositional information may have been less than optimal and, contrary to common (Ammann and Rizzoli, 2003; Turner, 2002), and it is belief, the ISS program was the first time a mandatory likely that a composite measure will eventually replace exercise program was instituted as part of a flight plan; BMD as the parameter of choice. (4) It is not known if a single daily concentrated “dose” of exercise in 0-g can effectively replace a “dose” that in 1-g The majority of current therapeutic interventions could be is distributed throughout the day; (5) The duration of classed as resorption-prevention drugs. A number of exercise programmed to date may not have been adequate advances in understanding how osteoclasts differentiate, to achieve the desired result; (6) There is considerable mature, and are activated have recently been made (Boyle debate in the scientific community about the optimal et al., 2003; Marx, 2004). Prevention of osteoclast loading strategy that will provide an osteogenic stimulus formation (osteoclastogenesis) and development has been to bone (Turner, 1998; Turner and Pavalko, 1998). a prime target through a number of different pathways Evidence in the literature ranges from a few intermittent (see below). On the formation side of the equation, large loads per day (Lanyon, 1996) to 18,000 small- preventing osteoblast cell death (apoptosis) is also of amplitude vibrations in a 10-minute period (Rubin et al., interest, and a number of other “anabolic” or bone- 2002a; Rubin et al., 2002b). There is also debate building drugs with uncertain mechanisms are also being regarding the relative role of force and rate of change of explored (Bisello et al., 2004; Deal and Gideon, 2003). force (Cullen et al., 2001; Linde et al., 1991; Mosley and Lanyon, 1998). SUPPLEMENTATION
Our own experiments using force-measuring insoles Traditionally, supplementation of daily intake of vitamin during exercise on the ISS (Rice et al., 2004) have D and calcium (current recommended daily allowances suggested that neither the load nor the duration of [RDAs] 400 IUs and 1500 mg, respectively) have been treadmill exercise in the current ISS exercise program is considered mainstays of osteoporosis prevention. adequate to replace 1-g exercise. Adequate calcium is needed for mineralization, and vitamin D plays a role in the regulation of calcium Only when all six issues noted above have been carefully deposition for bone mineralization. Both of these agents examined can the role of exercise as a countermeasure to have weak antiresorptive properties (compared, for in-flight bone loss be determined. Until such time, it is example, to bisphosphonates [Reginster, 2004]—see reasonable that the flight medicine community is looking below), but combined therapy for 18 months (1200 mg to explore the use in space of pharmacological options calcium plus 800 IU vitamin D3 [cholecalciferol]) has that are being used on Earth to prevent postmenopausal been shown to be effective in reducing hip fracture in osteoporosis. elderly women who were Vitamin D deficient (Chapuy et al., 1992). The bioavailability of the various forms of The remainder of this review will examine the cellular calcium used in supplementation (calcium carbonate, and molecular targets for such therapy, present the citrate, phosphate, lactate, and formate) have been shown currently available options, and discuss the limitations of to be different (Hanzlik et al., 2005). knowledge required for the implementation of these therapies in space. Since it is not always clear whether or not dietary intake of these agents is adequate, most drug trials routinely include calcium and vitamin D supplementation in control, placebo, and treatment arms. Astronaut diets can
Gravitational and Space Biology 18(2) June 2005 45 P.R. Cavanagh - Preventing Bone Loss in Space
Figure 4: Schematic of a bone multi-cellular unit (BMU). Osteoclasts resorb a cavity that is later occupied by osteoblasts that lay down new bone in the form of osteoid that subsequently undergoes mineralization (Deal and Gideon, 2003). Reprinted with the permission of The Cleveland Clinic Foundation.
be closely controlled, so inclusion of RDA and considered the regulator of the female skeleton and supplementation in diet can be easily accomplished. testosterone the male regulator. The discovery of mutations in the aromatase gene in men and concurrent HORMONE REPLACEMENT THERAPY abnormalities in skeletal metabolism (osteopenia and unfused epiphyses) have focused attention on the Estrogen, in the form of 17β-estradiol, has a complex importance of estrogen physiology. Aberrations in agonistic action on estrogen receptors (ERs) in the osteoclast acivity due to deficiency of inhibitors may nucleus of osteoblastsic cells (Riggs and Hartmann, attend the loss of estrogen with aging in both men and 2003), which in turn affect estrogen receptor elements women and cause increased bone turnover (Carani et al., (EREs) in target genes. In estrogen deficiency, resorption 1997; Khosla et al., 2002; Khosla et al., 2004). outpaces formation, resulting in net bone loss. Estrogen also stimulates breast epithelial cell production and has SELECTIVE ESTROGEN RECEPTOR been implicated in breast cancer risk (Riggs and MODULATORS (SERMS) Hartmann, 2003). Because of the side effects of HT, there has been Hormone replacement therapy (HT) using estrogen increased interest in this class of nonhormonal drugs that (unopposed HT) or estrogen-progestin (opposed HT) was target the ER. SERMs can have both agonist and widely recommended for postmenopausal women until antagonist effects in different tissues (e.g., tamoxifen the landmark Women’s Health Initiative (WHI) study [Tamofen], used in the treatment of ER-positive breast (Rossouw et al., 2002) demonstrated a number of adverse cancer, is an antagonist that slows the proliferation of responses (increased risk of coronary artery disease, tumor cells, whereas raloxifene [Evista] is a bone agonist stroke, thromboembolism, and breast cancer) in subjects that has an antiresorptive effect) (Riggs and Hartmann, using opposed HT. Riggs and Hartmann (2003) stated 2003). Different SERMs that have similar effects on bone that estrogen was the most widely prescribed drug in the (e.g., raloxifene and idoxifene [investigational]) appear to world and that it was taken by 38% of postmenopausal have their modes of action through different molecular women in the United States. In addition to reducing the pathways (Nuttall et al., 2000). Because raloxifene, risk for nonvertebral fractures (Torgerson and Bell-Syer, which is administered orally once per day, has a 2001), HT also had the added advantage of relieving a preferential effect on vertebral fracture risk reduction number of perimenopausal symptoms. Because of the (Ettinger et al., 1999), it is possible that there are increased risk of adverse side effects, HT is no longer differences between the action of SERMs on trabecular recommended for prevention or treatment of osteoporosis vs. cortical bone. (Kessel, 2004). There are some indications that raloxifene therapy Estrogen also has significant effects on skeletal decreases cardiovascular events in women with risk metabolism in men. Traditionally, estrogen was factors at baseline (Barrett-Connor et al., 2002) but carries
46 Gravitational and Space Biology 18(2) June 2005 P.R. Cavanagh - Preventing Bone Loss in Space with it a small increase in the risk for thromboembolism ANTIRESORPTIVE DRUGS (Daly et al., 1996). SERMs do not appear to alleviate postmenopausal symptoms (National Osteoporosis The largest class of antiresorptive drugs is the Foundation, 2002; Cranney et al., 2002). bisphosphonates (such as alendronate [Fosamax], etidronate [Didronel], ibandronate [Boniva], pamidronate The common risk for both therapies is that of deep vein [Aredia], risedronate [Actonel], zoledronate [Zometa], thrombosis, especially in conditions of clotting and tiludronate (Skelid)]. The drugs are distinguished by abnormalities. This risk is small but nonetheless present their potency, which is usually positively affected by the statistically. In other situations the drugs have divergent presence of a nitrogen atom (e.g., etidronate [low] to risks. Breast hyperplasia and breast cancer are not found zoledronate [high]), by their mode of delivery (e.g., with the SERM agents as they are with estrogen. intravenously for pamidronate and zoledronate, orally for Moreover, the SERM drugs do not cause cervical alendronate, orally, intravenously, or by injection for endometrial hyperplasia, menstrual bleeding, or cervical ibandronate), and by the frequency and size of dosing cancer. (e.g., 5 or 10 mg daily or weekly for alendronate, 2.5 mg daily for ibandronate, 10-90 mg annually for zoledronate). The SERM drugs may offer an option for treatment of An excellent review of these drugs is provided by prostate cancer. In the presence of decreasing androgens Reginster (2004). with aging, estrogen induces prostatic hyperplasia and neoplasia. Antiestrogens and SERMs suppress prostate Bisphosphonates are powerful and specific inhibitors of carcinogenesis. Some preliminary studies suggest that osteoclasts (Figure 5). They were originally thought to SERMs may not be useful as a general treatment for male exert their action via incorporation in the skeleton by osteoporosis, but there are some male patients, small in mimicking pyrophosphate and binding to the hydroxy- number, with the requisite balance of estrogen and apatite crystals in the bone matrix (Licata, 2005), testosterone for whom SERMs may be beneficial (Steiner especially at sites of remodeling, the bone multicellular and Raghow, 2003; Doran et al., 2001). units (BMUs) (Russell et al., 1999).
Figure 5: Schematic of bisphosphonate action (Rodan and Fleisch, 1996). Where bisphosphonate (BP in the diagram) has been incorporated into the bone matrix, osteoclastic resorption of bone cannot occur. Reprinted with permission.
Their actions have since been shown to be complex, function by “energy starving” the cell. Once incorporated, however. The amino bisphosphonates inhibit osteoclastic bisphosphonates remain bound at the bone surface and cholesterol synthesis and membrane function and increase exhibit extremely low serum concentrations, thus limiting cellular apoptosis. The non-amino bisphosphonates side effects. In general, the third generation (N2 produce ineffective ATP analogs and inhibit osteoclast containing) bisphosphonates have shown approximately Gravitational and Space Biology 18(2) June 2005 47 P.R. Cavanagh - Preventing Bone Loss in Space 40-50% reduction in the risk of vertebral and nonvertebral the upper gastrointestinal (GI) tract, constipation, fractures compared with placebo in postmenopausal flatulence, hypocalcemia, and diarrhea), but severe women (Black et al., 1996; Chesnut et al., 2004; Harris et esophageal reactions have been reported with alendronate al., 1999) and have also resulted in increased BMD in the (Schnitzer et al., 2000). Consequently, its use is not lumbar spine, total hip, and trochanter in women with and recommended for patients with a history of upper GI without osteoporosis (Cooper et al., 2003; Mortensen et complaints. al., 1998; Ravn et al., 1999). There is some concern that bone formed during the There are two bed rest studies involving bisphosphonates administration of bisphosphonates may not have the same that are relevant to the space program (LeBlanc et al., “quality” as normal bone, thus negatively affecting on the 2002; Watanabe et al., 2004). LeBlanc et al. (2002) mechanical integrity of the skeleton. Animal studies with administered 10 mg of alendronate daily to eight male bisphosphonates have shown a delay in fracture healing in subjects undergoing 17 weeks of horizontal bed rest. rats and rabbits and an increase in the presence and Compared with concurrent and historical controls, BMD persistence of microcracks and reduced remodeling, loss was significantly attenuated (or eliminated) in the suggesting a potential change in biomechanical factors (Li alendronate treatment group in the lumbar spine, femoral et al., 1999; Li et al., 2001; Mashiba et al., 2001; Lehman neck, trochanter, and pelvis (but not calcaneus). Most et al., 2004). In addition, it is notable that Ruggiero et al. markers of bone collagen breakdown and resorption (2004) have identified a cluster of patients on chronic (cross-linked N-teleopeptide of type I collagen [NTX], bisphosphonate therapy that had an associated risk of pyridinium [Pyd], and deoxypyridinium [D-Pyd]) in- osteonecrosis of the jaw. This condition is also seen in creased in both groups, but significantly less so in the the myeloma patients treated with i.v. zoledronic acid treated group than in controls. Markers of bone formation monthly (Lugassy et al., 2004), which is not the way it is (alkaline phosphatase, bone-specific alkaline phosphatase, used for treating osteoporosis. It is possible that such and osteocalcin) were unchanged in controls, but were patients may have immune compromise supporting local decreased in the treated group because of the reduced dental infection and subsequent bone destruction. bone turnover. These results demonstrate that the drug does not ablate the bone loss totally, thus the observed If sequential combination therapy of different drugs is clinical effects may require simultaneous mechanical planned, Gasser et al. (2000) showed in studies of rat stress. bone that the response to an anabolic drug (see below) was delayed in animals pretreated with bisphosphonates. Watanabe et al. (2004) administered 60 mg of pamid- However, in clinical studies, long-term use of alendronate ronate to seven male subjects 14 days before 90 days of 6- and risedronate for 7-10 years shows no similar findings. degree head-down bed rest. These authors also showed Both drugs still suppress fractures, which argues against that alendronates, in addition to their osteoprotective the adverse effects seen in animal models. Furthermore, properties, decrease the risk of renal stones. Compared histomorphometry shows no abnormal characteristics in with sedentary and resistance training controls, the patients after 3 or more years of use. pamidronate-treated subjects not only maintained significantly more bone in the proximal femur and lumbar ANABOLIC DRUGS spine, but also showed no evidence of urolithiasis (stones in the urinary tract). In the other groups, six subjects Drugs in this class exert their mode of action by were found to have radiographic evidence of stone increasing bone formation rather than by inhibiting formation during bed rest. All but one of these stone- resorption. The important role of parathyroid hormone forming subjects had baseline hypercalciuria (>250 mg (PTH) in regulating bone and mineral metabolism has per day). Such patterns of stone formation may be a been known for more than 70 years (Bisello et al., 2004), feature of all bed rest studies, and perhaps of long- but classical teaching identifies PTH as a powerful duration space flight, that has been previously mobilizer of skeletal calcium into the serum in the overlooked. However, it is extremely unusual for healthy presence of hypocalcemia (i.e., a state of secondary patients with no prior stone risk to become “at-risk” in hyperparathyroidism). Evidence from animal exper- such a short time, and these results, although cautionary, iments has shown that daily injection of PTH had need to be replicated. anabolic effects on bone, and recent work has resolved these apparently paradoxical effects by showing a Shapiro et al. (personal communication), in an as yet dependency on the pattern of exposure. Chronic elevation unpublished study, showed a reduction of bone loss in the of PTH (as in primary hyperparathyroidism) leads to lower extremities of patients with spinal cord injuries who increased bone resorption, whereas intermittent elevation had been administered intravenous zoledronate. The (as in once-daily injections with a short half-life) leads to paradigm of spinal cord injury has been suggested to be increased formation. The mechanism of action of PTH another analog of space flight, although the absence of appears to be the stimulation of existing osteoblasts via muscular action may tend to make it even more severe surface PTH receptors and interaction with RANK-L from a disuse point of view. (NF-κB; see below) (Deal and Gideon, 2003). It is The side effects of bisphosphonate therapy from the major known that the amino-terminal region of PTH (the first 34 study series have generally been mild (adverse effects in amino acids) is necessary and sufficient for full activity, 48 Gravitational and Space Biology 18(2) June 2005 P.R. Cavanagh - Preventing Bone Loss in Space and the only anabolic agent that is currently Food and activator of nuclear factor-κB ligand (RANK-L) ratio (see Drug Administration (FDA) approved for use in the below) (Locklin et al., 2001). treatment of osteoporosis is recombinant teriparatide (rhPTH [1-34] [Forteo]). RANK-L/OPG
Administration of teriparatide (daily subcutaneous In 1997, a new pathway regulating bone resorption was injection 20 µ g or 40 µg for 19 months) to women with identified (serendipitously) by a group looking for novel low bone mass and a history of prior fracture resulted in genes in the rat intestine (Simonet et al., 1997). The an almost 10% increase in vertebral BMD; treatment transgenic mouse overexpressing one particular gene was reduced the risk of a second vertebral fracture by found to have ostopetrosis and a deficiency of osteoclasts approximately 65% and that of a nonvertebral fracture by (Khosla, 2001) and the responsible protein was called approximately 50% compared with placebo. There is osteoprotegerin for its protective role in maintaining bone some concern that high doses of teriparatide (up to 60 mass. Simultaneously, Yasuda et al. (1998) found the times greater than approved human doses) have caused same protein in a targeted search for the signaling link osteosarcoma in rats, but not monkeys. No similar that had been previously hypothesized to exist between complications have been observed in human studies, but osteoclasts and ostoblasts (Rodan and Martin, 1982). The an initially promising trial conducted in men (Orwoll et pathway that has been identified as a result of these and al., 2003) was terminated because of concern regarding subsequent studies is shown in Figure 6. OPG is secreted the animal results. as a soluble protein from bone marrow stromal cells and appears to be a decoy receptor, which binds to RANK-L. It is also known that PTH-related peptide (PTHrP), a Since RANK-L is a major factor in osteoclast differ- protein with some homology to PTH that is produced by entiation, activation, and apoptosis inhibition, it follows tumors and leads to hypercalcemia, shares many of the that the binding of RANK-L to OPG, rather than to its actions of PTH but has receptors that are much more target RANK on the osteoclast precursor cell, will prevent widely distributed (Bisello et al., 2004). The authors have bone resorption. Because RANK-knockout mice also initial evidence from human studies that PTHrP has the exhibited osteopetrosis and absence of osteclasts (Li et al., potential to be a powerful anabolic agent, and clinical 2000), the existence of a new OPG/RANK/RANK-L trials to explore this possibility are ongoing. pathway in the control of bone resorption was confirmed. Several genetic mutations of this pathway are associated It is possible that the mechanism for the differential with bone diseases such as the family of hyperphos- effects of intermittent vs. continuous levels of PTH is in phatasias, Paget’s disease, and possible bone loss in the modulation of the osteoprotegerin (OPG)/receptor inflammatory arthritis (Boyle et al., 2003; Khosla, 2001).
Figure 6: Schematic of the OPG/RANK-L pathway (Khosla, 2001). Note that OPG acts as a decoy receptor preventing RANK from attaching to its ligand RANK-L and therefore inhibiting osteoclast differentiation. Copyright 2001, The Endocrine Society. Reprinted with permission.
Gravitational and Space Biology 18(2) June 2005 49 P.R. Cavanagh - Preventing Bone Loss in Space OPG was an obvious choice as a clinical therapeutic agent feature of calcitonin is that it can be administered by to prevent osteoporosis, and indeed two forms of the many routes, including nasally in the form of a daily (or protein were examined by Amgen in clinical trials of intermittently administered [Tekeoglu et al., 2005]) spray. osteoporosis (Bekker et al., 2001) and multiple myeloma and breast carcinoma (Body et al., 2003). A presumed BONE GENETICS combination of concerns regarding efficacy, safety, treatment duration and manufacturing factors has resulted In the last 5 years, early insights into some of the genetic in OPG’s no longer being examined for clinical use. OPG determinants of bone mass have been obtained. Ralston does, however, continue to be explored for the treatment (2003) and Recker (2004) have recently reviewed the of bone tumors (Wittrant et al., 2004). A fully human status of present knowledge in this area. Johnson et al. monoclonal antibody for RANK-L, AMG 162, is being (2004) have commented regarding “how little we really developed as an osteoporosis treatment instead (Bekker et know about the genes that control bone mass.” The al., 2004; McClung et al., 2004). Phase III clinical trials genetic basis for diseases caused by a defect in osteoclasts were initiated in late 2004 for AMG 162. is discussed by Helfrich (2003).
CALCITONIN Gong et al. (2001) found that that the LRP5 gene, which encodes the low-density lipoprotein receptor-related The peptide calcitonin exerts a complex inhibitory action protein 5, is important in bone mass accrual. They on osteoclast function (Kajiya et al., 2003). It has been reported that loss-of-function mutations in LRP5 caused used in trials of both men and women with low bone mass the autosomal recessive disorder osteoporosis- and has been shown to stabilize (or prevent) bone loss pseudoglioma syndrome and that Wnt-mediated signaling (Toth et al., 2005) and, in women, to decrease vertebral via LRP5 affects bone accrual during growth and peak fracture rate (Munoz-Torres et al., 2004). One attractive bone mass (Figure 7).
Figure 7: The Wnt signaling pathway that has been discovered through genetic studies of patients with high bone mass (Johnson et al., 2004). Adapted and reproduced from J Bone Miner Res 2004;19:1749-1757 with permission of the American Society for Bone and Mineral Research. Subsequently, mutations in the same gene were also Johnson et al. (2004). Genes regulating lipoxygenase are found to be associated with diseases in which there was also believed to influence bone mass (Klein et al., 2004). high bone mass (Boyden et al., 2002; Little et al., 2002). The lack of inhibitory action of the protein Dkk-1 on the There are indications that the Wnt signaling pathway is Wnt signaling pathway suggested this protein as a activated in response to mechanical loading (Johnson, potential therapeutic target for modulating bone mass. A 2004), and this may be a key element in the elusive review of LRP5 and Wnt signaling is presented by mechanotransduction that has long been hypothesized to exist. 50 Gravitational and Space Biology 18(2) June 2005 P.R. Cavanagh - Preventing Bone Loss in Space An alternative approach to the human linkage and Finally, the early discovery of the vitamin D receptor association studies described above is the use of mouse gene helped introduce the notion that bone mass had a models in quantitative trait locus (QTL) analysis (Liu et genetic basis (Eisman, 1995). al., 2003; Rosen et al., 2001). QTL is basically a statistical analysis, sometimes of the entire genome, to The complexity of BMD as a trait and the importance of identify which regions of the genome contain loci that gene-environment interactions have been emphasized in a influence the phenotype of interest. study of risk factors for low spine and hip BMD involving 12 candidate gene loci and lifestyle factors by Lau et al. The genes encoding type I collagen (COLIA1 and (2005). COLIA2) are mutated in osteogenesis imperfecta and may be useful markers of other osteoporotic phenotypes While these various studies of genetic influence on bone (Mottes et al., 1998). mass are in their early stages, there is a high likelihood that they will eventually identify new therapeutic targets. The estrogen receptor gene may regulate some aspects of bone density since the discovery of a male patient with a gene mutation and osteoporosis (Gennari et al., 2005).
Table 1. The major classes of osteoprotective therapeutic drugs
Drug Manufacturer Class Action Alendronate sodium Merck Bisphosphonate Inhibit osteoclasts (Fosamax) Zoledronic acid Novartis Bisphosphonate Inhibit osteoclasts (Zometa) Risidronate Procter & Gamble / Bisphosphonate Inhibit osteoclasts (Actonel) Aventis
Raloxifene Eli Lilly SERM Inhibit osteoclast (Evista) development
Estrogen (Several mfrs.) Sex steroid Inhibit osteoclast development
Calcitonin Novartis Peptide hormone Inhibit osteoclasts
Teriparatide Eli Lilly PTH fragment Anabolic (Forteo) PTHrP Osteotrophin PTH relative Anabolic
AMG 162 AMGEN RANK-L antibody Inhibit osteoclast development
SPECIAL CONSIDERATIONS FOR astronaut corps comprises primarily younger men (see THERAPEUTIC DRUG USE IN SPACE remarks above regarding the number of women astro- nauts/cosmonauts who have undergone long-duration The use of therapeutic drugs in space requires both the space flight), and such individuals are likely to be in good provider and the patient to accept a different set of bone health at the time of treatment. Such clinical trials standards, assumptions, and approvals compared with the typically take many years to accomplish (for example, the use of the same drugs on Earth. For example, the primary WHI study, mentioned above, was scheduled for 8.5 criterion that the FDA uses for approval of drugs designed years), and the time frame could slow the identification to treat osteoporosis is a demonstrated reduction of and application of effective therapies. fracture risk, usually hip or vertebral fracture. Such Given current NASA priorities, it is almost certain that evidence usually comes from a clinical trial of there will not be a sufficient number of astronauts to postmenopausal women with evidence of osteoporosis allow a placebo-controlled dose-ranging on-orbit trial that is blinded, placebo controlled, and randomized. This with sufficient statistical power to be mounted in the next approach may not be appropriate for decisions regarding decade. It is, therefore, likely that the decision to use a drugs for use in long-duration space flight, since the therapeutic drug for astronauts will be based on evidence Gravitational and Space Biology 18(2) June 2005 51 P.R. Cavanagh - Preventing Bone Loss in Space from a bed rest study supported by experience in a few Evaluation) randomized trial. Journal of the American individual volunteers who will take the drugs prior to Medical Association 287(7):847-57 and/or during space flight. Bekker, P.J., Holloway, D., Nakanishi, A., Arrighi, M., Among the questions that will need to be answered in Leese, P.T., and Dunstan, C.R. 2001. The effect of a these human trials are: (1) What is the bioavailability of single dose of osteoprotegerin in postmenopausal women. the various drug therapies in 0-g? (2) Are the dose- Journal of Bone and Mineral Research 16(2):348-60 response curves similar in 0-g to those established in 1-g? (3) What are the post-flight consequences for bone health Bekker, P.J., Holloway, D.L., Rasmussen, A.S., Murphy, of taking osteoprotective drugs? (4) If drugs need to be R., Martin, S.W., Leese, P.T., Holmes, G.B., Dunstan, taken on-orbit, how should they be stored for maximum C.R., and DePaoli, A.M. 2004. A single-dose placebo- effectiveness? (5) How will a drug’s effectiveness be controlled study of AMG 162, a fully human monoclonal determined on-orbit so that doses can be modulated? (6) antibody to RANKL, in postmenopausal women. Journal What is the best combination of drug and exercise of Bone and Mineral Research 19(7):1059-66 countermeasures? Biriukov, E.N., and Krasnykh, I.G. 1970. Changes in the SUMMARY AND CONCLUSIONS Optical Density of Bone Tissue an din teh Calcium Metabolism of the Astronauts. In: Kosmicheskaia This review has defined the current status of exercise and Biologiia i Meditsina. (Nikivaev, A.G., and Sevastianov, therapeutic drug countermeasures for bone loss during V.I. Eds) Moscow: pp. 42-45. long-duration space flight. The available data indicate that exercise countermeasures to date have not been Bisello, A., Horwitz, M.J., and Stewart, A.F. 2004. effective and crew members continue to lose significant Parathyroid hormone-related protein: an essential bone mass in the lower extremities and lumbar spine. physiological regulator of adult bone mass. Better-designed studies are needed to determine if the Endocrinology 145(8):3551-3 entire distributed daily dose of exercise that occurs in 1-g can be successfully replaced by short periods of high- Black, D.M., Cummings, S.R., Karpf, D.B., Cauley, J.A., intensity exercise on-orbit. Exercise dose on-orbit must Thompson, D.E., Nevitt, M.C., Bauer, D.C., Genant, also be quantified. H.K., Haskell, W.L., Marcus, R., Ott, S.M., Torner, J.C., Quandt, S.A., Reiss, T.F., and Ensrud, K.E. 1996. Drug therapeutics for bone have not yet been used in Randomised trial of effect of alendronate on risk of space, and, given the considerable experience using fracture in women with existing vertebral fractures. several classes of osteoprotective drugs on Earth (mostly Fracture Intervention Trial Research Group. Lancet in postmenopausal women with low bone mass), it seems 348(9041):1535-41 wise to explore such interventions for use during space flight. However, the many differences between the 1-g Body, J.J., Greipp, P., Coleman, R.E., Facon, T., Geurs, clinical studies and the 0-g individual prescription must F., Fermand, J.P., Harousseau, J.L., Lipton, A., Mariette, be carefully considered. Many new therapies can be X., Williams, C.D., Nakanishi, A., Holloway, D., Martin, expected in the future as investigators achieve a better S.W., Dunstan, C.R., and Bekker, P.J. 2003. A phase I understanding of the genetic regulation of bone mass, and study of AMGN-0007, a recombinant osteoprotegerin genetic screening may offer a means of selecting crew construct, in patients with multiple myeloma or breast members with a low susceptibility to bone loss. carcinoma related bone metastases. Cancer 97(3 Suppl):887-92 ACKNOWLEDGEMENTS Boyden, L.M., Mao, J., Belsky, J., Mitzner, L., Farhi, A., Supported by National Space Biomedical Research Mitnick, M.A., Wu, D., Insogna, K., and Lifton, R.P. Institute grants BL00401 and BL00402 through NASA 2002. High bone density due to a mutation in LDL- NCC 9-58. The assistance of Ted Bateman, Ph.D., was receptor-related protein 5. New England Journal of appreciated. Medicine 346(20):1513-21
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58 Gravitational and Space Biology 18(2) June 2005
CONSEQUENCES OF CARDIOVASCULAR ADAPTATION TO SPACEFLIGHT: IMPLICATIONS FOR THE USE OF PHARMACOLOGICAL COUNTERMEASURES Victor A. Convertino, PhD U.S. Army Institute of Surgical Research, Fort Sam Houston, Texas 78234-6513
ABSTRACT hemodynamic and autonomic functions have been considered or tested. There is little evidence obtained from space flight to support the notion that occurrence of cardiac dysrhythmias, impaired cardiac In 2000, the National Aeronautics and Space and vascular function, and manifestation of asymptomatic Administration (NASA) published their first draft of the cardiovascular disease represent serious risks during space Bioastronautics Critical Path Roadmap (BCPR) with the flight. Therefore, the development of orthostatic hypotension purpose of defining areas of biomedical research required and instability immediately after return from spaceflight probably reflect the most significant operational risks associated for future long duration space flight. Specifically, the with the cardiovascular system of astronauts. Significant objective of the BCPR for human health and reductions in stroke volume and lower reserve for increasing countermeasures was to focus on “understanding, peripheral vascular resistance contribute to ineffective characterizing, and counteracting the whole body’s maintenance of systemic arterial blood pressure during standing adaptation to microgravity, enabling healthy astronauts to after spaceflight despite compensatory elevations in heart rate. accomplish mission objectives and return to normal life The primary mechanism underlying reduced stroke volume following a mission”. The BCPR outlined specific critical appears to be a reduction in preload associated with less risks of serious adverse health or performance circulating blood volume while inadequate peripheral vasoconstriction may be caused partly by hyporeactivity of consequences that would result from space flight. The receptors that control arterial smooth muscle function. A focus priority for cardiovascular risks identified by the BCPR for development of future countermeasures for hemodynamic included 1) occurrence of serious cardiac dysrythmias; 2) responses to central hypovolemia includes the potential diminished cardiac function; 3) manifestation of application of pharmacological agents that specifically target previously asymptomatic cardiovascular disease; 4) and restore blood volume (e.g., fludrocortisone, electrolyte- impaired cardiovascular response to orthostatic stress; and containing beverages) and reserve for vasoconstriction (e.g., 5) impaired cardiovascular response to exercise stress. In midodrine, vasopressin). Based on systematic evaluations, acute 2004, a revised version of the BCPR reduced the physical exercise designed to elicit maximal effort or inspiratory identified priority for cardiovascular risks to include only resistance have shown promise as successful countermeasures the occurrence of serious cardiac dysrythmias and that provide protection against development of orthostatic hypotension and intolerance without potential risks and side diminished cardiac and vascular function. effects associated with specific pharmacological interventions. The purpose of this paper is to provide an assessment of Key words: blood volume; blood pressure; heart rate; proposed risk(s) to the cardiovascular system during stroke volume; cardiac output; peripheral vascular resistance space flight based on a critical review of data documented in the literature. An emphasis will be placed on the INTRODUCTION efficacy of specific pharmacological treatment of mechanisms associated with cardiovascular adaptations The effects of extended exposure to microgravity that lead to compromised operational performance of environments on the cardiovascular system are well astronauts. An attempt will be made to provide documented (Convertino, 2002). Although cardiovascular perspectives on limitations and interpretations of these adaptations appear benign during a space mission, they data in an effort to present future directions for have been manifested in reduced physiological or development and/or implementation of effective physical function upon return to Earth. As a result, a pharmacological and non-pharmacological major focus of space-related research has been directed to countermeasures for cardiovascular adaptation to space the systematic development and evaluation of potential flight. countermeasures. Among numerous treatments, specific pharmacological agents designed to enhance ASSESSMENT OF RISK TO THE ______CARDIOVASCULAR SYSTEM DURING SPACE * Correspondence to: Victor A. Convertino, Ph.D. FLIGHT
U.S. Army Institute of Surgical Research Occurrence of serious cardiac dysrhythmias. Despite 3400 Rawley E. Chambers Ave. Bld. 3611 numerous anecdotal reports, there is little evidence of a Fort Sam Houston, Texas 78234-6513 Email: [email protected] potential for occurrence of heart rhythm disturbances Phone: (210) 916-5633; Fax: (210) 916-5992 during space flight that may result in a serious cardiac event. For instance, no arrythmias were reported in a group of healthy astronauts during long-duration space missions despite a prolongation in QT interval (D’Aunno Gravitational and Space Biology 18(2) June 2005 59 V. Convertino — Spaceflight and Cardiovascular Countermeasures et al., 2003). No increase in cardiac dysrhythmias were revealed an average 14% reduction in left ventricular reported from electrocardiogram (ECG) tracings collected mass (Perhonen et al., 2001). These data were the first on astronauts while performing their routine tasks and obtained from humans to offer evidence that there is a extravehicular activities (EVA) during short- (<14 d) or possibility for cardiac remodeling during space missions long-duration space missions (>14 d) (Fritsch-Yelle et al., that might compromise myocardial function and 1996a; Rossum et al., 1997; Goldberger et al., 1994). contribute to lower stroke volume. In addition, there is Although a single isolated episode of a non-sustained, evidence from ground simulation experiments that asymptomatic 14-beat ventricular tachycardia (VT) diminished cardiac compliance might reduce diastolic episode was reported in an astronaut during the second function and compromise cardiac filling (Levine et al., month of a mission on the Russian MIR space station 1997). However, recent evidence generated from ground- (Fritsch-Yelle et al., 1998), further analysis raised the based and flight experiments on animals suggests that possibility that this VT episode might represent a normal smaller cardiac size simply may reflect the impact of variant if the ST elevation existing in the ECG tracing negative caloric balance and reduction of body mass was also seen in the astronaut’s previous resting tracing routinely observed in astronauts during space flight and (Ellestad, 1998). Finally, “no pathology in the myocardial results in a constant cardiac mass to body mass ratio (Ray bioelectrical activity” was reported in 59 cosmonauts et al., 2001). Regardless of any evidence for cardiac during MIR space missions of greater than 6-month remodeling, measures of myocardial function curves duration (Golubchikova et al., 2003). Taken together, before and after the 84-day U.S. Skylab mission (Henry et there is little evidence to suggest that the occurrence of al., 1977), ejection fractions measured before and during serious cardiac dysrhythmias is a high risk to the health the 237-day Russian Salyut-7 mission (Atkov et al., and well-being of astronauts during short- or long- 1987), and arterial pulse wave velocities measured before duration space missions. and during the Russian 23-day Salyut-1 and 63-day Salyut-4 missions (Convertino, 1990) all suggest that Manifestation of previously asymptomatic cardiovascular there is little impact of long-duration exposure to disease. The basis of the hypothesis that long-duration microgravity on cardiac function. The space flight data space flight may exacerbate previously undetected probably reflect the effectiveness and importance of cardiovascular disease (e.g., coronary artery disease) is performing current intense exercise countermeasures in dependent upon the existence of evidence that supports the maintenance of normal cardiac function. Therefore, one or both of two premises: 1) there have been cases the current evidence suggests that the risk of diminished within the astronaut community of undetected cardiac function during or following space flight appears cardiovascular disease that existed before space flight; negligible in the presence of the current effective exercise and/or 2) extended exposure to microgravity in some way space flight countermeasures. aggravates pre-existing cardiovascular disease. Unfortunately, there are no published data to support the Diminished vascular function. Hemodynamic responses occurrence of either condition and, therefore, no evidence during stand tests conducted on 14 astronauts following 9- to suggest that conditions of space flight might cause a 14 days of space flight revealed that the distinguishing pre-existing cardiovascular disease to become feature between astronauts who could (finishers) or could symptomatic or accelerate the progression of the disease. not (nonfinishers) complete 10 minutes of standing after Likewise, there is no published documentation to suggest these space missions was a significantly lower that any astronauts have displayed the presence of vasoconstrictor response in nonfinishers (Buckey et al., asymptomatic cardiovascular disease prior to long 1996). The relationship between low vasoconstrictive duration missions. Therefore, in the currently selected response and failure to complete stand tests has been astronaut population who undergo extensive medical corroborated in an additional 87 astronauts after space screening prior to selection and mission in an effort to flight (Fritsch-Yelle et al., 1996b; Meck et al., 2004; exclude the existence of clinical conditions, the risk of Waters et al., 2002). These results obtained from exacerbating a pre-existing asymptomatic cardiovascular astronauts following space missions have advanced the disease appears to be very low. hypothesis that diminished vascular function may represent a significant cardiovascular risk of space flight. Diminished cardiac function. It is clear from space flight This hypothesis may be further supported by evidence of experiments that stroke volume is significantly reduced reduced vascular smooth muscle contraction with upon return to earth (Buckey et al., 1996; Bungo et al., associated atrophic and morphological alterations 1987; Convertino, 1990, 1995; Henry et al., 1977; Levine generated from ground base animal models (Delp et al., et al., 1996, 2002; Mulvaugh et al., 1991). 1993, 1999, 2000; Zhang et al., 1997; Zhang, 2001). Echocardiographic data demonstrated that the lower stroke volume was associated with reduced cardiac size In contrast to attenuated vasoconstrictive responses (Bungo et al., 1987; Mulvaugh et al., 1991). Although a reported in pre-syncopal astronauts, astronauts who reduction in circulating plasma and blood volume that display orthostatic stability after spaceflight exhibit occurs with space flight is associated with less cardiac elevated vascular resistance compared to preflight filling, data obtained from magnetic resonance imaging (Buckey et al., 1996; Fritsch-Yelle et al., 1996; Meck et measurements obtained from 4 astronauts who al., 2004; Waters et al., 2002; Levine et al., 2002). These participated in the 10-d D-2 NASA space mission results have been corroborated in subjects exposed to 60 Gravitational and Space Biology 18(2) June 2005 V. Convertino — Spaceflight and Cardiovascular Countermeasures ground simulations of microgravity (Convertino et al., associated with presyncope during stand tests following 1994; Kimaya et al., 2004). Therefore, there appears to be exposure to simulated and actual microgravity a discrepancy in vasoconstrictive response between non- (Convertino et al., 1990; Fritsch et al., 1992). Although presyncopal and presyncopal astronauts. A reduction in heart rate increased with standing in syncopal subjects, the vasoconstrictive reserve has been identified as a their tachycardia was less than half that observed in the mechanism that contributes to orthostatic intolerance (Fu nonsyncopal subjects. These data provided the first et al., 2004) and thus may provide an alternative evidence that attenuated carotid-cardiac baroreflex explanation for a limitation in vascular function following function may impair the capacity of tachycardic adaptation to spaceflight. The vasoconstrictive reserve is mechanisms to maximize elevations in heart rate, and defined as the difference between baseline and maximum subsequently cardiac output, during standing. Therefore, vasoconstriction (Engelke et al., 1996). Both spaceflight attenuation of baroreflex-mediated cardiac chronotropic and ground experiments have provided evidence that responses induced by exposure to microgravity may vasoconstriction during supine rest after exposure to represent a cardiovascular risk of limiting reflex microgravity is increased and associated with compensatory tachycardic responses necessary to hypovolemia (Convertino, 1996, 1999; Convertino et al., maintain adequate cardiac output. 1994; Engelke et al., 1996; Gabrielsen et al., 1995). Increased peripheral vasoconstriction after return from Impaired cardiovascular response to orthostatic stress. space flight reflects a sympathetically-mediated reflex Orthostatic hypotension and compromise following return compensatory response to a reduction in stroke volume from space flight has been well documented since the and cardiac output (Convertino et al., 2004a). A linear U.S. Gemini program (Hoffler, 1977) and presyncopal relationship between increased muscle sympathetic nerve symptoms have been reported in 28% to 65% of mission activity and stroke volume was maintained between pre- specialists or scientists studied during stand or tilt test and post-space flight tilt tests, suggesting a tight coupling after returning from specific life science space missions (signaling) between stroke volume and sympathetic nerve (Buckey et al., 1996; Fritsch-Yelle et al., 1996; Meck et activity (Levine et al., 2002). Since maximal al., 2004; Waters et al., 2002). Impaired orthostatic vasoconstriction is finite, the elevated resting performance in astronauts following their return from vasoconstriction associated with low circulating vascular space flight has been associated with lower circulating volume and stroke volume represents a reduction in blood volume, decreased stroke volume and cardiac vasoconstrictive reserve and lowers the capacity to buffer output, and limited capacity to elevate peripheral vascular orthostatic hypotension (Convertino, 1999; Engelke et al., resistance (Buckey et al., 1996; Convertino, 1996; 1996). It is therefore unclear based on the current Fritsch-Yelle et al., 1996; Meck et al., 2004; Waters et al., evidence that any cardiovascular risk associated with 2002). It is clear that the inability of an astronaut to stand space flight reflects a diminished vascular function or and perform an emergency egress from a spacecraft after simply lower vasoconstrictive reserve secondary to landing could result in a life-threatening event. Thus, hypovolemia. impaired cardiovascular response to standing after return from space may represent one of the highest risks to the Impaired cardiovascular autonomic functions. safety, well-being, and performance of astronauts. Adaptations of autonomically-mediated baroreflex mechanisms that control cardiac chronotropic responses Impaired cardiovascular response to exercise stress. and peripheral vascular resistance may contribute to Numerous experiments using human subjects exposed to inadequate blood pressure regulation after exposure to ground simulations of microgravity have demonstrated microgravity. Hypoadrenergic responsiveness has been significant reduction in aerobic capacity (Convertino, hypothesized as a contributing mechanism to post-flight 1995). More recently, a 22% reduction in aerobic capacity orthostatic intolerance as evidenced by a relationship was demonstrated in 6 astronauts following only 9 or 14 between low blood norepinephrine and less vascular days of space flight and was associated with reduced resistance in presyncopal astronauts (Fritsch-Yelle et al., stroke volume (Levine et al., 1996). It is also clear that the 1996; Waters et al., 2002). Since sympathetic nerve reduced stroke volume during physical work in space is activity, circulating norepinephrine and peripheral affected directly by lower cardiac filling, i.e., end- vascular resistance are all elevated in orthostatically- diastolic volume (Atkov et al., 1987). The relative stable astronauts after space flight (Levine et al., 2002; reduction in maximal oxygen uptake following Fritsch-Yelle et al., 1996; Waters et al., 2002), cardiovascular adaptation to ground simulations of sympathetic withdrawal that occurs at the point of microgravity is correlated highly with the relative presyncope (Cooke & Convertino, 2003; Iwase et al., magnitude of reduced circulating blood volume 2000) in addition to blood sampling in the supine posture (Convertino, 1995), suggesting a close coupling between of only the presyncopal astronauts (Fritsch-Yelle et al., blood volume and cardiac filling. However, there is no 1996; Meck et al., 2004) may offer an explanation other evidence in the literature to suggest that a loss of 20% to than hypoadrenergic responsiveness for lower circulating 25% of aerobic capacity has impaired operational norepinephrine reported in presyncopal astronauts. performance during or after space flight.
Attenuation of cardiac vagal nerve traffic withdrawal Summary of cardiovascular risks associated with space induced by carotid baroreceptor stimulation was flight. There is little evidence obtained from space flight Gravitational and Space Biology 18(2) June 2005 61 V. Convertino — Spaceflight and Cardiovascular Countermeasures to indicate that occurrence of cardiac dysrhythmias, elevating peripheral vascular resistance by inducing impaired cardiac function, and manifestation of arteriolar constriction (Benditt et al., 1999; Raviele et al., asymptomatic cardiovascular disease represent serious 1996; Robertson & Davis, 1995). Based on operational risks during space flight. Data from the literature provide efficacy, the discussion of pharmacological agents used as the most convincing argument that impaired potential countermeasures against deleterious effects of cardiovascular responses to orthostatic and exercise cardiovascular adaptation(s) to space flight will focus on stresses represent the primary operational risks to specific experimentation and testing of blood volume astronaut health, safety and performance following space expanders and vasoconstrictors. flight. Figure 1 illustrates the changes and interactions of mechanisms underlying the effect of cardiovascular adaptation to microgravity on orthostatic and exercise Pharmacological expansion of circulating blood volume. performance. It is clear from Figure 1 that the Microgravity-induced hypovolemia contributes to development of countermeasures should focus on orthostatic compromise after space flight. To counter this restoring central blood volume, stroke volume and reserve effect, U.S. astronauts currently adhere to a regimen of for increasing peripheral vascular resistance. consuming a maximum of eight 1-g salt tablets with approximately 912 ml of fluid designed to make an isotonic saline drink approximately 2 h prior to reentry in an effort to restore blood volume (Bungo et al., 1985). Although a reduced orthostatic tachycardia following short duration space missions was encouraging during the initial use of saline loading (Bungo et al., 1985), exposure to microgravity for longer than 7 days failed to ameliorate orthostatic compromise in astronauts (Vernikos & Convertino, 1994; White et al., 1991). Despite the continued use of saline loading by astronauts in the U.S. space program, there is little evidence to suggest that taken alone it is effective against the development of post- flight orthostatic intolerance (Buckey et al., 1996).
Figure 1. Diagram outlining adaptations in underlying The use of the mineralcorticoid fludrocortisone has been mechanisms of cardiovascular functions and their impact on used clinically for nearly 40 years with some success to operational functions in astronauts that result from exposure to treat orthostatic hypotension, particularly in patients with microgravity (spaceflight). an etiology linked to hypovolemia (Benditt et al., 1999; Robertson & Davis, 1995). Fludrocortisone acts to PHARMACOLOGICAL COUNTERMEASURES enhance sodium and fluid retention and has been reported FOR CARDIOVASCULAR ADAPTATION TO to sensitize alpha-adrenergic receptors (Benditt et al., SPACE FLIGHT 1999). However, fludrocortisone appears to be most effective when consumed over days to weeks rather than Extensive experiments conducted in both space flight and on the day it is first administered (Robertson & Davis, ground simulations provide a compelling argument that 1995). Consequently, in a preliminary investigation, the most effective pharmacological countermeasures for Vernikos and co-workers (1991) were the first to report protection of orthostatic and physical work performance that the administration of fludrocortisone with three doses should target plasma and/or blood expansion, autonomic over the final 24 hours of exposure to 7 days of simulated dysfunction, and/or impaired vascular reactivity. Current microgravity restored plasma volume in all subjects and clinical practices include the use of agents such as protected orthostatic tolerance in 4 of 7 subjects who had fludrocortisone or electrolyte containing beverages that previously become syncopal after head-down bed rest. In expand circulating blood volume (Benditt et al., 1999; a subsequent investigation (Vernikos & Convertino, Raviele et al., 1996; Robertson & Davis, 1995); beta- 1994), a more rigorously controlled experiment was adrenergic blockers such as propranolol, metoprolol, conducted to compare the effectiveness of the current atenolol, nadolol, and esmolol in an effort to diminish the astronaut saline loading regimen to fludrocortisone as degree of cardiac mechanoreceptor activation or oppose countermeasures for reduced plasma volume and peripheral vasodilatory effects of epinephrine (Benditt et orthostatic intolerance after spaceflight. Eleven healthy al., 1999; Raviele et al., 1996); disopyramide in an effort male subjects underwent a 3-day ambulatory baseline to avoid vasovagal responses by counteracting period followed by exposure to 7 days of 6° head-down parasympathetic activity (Benditt et al., 1999; Raviele et bed rest. Treatments consisted of two volume expansion al., 1996); serotonin reuptake blockers such as fluoxetine groups. One group (5 subjects) consumed 8 salt tablets (1 hydrochloride and verlafaxine hydrochloride in an effort g NaCl per tablet) and 960 ml of water 2 hours prior to to reduce the effects of serotonin-mediated vasodepressor ambulation. The second group (6 subjects) consumed 0.2 effects (Benditt et al., 1999); alpha-adrenergic agonists mg oral dose of fludrocortisone at 0800 and 2000 h the such as ephedrine, etilephrine or midodrine in an effort to day before and 0800 h the day the subjects got out of bed increase venous tone and venous return as well as (2 hours before standing). After treatments, all subjects 62 Gravitational and Space Biology 18(2) June 2005 V. Convertino — Spaceflight and Cardiovascular Countermeasures attempted a 15-min unsupported stand test. Plasma volume decreased by 12% on day 7 of bed rest, and was restored by fludrocortisone but not by saline load (Fig. 2). Despite similar elevation in heart rate between the two groups, the group treated with saline loading experienced significant orthostatic hypotension compared to the fludrocortisone group (Fig. 3). Protection of arterial blood pressure during standing with fludrocortisone treatment was associated with restored vasoconstriction reserve and cardiac baroreflex function. Only 1 of 6 subjects showed syncopal symptoms in the fludrocortisone-treated group, whereas 4 of 5 subjects did so in the saline-load group. Acute fludrocortisone treatment appeared to have distinct advantages as a protective measure for orthostatic intolerance after exposure to ground simulation of Figure 2. Plasma volume before and at the end of head-down microgravity. bed rest, and after treatment with saline (open circles and broken lines) and fludrocortisone (closed circle and solid line). Symbols represent mean (± SE). Asterisk indicates P < 0.05 compared to pre-bed rest baseline level. Data are modified from Vernikos and Convertino [1994].
Figure 3. Responses of heart rate (top panels) and mean arterial pressure (bottom panels) in subjects during supine baseline and at the end of 15 min of standing before (left panels) and after (right panels) bed rest after treatments with saline (closed circles and solid lines) and fludrocortisone (closed circles and broken lines). Symbols represent mean (± SE). Asterisk indicates P < 0.05 compared to saline treatment. Data are modified from Vernikos and Convertino [1994].
Subsequently, the fludrocortisone countermeasure was of 0.2 mg taken 3 times during 24 hours prior to standing tested on 7 male astronauts whose orthostatic responses in the ground experiment was altered to a single dose of were compared to 18 astronauts who received a placebo 0.3 mg taken 7 hours prior to landing in conjunction with (Shi et al., 2004). Astronauts took either 0.3 mg the operational saline fluid loading countermeasure that fludrocortisone or placebo orally 7 hours prior to landing. was taken approximately 5 to 6 hours before landing. The Treatment with this single dose of fludrocortisone difference in results of fludrocortisone application resulted in some protection of plasma volume but no between ground and space may simply reflect significant protection of orthostatic tolerance. In the transition to alterations in the operational transition from the ground spaceflight operational implementation, an effective dose experiment to spaceflight testing. In the end, the use of Gravitational and Space Biology 18(2) June 2005 63 V. Convertino — Spaceflight and Cardiovascular Countermeasures fludrocortisone as a countermeasure for post-spaceflight continued successful implementation to future spaceflight orthostatic intolerance was discontinued. missions.
Use of adrenergic-receptor agents. The association of impaired peripheral vascular constriction with development of post-spaceflight orthostatic hypotension and syncope motivated consideration for the use of pharmacological agents that target the response of vascular adrenoreceptors. The administration of the non- specific β–adrenoreceptor antagonist propranolol has been proposed as a countermeasure targeted at increasing peripheral vascular resistance by inhibition of vasodilatory effects of circulating epinephrine on vascular smooth muscle (Sandler et al., 1985). However, this approach was abandoned when benefits of peripheral vasoconstriction were overridden by inhibitory
chronotropic and inotropic effects that led to reduced Figure 4. Responses of systolic (top 2 lines) and diastolic orthostatic tolerance in ground experiments. (bottom 2 lines) arterial blood pressures in a female astronaut during a stand test following her first space mission (9 days) Most recently, the α1-agonist drug midodrine was without midodrine (solid circles and solid lines) and during a tilt administered to 6 subjects one hour before a tilt stand test test following her second space mission (11 days) with after they had completed exposure to 16 days of 5° head- midodrine (open circles and broken line). Data are modified down tilt (Ramsdell et al., 2001). Midodrine stimulates from Platts et al. [2004]. both arterial and venous constriction. Compared to the responses of 4 control subjects who received a placebo, midodrine significantly ameliorated development of Potential limitations and side effects of pharmacological hypotension and presyncope during the tilt test. intervention. The primary concern for using Subsequently, this countermeasure was tested on a female pharmacological intervention for space flight astronaut who had become hypotensive and presyncopal countermeasures is timing of drug administration and side during a stand test following her first 9-day space mission effects. In the operational space flight environment, the (Platts et al., 2004). After a second 11-day space flight, time that the drug is administered is critical to its this astronaut received a single 10-mg dose of midodrine effectiveness. For instance, most of the blood pressure administered orally 1 hour prior to a tilt test. Her raising effect of fludrocortisone results from sodium hemodynamic responses to the post-spaceflight retention that develops over several days, with the full orthostatic tests were compared. Compared to the supine pressor action being observed in 1 to 2 weeks (Robertson posture, midodrine treatment was associated with stable & Davis, 1995). When attempts were made to administer systolic, diastolic and pulse pressures in contrast to fludrocortisone to astronauts daily over the final 3 to 5 dramatic reductions in these pressure in the absence of days of the mission, crewmembers complained of painful midodrine (Fig. 4). Stabilization of upright blood pressure pressure behind the eyes and discontinued use of the drug with midodrine was associated with attenuated reductions [Shi et al., 2004]. This should not be unexpected since in stroke volume and cardiac output (Fig. 5). Despite the headaches in addition to hypokalemia are a common side effect of midodrine on arterial vasoconstriction, the effect of fludrocortisone (Robertson & Davis, 1995). elevation of peripheral vascular resistance during upright When applied to astronauts only on the final day of flight, posture after space flight with midodrine treatment was there was no effect on orthostatic responses. Thus, the similar to the preflight response and dramatically lower combination of administration schedule and side effects compared to the astronaut’s post-spaceflight response have rendered fludrocortisone an ineffective following her initial mission when she experienced countermeasure. hypotension and presyncope (Fig. 5, lower right panel). Rather than arterial vasoconstriction, the lower peripheral vascular resistance in the presence of higher stroke Since plasma concentrations of the most potent metabolite volume suggests that the primary effect of midodrine after of midodrine peak at 1 hour (Robertson & Davis, 1995), space flight in this astronaut was restoration of the drug would be most effective operationally if taken vasoconstrictive reserve by the improvement of central before re-entry. With an α1-adrenoreceptor agonist action, blood volume and cardiac filling (i.e., enhanced the primary side effect of midodrine is hypertension, venoconstriction and venous return). These results may particularly in the supine (non-orthostatic) posture. It is underscore the importance of central blood volume rather therefore likely that the administration of midodrine than autonomic dysfunction(s) as a primary mechanism of before re-entry would result in high blood pressure while postflight orthostatic intolerance. The use of midodrine as in orbit. a potential countermeasure for prevention of orthostatic intolerance following spaceflight awaits the results of
64 Gravitational and Space Biology 18(2) June 2005 V. Convertino — Spaceflight and Cardiovascular Countermeasures
Figure 5. Heart rate, stroke volume, cardiac output and peripheral vascular resistance responses from supine to upright postures preflight (open triangles and dotted line), postflight without midodrine (open circles and broken line) and postflight with midodrine (solid circles and solid lines). Data are modified from Platts et al. [2004].
NON-PHARMACOLOGICAL landing during space flight missions to test this COUNTERMEASURES FOR CARDIOVASCULAR countermeasure as a possible treatment for post-space ADAPTATION TO SPACE FLIGHT flight orthostatic hypotension and intolerance (Moore et al., 2001). Echocardiographic measurements made on the Use of a single bout of maximal exercise. The use of astronauts involved in the testing of the maximal exercise physical exercise as a potential countermeasure against countermeasure demonstrated that stroke volume and post-space flight orthostatic intolerance has been long cardiac output were restored to pre-flight levels in the considered because of the recognized effect of physical exercise group during post-space flight standing, but fell activity on circulating blood volume and baroreflex in the control group in a similar fashion as that reported in functions. More than a decade ago, specific attention was the ground investigation (Convertino, 2002). Therefore, given to a single exposure of graded exercise designed to this exercise regime was successful in targeting the elicit maximal effort performed within 24 hours before re- primary mechanisms associated with post-space flight entry from a space mission. In addition to the potential orthostatic intolerance. It is also operationally attractive benefits of protecting aerobic capacity (Convertino, because it could be performed within 24 hours before the 1987), orthostatic tolerance was restored following 16 end of a mission and required minimal time of the days exposure to a ground simulation of microgravity astronauts (less than 20 minutes only once). when tested 24 hours after a maximal exercise countermeasure was applied (Engelke et al., 1996). Use of an impendance threshold device (ITD). Recent Improved physiological functions affected within 24 investigations have focused on the application of a simple hours by acute maximal exercise and associated with concept that central blood volume may be increased blood pressure regulation included restoration of blood acutely by transforming the thorax into a more active volume (Convertino et al., 1996), vasoconstrictive reserve vacuum and drawing venous blood from extrathoracic (Engelke et al., 1996), and cardiac baroreflex sensitivity cavities into the heart and lungs. Building on this concept, (Engelke et al., 1996). Subsequent to ground experiments, an inspiratory impedance threshold device (ITD) designed a single bout of maximal cycle ergometer exercise was to elevate intrathoracic negative pressure, i.e., create a performed by astronauts within 18 to 24 hours prior to vacuum, within the chest each time the chest expands
Gravitational and Space Biology 18(2) June 2005 65 V. Convertino — Spaceflight and Cardiovascular Countermeasures during the inspriatory phase of breathing has been flow (Convertino et al., 2005); and (e) reduce orthostatic described (Convertino et al., 2005). With the use of an symptoms (Convertino et al., 2005). Again, the ITD ITD, we have demonstrated in several human experiments represents a successful approach to targeting the primary that inspiratory resistance can: (a) reset cardiac baroreflex mechanisms associated with post-space flight orthostatic function to a higher operating range for blood pressure intolerance. It is also operationally attractive because it (Convertino et al., 2004b); (b) increase stroke volume, requires little training and easily could be used by an cardiac output and arterial blood pressure in astronaut to avoid cardiovascular collapse associated with normovolemic (Convertino et al., 2004c) and orthostatic post-flight orthostatic instability. Currently the ITD is (Convertino et al., 2005) subjects; (c) reduce peripheral under consideration for placement in the flight medical vascular resistance (i.e., increase vasoconstrictive reserve) bag and on the International Space Station. (Convertino et al., 2004c); (d) increase cerebral blood
Table 1. Qualitative summary of the effects of microgravity and five pharmacological and non-pharmacological countermeasures on cardiovascular functions associated with risk to astronaut health, safety and performance after a space mission.
SUMMARY intolerance, there is compelling evidence that intense physical exercise or application of mechanical devices Impaired cardiovascular responses during standing and can provide similar physiological effects on acute performing physical work represent operational risks to expansion of baroreflex function(s), central blood volume astronaut health, safety and performance following space and vasoconstrictive reserve (Table 1). Since the flight. Cardiovascular functions associated with these possibility of side effects or interaction with other drugs operational risks include attenuated autonomic baroreflex exists, the use of pharmacological agents as functions, lower central blood volume, stroke volume and countermeasures to cardiovascular dysfunctions following cardiac output, and vasoconstrictive reserve (Table 1). space flight should be considered only after the Plasma volume expanders and vasoconstrictors represent application of more physiologically natural techniques are the primary pharmacological treatments that have been exhausted. tested for management of post-flight orthostatic intolerance. Table 1 summarizes the effects of three pharmacological countermeasures in relation to ACKNOWLEDGEMENTS cardiovascular alterations induced by adaptation to microgravity. Saline loading has had little success in The views expressed herein are the private views of the reversing adverse cardiovascular adaptations. author and are not to be construed as representing those of Fludrocortisone showed promise in ground experiments, the Department of Defense or Department of the Army. but alterations in dose implementation for operational use in space missions showed little positive effects. REFERENCES Midodrine was successful in improving orthostatic tolerance in ground experiments and in one application to Atkov, O.Y., Bednenko, V.S., and Fomina, G.A. 1987. space flight. Although pharmacological intervention Ultrasound techniques in space medicine. Aviat. Space offers an alternative to treatment of post-flight orthostatic Environ. Med. 58(suppl 9):A69-A73. 66 Gravitational and Space Biology 18(2) June 2005 V. Convertino — Spaceflight and Cardiovascular Countermeasures
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Gravitational and Space Biology 18(2) June 2005 69 V. Convertino — Spaceflight and Cardiovascular Countermeasures
70 Gravitational and Space Biology 18(2) June 2005 DIET AS A FACTOR IN BEHAVIORAL RADIATION PROTECTION FOLLOWING EXPOSURE TO HEAVY PARTICLES Bernard M. Rabin1, Barbara Shukitt-Hale2, James Joseph2 and Paul Todd3 1University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250 2HNRC on Aging at Tufts, 711 Washington St., Room 713, Boston, MA 02111 3SHOT, Inc., 7200 Highway 150, Greenville, IN 47124
ABSTRACT radiation health research and protection then summarizes recent research related[ to a specific countermeasure Major risks associated with radiation exposures on deep space (dietary antioxidants) to a specific risk (neurological missions include carcinogenesis due to heavy-particle exposure performance decrement). of cancer-prone tissues and performance decrements due to neurological damage produced by heavy particles. Because Many things come in threes. Omniae homiliae in tres exposure to heavy particles can cause oxidative stress, it is partes divisae sunt. possible that antioxidants can be used to mitigate these risks (and possibly some health risks of microgravity). To assess the In this section, briefly, we introduce capacity of antioxidant diets to mitigate the effects of exposure to heavy particles, rats were maintained on antioxidant diets • Three kinds of space radiation containing 2% blueberry or strawberry extract or a control diet • Three space radiation risks for 8 weeks prior to exposure to1.5 or 2.0Gy of accelerated iron • Three forms of radiation risk management particles at Brookhaven National Laboratory. Following • Three forms of biological countermeasures irradiation rats were tested on a series of behavioral tasks: amphetamine-induced taste aversion learning, operant THREE KINDS OF SPACE RADIATION responding and spatial learning and memory. The results indicated that the performance of the irradiated rats maintained on the antioxidant diets was, in general, significantly better than Space radiations consist of (1) energetic protons from the that of the control animals, although the effectiveness of the sun, (2) protons and electrons from the sun that are diets ameliorating the radiation-induced deterioration in trapped in the earth’s magnetic field, and (3) cosmic rays performance varied as a function of both the specific diet and that include energetic nuclei of H, He, C, N, O and Fe the specific endpoint. In addition, animals fed antioxidant diets atoms. prior to exposure showed reduced heavy particle-induced tumorigenesis one year after exposure compared to the animals Solar Particles. When the sun is very active, such as just fed the control diet. These results suggest that antioxidant diets before and just after sunspot maxima (every 11 years) have the potential to serve as part of a system designed to provide protection to astronauts against the effects of heavy magnetohydrodynamic effects allow the escape of intense particles on exploratory missions outside the magnetic field of clouds of energetic protons that can deliver doses of 0.3 to the earth. 3.0 Gy over a period of about 3 days (Townsend et al., 1991; Parsons et al., 1999). Otherwise the sun is constantly releasing lower energy protons that pass from INTRODUCTION TO SPACE RADIATION RISKS the solar magnetosphere to Earth's magnetosphere. AND THEIR MANAGEMENT Trapped Radiations. Energetic electrons and protons arriving at Earth from the sun are trapped in Earth's The field of space radiation health has recently been magnetic flux lines, where they spiral back and forth singled out as one of two major initiatives within the between the north and south magnetic poles, in which research programs of the U. S. National Aeronautics and case they are said to comprise “trapped radiation belts”. Space Administration (NASA, 2002). The goals of space radiation health research are to understand qualitatively Galactic Cosmic Rays. The presumed celestial origin of and quantitatively the ionizing radiations present in the the high-energy, high-charge particles causes them to be space environment, identify qualitatively and called "galactic cosmic rays”, or GCR. They are also called "HZE" particles owing to their high charge and quantitatively the risks associated with these radiations, 2 and discover countermeasures to mitigate these risks energy. A few such particles pass every cm every few (Tobias and Todd, 1974). This article first introduces the seconds above the Earth's atmosphere, but these are basic science and definitions of quantities underlying attenuated by the Earth’s magnetic field and atmosphere and never reach sea level. All of these three categories of ______radiations produce secondary radiations such as neutrons * Correspondence to: Bernard M. Rabin and gamma rays when they interact with matter. This fact University of Maryland Baltimore County is relevant to problems of shielding spacecraft and their 1000 Hilltop Circle contents; not only is shielding heavy, it could generate Baltimore, MD 21250 more dangerous radiation. Email: [email protected] Phone: (410)455-2430; Fax: (410) 455-1055
Gravitational and Space Biology 18(2) June 2005 71 B. Rabin — Diet as a Factor in Behavioral Radiation Protection THREE SPACE RADIATION RISKS Neurological Effects of Heavy Ions. The earliest biological studies with cyclotron beams targeted neural The risks presented to space travelers by these radiations tissue damage and the accompanying physiological include (1) cancer due to chronic proton and cosmic-ray effects (Malis et al., 1957). The general thought was that exposure, (2) immune and/or hematopoietic failure due to heavy ions, due to the geometry of their energy loss, were high-dose solar proton storms, and (3) possible capable of directly killing non-dividing neurons and vital neurological effects caused by single tracks of cosmic-ray glial cells whereas only very large doses of x or gamma heavy nuclei. Other well-known effects of heavy ions, rays had this capability (Zeman et al., 1961). A wide such as cataracts and retinal flashes, are not considered variety of morphological and physiological changes in mission- or life-threatening. Until more is known about neural systems have been reported following heavy-ion the biological effects of chronic heavy-ion exposure it irradiation, usually at rather high doses, typically remains to be determined which of these is the most exceeding 1 Gy (Joseph et al., 1998). Recalling that serious biological risk or whether these three risks need to about half of all cells are hit by at least one cosmic ray in be considered in parallel, independently. a 2-3-year deep-space mission although the dose is only a few cGy, it is still necessary to ask if the neurological Cancer and cellular effects due to heavy-ion exposure. It functioning of crew members will be impaired to such an has been noted that (Curtis and Letaw, 1989; Setlow, extent as to jeopardize a mission and/or the lives of the 1999) on a round trip to Mars, the nuclei of about half of crew members. Critical switchyards in the central nervous all of a crew member's cells will have been traversed by at system, sparsely populated with neurons (hippocampus, least one energetic multiply charged cosmic-ray particle corpus callosum, etc.) are particularly vulnerable points unless extraordinary shielding measures are implemented. for the induction of behavior decrements due to the The irradiation of cells in vitro with heavy ions has for destruction of a small number of cells. Laboratory animal several decades served as a model for studying the studies are now underway to determine, via quantitative potential effects of cosmic-ray particles on cells. Most of neurochemistry and behavior analysis, the nature and our earliest understanding of high LET particle level of effects of nervous function (Rabin et al., 2000), radiobiology was derived from studies of cell killing, since functional effects constitute end-points of relevance carcinogenesis and mutagenesis in vitro. It has been to mission performance while morphological effects may amply demonstrated that surviving cells traversed by or may not relate to critical functions. This risk is the heavy particles are transformed to malignancy (Yang, subject of this article. 1985; Kronenberg, 1994) or mutated (Evans et al., 2001). Modern molecular methods of studying mutagenesis and in vitro and in vivo carcinogenesis are being applied to THREE FORMS OF RADIATION RISK this problem with the discovery of interesting, previously MANAGEMENT unappreciated phenomena such as genome instability and "bystander effects" on non-hit cells (Deshpande et al., With three categories of radiation and three categories of 1996). Delayed effects on the progeny of irradiated cells, biological response radiation health in space is more such as mutation, carcinogenesis and impaired growth complicated than that on Earth; therefore, one of the goals rate are not expected to be reflected in human responses of space radiation health research is the reduction of during a deep space mission; these processes carry post- uncertainties. However, there are predictable (trapped flight risks to individual health and constitute the most protons, galactic cosmic rays) and stochastic (solar event significant risk only if the other two risks are less protons) radiation sources. Likewise the biological important or less probable. responses include stochastic (carcinogenesis, neurological effects) and those with predictable dose-response curves. Immune/Hematopoeitic System Failure due to High-Dose These combine to create risk management dilemmas Proton Storms. The most efficient action of ionizing (Todd, 2003). radiation is the killing of cells. A hit cell is typically 10,000 times as likely to undergo reproductive death as it Potential countermeasures have been classified into three is to be transformed to malignancy or to express an categories (Cucinotta et al., 2001). These are (1) assayable mutation. The probability of cell death per unit operations (which establish flight schedules and orbital dose increases with LET up to a maximum (Barendsen et strategies), (2) shielding (which increases spacecraft up- al., 1960; Todd, 1967). Granulopoietic and mass), and (3) biological, such as medication consisting lymphopoietic cells in the bone marrow are exquisitely of radical scavengers (that must be taken immediately sensitive to ionizing radiation, and the reduction of their before exposure), anti-oxidant consumption (which must progeny in the circulation to about 2% of normal cell be maintained continuously), cytokines (which may counts is considered life threatening (Bond et al., 1965). ameliorate immune and hematological effects specifically In this context, a proton dose as low as 2.0 Gy could be after exposure), and cell transplants (which should be life threatening, and, considering the compromising isologous). effects of space flight, including low gravity, on the immune system (Konstantinova, 1991), even lower doses Operations. It is unwise to be in the wrong place at the should be considered dangerous (Todd et al., 1999). wrong time if this can be avoided. Scheduling deep-space missions to miss periods of solar proton storm activity 72 Gravitational and Space Biology 18(2) June 2005 B. Rabin — Diet as a Factor in Behavioral Radiation Protection and choosing trajectories and spacecraft orientations are the length of time rats can maintain their grip on a wire examples of risk management by operations. suspended above the ground (Joseph et al., 1992). Similarly, exposure to low doses of HZE particles will Shielding. In the case of shielding, several approaches prevent the acquisition of a conditioned taste aversion have been considered, some very creative. In addition to (CTA) produced by the dopamine agonist amphetamine the skin of a spacecraft the normal contents of space (Rabin et al., 1998). A CTA is produce by pairing a novel vehicles (water tanks, waste containers, avionics taste solution (10% sucrose) with an unconditioned instruments) also constitute shielding. Active shielding, stimulus (amphetamine). As a result of this pairing the rat such as with a plasma or strong magnetic fields, has been will avoid ingestion of the solution at a subsequent entertained for several years. presentation. Because amphetamine is a dopamine agonist, the development of an amphetamine-induced Biological Countermeasures. In the case of biological CTA requires an intact dopamine system. In this regard, countermeasures, all of the alternatives mentioned below the effects of exposure to 56Fe particles are similar to are under exploration. those produced by the dopamine antagonist haloperidol (Rabin et al., 1998) in that both treatments disrupt THREE FORMS OF BIOLOGICAL dopaminergic function and interfere with the acquisition COUNTERMEASURES of an amphetamine-induced CTA.
Biological countermeasures against near-term and late Exposure to HZE particles can also affect cognitive effects of ionizing radiations consist of (1) radical performance (Shukitt-Hale et al., 2000). The Morris scavengers, (2) cytokine treatment and (3) water maze is a standard test of cognitive ability in which pharmacological and nutritional countermeasures against rats are required to use spatial cues to locate a platform reactive oxygen species (ROS). At least 2 of these placed just below the surface of the water. There are no categories might also mitigate microgravity effects. differences in performance between the non-irradiated controls and the irradiated rats in the initial acquisition of Radical scavengers. These consist of compounds, the task. However, when the platform is moved to a typically alcohols and sufhydryls that function at the different location in the maze the irirradiated rats show moment of irradiation to chemically react with free significantly poorer performance than the control rats. radical species produced in the radiation’s path. Most Similarly, when the platform is absent during probe trials, radical scavengers have been tested in vitro, and most of the irradiated rats spend significantly less time in the them have been found toxic in vivo. quadrant in which the platform had been located than do the non-irradiated control rats. These results indicate that Cytokine treatment. The radiosensitive leukopoietic and the irradiated rats are deficient in their ability to perform a hematopoietic systems are known to be responsive to task requiring the use of spatial cues. interleukins and hematopoietic factors. These are relevant in managing both space-flight stress (low gravity) and A second cognitive behavior which is affected by radiation damage (cell killing and carcinogenesis) exposure to HZE particles is operant conditioning (Rabin et al., 2002), in which the organism learns to make a Pharmacological and Nutritional countermeasures response in order to obtain reward or avoid punishment. against persistent ROS. Research over the past several Operant conditioning is broadly construed to include all years has been conducted to discover biological counter- forms of complex learning. The specific task that was measures to the above three risks: carcinogenesis by HZE utilized was responding on a fixed-ratio (FR) particles, immune system effects due to high doses plus reinforcement schedule. On an FR schedule, a rat is life in low gravity, and subtle neurological and behavioral required to make a fixed number of responses (level effects that might jeopardize a deep-space mission. The presses) in order to secure a reinforcement (45 mg food general findings to date indicate that dietary antioxidants pellet). On an FR-1 schedule, the rat is rewarded for can constitute a line of defense against all of these every lever press, while on an FR-35 reinforcement radiation risks. schedule the rat is required to make 35 responses in order to be rewarded with a single food pellet. When tested 3 NEUROBEHAVIORAL EFFECTS OF EXPOSURE months following exposure to 56Fe particles, only the rats TO HZE PARTICLES exposed to 2.0 Gy (but not 1.0 or 1.5 Gy) showed a disruption of responding at schedules of reinforcement of Exposing rats to HZE particles can affect performance on FR-20 or greater. There were no effects of irradiation at a variety of neurobiological and behavioral endpoints. schedules less that FR-15. When tested 8 months later Following exposure to 56Fe particles rats show a reduction (11 months post-irradiation) all irradiated groups showed in potassium-stimulated dopamine release and in the significantly decreased performance compared to the non- behaviors that are dependent upon the integrity of the irradiated controls. dopaminergic system (Joseph et al., 1992). Deficits have been observed in both motor and cognitive behaviors. The effect of exposure on motor behavior is shown as a decrease in upper body strength, measured by Gravitational and Space Biology 18(2) June 2005 73 B. Rabin — Diet as a Factor in Behavioral Radiation Protection RELATIONSHIP TO AGING AND OXIDATIVE depend upon the integrity of the dopaminergic system STRESS (Bickford et al., 2000; Joseph et al., 1998, 1999).
The neurochemical and behavioral deficits detailed above DIETARY COUNTERMEASURES AGAINST are also observed in old rats. As a result, it has been SPACE RADIATION RISKS proposed that exposing rats to HZE particles produces “accelerated aging” (Joseph et al., 1992). Specifically, To the extent that oxidative stress mediates the old rats show deficits in potassium-stimulated dopamine neurobehavioral effects of exposure to HZE particles, release and related deficits in motor behavior (Joseph et then antioxidant treatments may function to ameliorate al., 1978). Similarly there are significant decreases in the the effects of irradiation. Given the similarity in the performance of old rats on the Morris water maze, neurobehavioral effects of aging and irradiation, it is indicating decreased ability to utilize spatial cues in a possible that maintaining rats on diets containing learning task (Shukitt-Hale at al., 1999). In addition, the flavonoid antioxidants may also counteract the effects of partial loss of dopaminergic neurons produced by exposure to HZE particles. treatment with the neurotoxin 6-hydroxydopmine which does not affect the performance of young rats on an Blueberries and strawberries contain a variety of ascending fixed-ratio schedule does cause a significant compounds which may function as antioxidants. The impairment in the performance of older rats (Lindner et polyphenolics contained in fruits include the al., 1999). hydroxycinnamates and the flavonoids such as the anthocyanins and flavonols. As indicated by HPLC Previous research has shown that maintaining rats on analysis (Joseph, unpublished), the relative amounts of antioxidant diets containing blueberry or strawberry these compounds in different fruits vary, which may extract can ameliorate the neurochemical and behavioral account for differences in the antioxidant capacity. changes that are characteristic of the aging process, This Additional work will be needed in order to determine the observation is consistent with current theories which active compounds and their effects on specific suggest that oxidative stress and the production of neurobehavioral endpoints. Nonetheless, as summarized reactive oxygen species (ROS) are key factors in the below, maintaining rats on diets containing either aging process (Finkel and Holbrook, 2000). blueberry or strawberry extract can ameliorate the effects of exposure to HZE particles on specific neurochemical Oxidative stress occurs when endogenous and exogenous and behavioral endpoints. sources of ROS exceed the capacity of the antioxidant systems to remove them. In addition to the production of When rats are maintained on diets containing 2% ROS by endogenous sources, such as the aerobic blueberry or strawberry extract for two months prior to metabolism of mitochondria and the destruction of exposure to 56Fe particles (1.5 Gy, 1 GeV/n), the dopamine by monoamine oxidase, oxidative stress can radiation-induced decrease in potassium-stimulated also be produced by exogenous sources such as exposure dopamine release in the striatum is prevented (Joseph et to ionizing radiation, including exposure to HZE particles al., unpublished). These results are similar to those (Denisova et al., 2002). Acting to mitigate the effects of obtained with aged rats maintained on identical diets oxidative stress are a variety of endogenous antioxidant (Joseph et al., 1998, 1999). defense systems (superoxide dismutase and glutathione peroxidase) and exogenous sources of antioxidants Concordant with the neurochemical effects, antioxidant (vitamins and flavonoid antioxidants). Where the diets also ameliorate the cognitive/behavioral deficits production of ROS exceeds the antioxidant capacity of produced by exposure to HZE particles, although the these systems, the consequences of oxidative stress effectiveness of the blueberry and strawberry diet varies include aging (Finkel and Holbrook, 2000), as a function of the specific endpoint. For CTA learning, carcinogenesis (Oberly, 2002) and a variety of the rats maintained on either the blueberry or strawberry neurodegenerative disorders such as Parkinson’s and diet failed to show the 56Fe particles-induced disruption of Alzheimer’s diseases (Halliwell, 2001). an amphetamine-induced CTA (Rabin et al., 2002). Following exposure to either 1.5 Gy or 2.0 Gy the One treatment that has been effective in ameliorating the irradiated rats maintained on either diet for two months neurobehavioral effects of aging has been the use of prior to exposure showed the acquisition of a CTA dietary antioxidants, such as are found in fruits and following injection of the dopamine agonist berries. Measured as Oxygen Radical Absorbance amphetamine. As shown previously (Rabin et al., 1998, Capacity, the free radical scavenging capacity of 2000), the irradiated rats fed a control diet failed to blueberries and strawberries is much higher than that of acquire an amphetamine-induced taste aversion. vitamin E (Prior et al., 1998; Wang et al., 1996). Research using old animals has shown that maintaining Similar results were obtained in the initial test of spatial rats on diets containing 2% blueberry or strawberry learning and memory using the Morris water maze extract prevents the age-related changes in potassium- (Shukitt-Hale, unpublished). The irradiated rats stimulated dopamine release and in the behaviors that maintained on either the blueberry or strawberry diets showed a significant reduction in the latency to find the 74 Gravitational and Space Biology 18(2) June 2005 B. Rabin — Diet as a Factor in Behavioral Radiation Protection location of the submerged platform on the probe trial on generation of ROS, diets containing blueberry or Day 2 compared to the irradiated rats fed a control diet. strawberry extract may provide necessary protection By Day 3, however, the performance of the irradiated rats against the deleterious effects of exposure to GCR, while fed the strawberry diet was not significantly different also potentially mitigating other space flight stresses, and from that of irradiated rats fed the control diet, whereas enabling astronauts to successfully fulfill mission the irradiated rats fed the blueberry diet continued to requirements. show a reduced latency compared to the irradiated rats fed the control diet. ACKNOWLEDGMENTS The effects of antioxidant diets on operant responding also varied as a function of the specific diet. Seven This research was supported by Grants NAG9-1190 and months following exposure to 1.5 Gy of 56Fe particles, NAG9-1529 from National Aeronautics and Space there was no effect of irradiation on operant responding. Administration to BMR. When the rats were tested eleven months following irradiation, the animals fed either the control or blueberry NOMENCLATURE AND GLOSSARY diets showed significantly poorer performance on an ascending fixed-ratio reinforcement schedule than the GCR Galactic Cosmic Rays, high energy nuclei from non-irradiated rats (Rabin et al., in press). The outer space performance of the rats fed the strawberry diet was not Gy Gray, the SI unit of absorbed ionizing radiation significantly different from that of the non-irradiated dose (1 J/kg) controls and significantly better than that of the irradiated HZE High-charge, high energy particles (GCR) rats fed the blueberry diet. Similar results were obtained LET Linear Energy Transfer, energy deposited per following exposure to 2.0 Gy of 56Fe particles, except that unit distance in particle track the effects of irradiation and diet were observed when the QF Quality factor, for adjusting dose to equivalent rats were first tested five months following irradiation dose based on radiation quality (LET) (Rabin et al., submitted). rad Radiation absorbed dose, cgs units (100 erg/g; 0.01 Gy) As indicated above, exposure to HZE particles can rem Biological equivalent cgs dose unit, rad x RBE produce cancer. Current theories ascribe a role for (0.01 Sv) oxidative stress in carcinogenesis (Oberly, 2002). To the RBE Relative Biological Effectiveness extent that oxidative stress does play a role in tumor ROS Reactive Oxygen Species development, the use of antioxidant diets should reduce Sv Sievert, the SI unit of biologically equivalent the development of tumors following irradiation. dose (Gy x RBE) Because the rats that were tested on the operant conditioning task were observed for up to 12 months REFERENCES following exposure to 1.5 Gy or 2.0 Gy of 56Fe particles, data were also collected on the effects of diet on tumor Barendsen, G. W., Beusker, T. L., Vergroesen, A. J., development. Preliminary analyses indicate that both Budke, L. 1960. Effects of different ionizing radiations blueberry and strawberry diets significantly reduced the on human cells in tissue culture. II. Biological occurrence of radiation-induced tumors (Joseph, in experiments. Radiat. Res. 13, 841-849. preparation). Bickford, P. Gould, T., Briederick, L., Chadman, K., CONCLUSIONS Pollock, A., Young, D., Shukitt-Hale, B., Joseph, J. 2000. Antioxidant-rich diets improve cerebellar physiology and Overall, the observations presented in this review indicate motor learning in aged rats. Brain Res. 866, 211-217. that exposing the heads of laboratory animals to doses of HZE particles can have deleterious effects on cellular and Bond, V. P., Fliedner, T. M., Archambeau, J. O. 1965. systemic functioning. On a cellular level, irradiation can Mammalian Radiation Lethality, A Disturbance in Cell produce mutagenesis or cell death. On a systemic level, Kinetics. Academic Press, New York. there are changes in signal transduction processes in the central nervous system and related decrements of motor Cucinotta, F. A., Schimmerling, W., Wilson, J. W., and cognitive performance which have the potential to Petersen, L. E., Badhwar, G. D., Saganti, P. B., Dicello, J. affect the capacity of astronauts to successfully meet F. 2001. Space Radiation Risk Projections for Exploration mission requirements. There are a number of strategies Missions: Uncertainty Reduction and Mitigation. NASA that may be used to counteract the effects of exposure to Report JSC-29295. HZE particles. These include scheduling missions during times of reduced GCR flux or increasing the level of Curtis, S. B., and Letaw, J. R. 1989. Galactic cosmic rays shielding. An alternative approach which may hold and cell-hit frequencies outside the magnetosphere. Adv. promise involves using dietary manipulations to reduce Space Res. 9, 293-298. the levels of oxidative stress produced by exposure to HZE particles. By reducing oxidative stress and the Gravitational and Space Biology 18(2) June 2005 75 B. Rabin — Diet as a Factor in Behavioral Radiation Protection Denisova, N., Shukitt-Hale, B., Rabin, B. M., Joseph, J. A. 2002. Brain signaling and behavioral responses Lindner, M. D., Cain, C. K., Plone, M. A., Frydel, B. R., induced by exposure to 56Fe radiation. Radiat. Res. 158: Blaney, T. J., Emerich D. F., Hoane, M. R. 1999. 725-734. Incomplete nigrostriatal dopaminergic cell loss and partial reductions in striatal dopamine produce akinesia, rigidity, Deshpande, A., Goodwin, E. H., Bailey, Marrone, S. M., tremor and cognitive deficits in middle-agrd rats. Behav. Lehnert. B. E. 1996. Alpha-particle-induced sister Brain Res. 102, 1-16. chromatid exchange in normal human lung fibroblasts: evidence for an extranuclear target. Radiat. Res. 145, 260- Malis, L. I., Loevenger, R., Kruger, L., Rose, J. E. 1957. 267. Production of lesions in the cerebral cortex by heavy ionizing radiations. Science 126, 302. Evans, H. H., Horng, M.-F., Evans, T. E., Jordan, R., Schwartz, J. L. 2001. Genotoxic effects of high-energy McDonald, F. B. 1965. Review of galactic and solar iron particles in human lymphoblasts differing in cosmic rays. In Second Symposium on Protection against radiation sensitivity. Radiat. 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Halliwell, B. 2001. Role of free radicals in the Prior, R. L., Cao, G., Martin, A. Sofic, E., McEwen, J., neruodegenerative diseases; therapeutic implications for O=Brien, C.m Lischner, N., Ehlenfeldt, M., Kalt, W., antioxidant treatment. Drugs & Aging 18, 685-716.. Krewer, Mainland, M. 1998.. Antioxidant capacity as influenced by total phenolic and anthocyanin content, Joseph, J. A., Erat, S., Rabin, B. M. 1998. Selective maturity and variety of Vaccinium species. J. Agric. Food efficacy of space-like radiation effects (56Fe particles) on Chem.46, 2586-2593. muscarinic neurotransmitter sensitivity and motor Rabin, B. M., Joseph, J. A., Erat, S. 1998. Effects of behavior. Adv. Space Res. 22, 216-224. exposure to different types of radiation on behaviors mediated by peripheral or central systems. Adv. Space Joseph, J. A., Berger, R. E., Engel, B. T., Roth, G. S. 1978. Res. 22, 217-225 Age-related changes in the nigrostriatum: A behavioral and biochemical analysis. J. Gerontol., 33, 33-35. Rabin, B. M., Joseph, J. A., Shukitt-Hale, B., McEwen, J. 2000. Effects of exposure to heavy particles on a behavior Joseph, J. A., Hunt, W. A., Rabin, B. M., Dalton, T. K. mediated by the dopaminergic system. Adv. Space Res. 1992. Possible "accelerated aging" induced by 56Fe heavy 25, 2065-2074. particle irradiation: Implications for manned space flights. Radiat. Res., 130, 88-93. Rabin, B. M., Buhler, L. L., Joseph, J. A., Shukitt-Hale, B., Jenkins, D. G. 2002. Effects of exposure to 56Fe Joseph, J. A., Shukitt-Hale, B., Denisova, N. A., Bielinski., particles or protons on fixed-ratio operant responding in D., Martin, A.,. McEwen, J. J., Bickford, P. C. 1999. rats. J. Radiat. Rese, 43 (Suppl.): S225-S228. Reversals of age-related declines in neuronal signal transduction, cognitive and motor behavioral deficits with Rabin, B. M., Shukitt-Hale, B., Szprengiel, A., Joseph, J. diets supplemented with blueberry, spinach or strawberry A. 2002. Effects of heavy particle irradiation and diet on dietary supplementation. J. Neurosci. 19, 8114-8121. amphetamine- and lithium chloride-induced taste avoidance learning in rats. Brain Res. 953: 31-36. Joseph, J. A., Shukitt-Hale, B., Denisova, N. A., Prior, R. L., Cao, G., Martin, A., Taglialatela, G., Bickford, P. C. Rabin, B. M., Joseph, J. A. Shukitt-Hale, B. Effects of 1998 Long-term dietary strawberry, spinach, or vitamin E age and diet on the heavy particle-induced disruption of supplementation retards the onset of age-related neuronal operant responding produced by a ground-based model signal-transduction and cognitive behavioral deficits. J. for exposure to cosmic rays. Brain Res., in press. Neurosci. 18, 8047-8055. Riley, P A. 1994. Free radicals in biology: Oxidative stress Konstantinova, I. 1991. Immune resistance of man in and the effects of ionizing radiation. Int. J. Radiat. Biol. 65, space flights. Acta Astronautica 23, 123-127. 27-33.
Kronenberg, A. 1994. Mutation induction in human Setlow, R. B. 1999. The U. S. National Research lymphoid cells by energetic heavy ions. Adv. Space Res. Council's views on the radiation hazards in space. 14, 339-346. Mutation Res. 430, 169-175. 76 Gravitational and Space Biology 18(2) June 2005 B. Rabin — Diet as a Factor in Behavioral Radiation Protection Yang, T. C., Craise, L. M., Mai, M.-T., Tobias, C. A. Shukitt-Hale, B., Mouzakis, G., Joseph, J. A. 1998. 1985. Neoplastic transformation by heavy charged Psychomotor and spatial memory performance in aging particles. Radiat. Res. 104, Suppl. 8, S177-S. male Fischer 344 rats. Exp. Gerontol. 33, 615-624. White, M. G., Isaila, M. Prelec, K., Allen, H. L. 1971. Shukitt-Hale, B., Casadesus, G., McEwen, J. J., Rabin, B. Acceleration of nitrogen ions to 7.4 GeV in the Princeton M., Joseph, J. A. (2000). Spatial learning and memory Particle Accelerator. Science 174, 1121-1123. deficits induced by 56Fe radiation exposure. Radiat. Res. 154, 28-33. Zeman, W., Curtis, H. J., Baker, C. P. 1961. Histopathological effect of high-energy-particle Sinclair, W. K. 1964. X-ray induced heritable damage microbeams on the visual cortex of the mouse brain. (small colony formation) in cultured mammalian cells. Radiat. Res. 15, 496-514. Radiat. Res. 21, 584-611.
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Todd, P. 1983. Unique biological aspects of space radiation hazards--an overview. Adv. Space Res. 3(8), 187-194.
Todd, P., Schroy, C. B., Vosburgh, K. G., Schimmerling, W. 1971. Spatial distribution of biological effect in a 3.9 GeV nitrogen ion beam. Science 174, 1127-1128.
Todd, P., Pecaut, M. J., Fleshner, M. 1999. Combined effects of space flight factors and radiation on humans. Mutation Res. 430, 211-219.
Todd, P., Wood, J. C. S., Walker, J. T., Weiss, S. J. 1985. Lethal, potentially lethal, and nonlethal damage induction by heavy ions in cultured human cells. Radiat. Res. 104, S5-S12.
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78 Gravitational and Space Biology 18(2) June 2005
Short Papers
Advanced Life Support and Biotechnology (page 81) Animal Development, Physiology and Gravity Response (page 91) Cell Biology (page 99) Plant Development and Gravity Response (page 113)
Gravitational and Space Biology 18(2) June 2005 79
80 Gravitational and Space Biology 18(2) June 2005
EVALUATION OF A SILANE QUATERNARY AMMONIUM SALT AS AN ANTIMICROBIAL SURFACE TREATMENT Daniel Blustein1, Nona Hinkle2 and Ami Smith2 1Kalamazoo College, MI; 2 The Bionetics Corporation, Kennedy Space Center, FL, 32899 USA
NASA guidelines for planetary protection aim to preserve Culture Preparation: Cultures of Bacillus subtilis, the unaltered environments of both our own planet and all Escherichia coli, and Staphylococcus epidermidis were other planetary bodies in our solar system. Preventing the prepared in Tryptic Soy Broth (TSB) overnight at 35 ºC. spread of Earth-based life forms to other planets and the The broth was diluted to a transmittance of 72% on a 8 contamination of our own planet by extraterrestrial life Vitek Colorimeter (1.5-3.0 x 10 CFU/mL). This forms is an important consideration when planning space bacterial solution was mixed with 0.003 M KH PO 2 4 missions. Even with extensive sterilization techniques 5 during the assembly of spacecraft, bacteria, mainly buffer to a 1:1000 dilution (1.5-3.0 x 10 CFU/mL). 1,2 extremophiles, still survive . A company protocol was followed for testing the Another pressing concern when discussing the control 4 of microbes in space is the maintenance of astronaut effectiveness of the QAS. health. Astronauts traveling in spacecraft are subject to close-quarter living for an extended period of time. Ideal conditions for microbe growth often arise as a result of irregularity in temperature and humidity control onboard the spacecraft. An impaired human immune system during spacflight and the lack of available medical care 3 necessitate bacterial growth prevention measures . To prevent the survival and growth of microbes and to decrease the pathogenic stress on crewmembers, we tested whether the ÆGIS Microbe Shield™ could be used as an antimicrobial surface treatment for materials in and on spacecraft using a dynamic contact bacterial solution 4 Figure 2. Aluminum coupon used in surface treatment testing. method. Surface Testing: The bacterial suspension was transferred into 9 flasks in 50 mL portions; 4 flasks contained treated O surface, 4 flasks untreated surface, and 1 flask no surface to act as a growth control. A sample was taken from the N+ growth control at time=0 and plates were poured. The nine flasks were placed in a horizontal shaking water bath at 35 ºC and ~120 RPM (Rate 6 on Lindberg/Blue M Si Agitator). After one hour, samples were taken from every flask, plated, and incubated at 35 ºC overnight. Bacterial colonies were counted on the plates, population densities were calculated across all trials, and t-tests were completed. This information was used to determine the
effectiveness of the QAS by examining its toxicity Figure 1. Chemical makeup of the ÆGIS Microbe Shield™ with towards bacteria. silicone base, oxygen atoms, nitrogen ion and hydrocarbon Aluminum coupons, treated and untreated, were 5 ‘spear’ . autoclaved, rinsed with deionized water, and reused for the next trial. A confidence level of 95% was used to test the significance of the logarithmic data.
Surface Preparation: Four aluminum coupons (1.7 cm x For each of the species tested, the numbers of live 5.5 cm x 0.2 cm) were treated with a 1:100 water dilution bacteria were significantly reduced on the coupons treated of silane QAS (42% in methanol; AEM 5700) to 0.42% with QAS according to t-tests of the logarithmic data (Figure 2). The pieces were flipped after 7.5 minutes and (Figure 3). The exception was the second trial of B. soaked for a total of 15 minutes. The surfacing was subtilis in which a p- value of 0.428 was obtained. The TM performed in a Nuaire Biological Safety Cabinet at effectiveness of the QAS on E. coli was less than that room temperature. The coupons were hung on a wire in obtained for the other bacterial species. the safety cabinet to dry overnight.
Gravitational and Space Biology18(2) June 2005 81 D. Blustein et al. – Evaluation of a Silane Quaternary Ammonium Salt as an Antimicrobial Surface Treatment
The results, except from the fourth use of the metal (second trial of B. subtilis), correlated with the expected antimicrobial effects of the QAS. However, company claims of the permanency of the product were not supported by results of the experiments. E. coli, expected to be particularly vulnerable to the QAS from previous testing and because it is non-spore-forming, was less affected by the treatment compared to the other species. Since E. coli was used in the final two trials, a degrading (CFU/mL) microbe shield would explain this result possibily attributed to the effect of autoclaving and rinsing the
Population Density coupons between uses. The surface darkened after autoclaving in the spent media, suggesting that some sort E. coli S. epidermidis B. subtilis of change occurred to the metal. Autoclaving could have Treated 2,52E+05 5,46E+03 6,15E+03 simulated the effect of burning, which the product is not Untreated 4,46E+05 2,19E+05 5,06E+04 designed to withstand. Even with the evidence of the loss of effectiveness over Control 2,14E+05 1,09E+05 3,94E+04 time, the results suggested that this product may be of some use in controlling microbes in future space travel. If Figure 3. Logarithmic comparison of bacterial growth on aluminum coupons treated and untreated with the QAS for each surfacing techniques could be refined and the scope of the bacteria species. product better understood, this antimicrobial shield could be a very important tool in the prevention of bacterial growth on and in spacecraft of the future. According to the claim of the manufacturer (ÆGIS Since this was a preliminary analysis of the product Environments; Midland, MI.) that the QAS is permanent, only demonstrating effectiveness of the QAS in a solution repeated use of the treated surfaces was not expected to of bacteria under dynamic contact conditions, much work have an effect on the outcome. The microbe shield was still needs to be completed. While possible applications also intended to withstand temperatures well over the 6 such as using the shield within water-filled pipes could autoclaving temperature of 121 °C . The reduction in arise from this testing, the ability of the microbe shield to activity of the product after autoclaving and rinsing prevent bacterial colonization in other spacecraft increased the difficulty of comparing the QAS environments needs to be explored. Testing of bacteria effectiveness across species of bacteria (Figure 4). using newly treated metal should be performed and the durability of the surfacing agent should be explored.
100 Other investigations should include determining the concentrations of bacteria against which the product is 90 effective and in what concentration the substance must be 80 applied to the surface to retain antimicrobial properties.
70 REFERENCES 60 1. La Duc MT, W Nicholson, R Kern, K Venkateswaran. “Microbial characterization of the Mars Odyssey 50 spacecraft and its encapsulation facility.” Environ
40 Microbiol. 2003 Oct; 5(10): 977-85. 2. La Duc MT, M Satomi, K Venkateswaran. “Bacillus 30 odysseyi sp. nov., a round spore-forming bacillus isolated from the Mars Odyssey spacecraft.” Int J Syst Evol 20
Percent Inhibition (%) Microbiol. 2004 Jan; 54(Pt 1): 195-201. 10 3. Sonnenfeld G, JS Butel, WT Shearer. “Effects of the space flight environment on the immune system.” Rev 0 0 First Second Third Fourth Fifth Environ Health. 2003 Jan- Mar; 18(1): 1-17. (S. epi.) (B. sub.) (S. epi.) (B. sub.) (E. coli) (E. coli) 4. Standard Test Method for Determining Antimicrobial Repeated Inoculation Activity of Immobilized Antimicrobial Agents Under Dynamic Contact Conditions. E 2149-01. ASTM, Figure 4. The effectiveness of the QAS after repeated International; West Conshohocken, PA. Aug 2001. inoculations of Escherichia coli, Staphylococcus epidermidis, TM and Bacillus subtilis. The red arrows mark the points at which 5. ÆGIS Microbe Shield Technical Bulletin. ÆGIS the treated metal coupons were autoclaved and rinsed. Environments; Midland, MI. 7/2002. 6. Woodard, Daniel. Personal Communication. 7/6/2004.
82 Gravitational and Space Biology 18(2) June 2005
SCREENING AND IDENTIFICATION OF CRYOPRESERVATIVE AGENTS FOR HUMAN CELLULAR BIOTECHNOLOGY EXPERIMENTS IN MICROGRAVITY Todd F. Elliott1, Gokul C. Das2, Dianne K. Hammond3, Robert J. Schwarzkopf1, Leslie B. Jones1, Tacey L. Baker1 and John E. Love4 1Wyle Life Sciences, Houston, TX; 2Muniz Engineering, Inc., Houston, TX; 3EASI, Houston, TX; 4Biological Systems Office, Johnson Space Center, NASA, Houston, TX
The field of cryobiology originated in 1949, when Clonogenic Assay: Cells were seeded in 35-mm tissue Polge, et al., described the cryoprotective property of culture plates and cultured for 10-12 days. Individual glycerol in the freezing of spermatozoa (1). Lovelock and colonies were counted after staining with crystal violet. Bishop reported the cryoprotective ability of dimethyl Binary Cryoprotectant: Suboptimal levels of ethylene sulfoxide (DMSO) on red blood cells in 1959 (2). Forty- glycol and raffinose were combined to cryopreserve cells. two years later, DMSO was used to preserve the first cryogenically-stored cells launched onboard STS-105 for 100 100 80 80 use on the first human cellular biotechnology payload, 60 1ºC/min 60 1ºC/min 40 10ºC/min 40 10ºC/min Cellular Biotechnology Operations Support System 20 20 (CBOSS-01), on the International Space Station (ISS). Percent Viable 0 Percent Viable 0 7.5% 1% 4% 10% 21% 1% 4% 10% 25% DMSO is the most common cryoprotective agent used Dimethyl Sulfoxide (v/v) Glycerol (v/v) in the laboratory. While DMSO is easily eliminated in 100 100 ground-based experiments, its removal in flight-based 80 80 60 1ºC/min 60 1ºC/min experiments is more difficult due to microgravity, 40 10ºC/min 40 10ºC/min 20 20 hardware limitations, and on-orbit constraints. Because Viable Percent Viable Percent 0 0 of the deleterious effects of DMSO on cells at non- 1% 3% 10% 17% 1% 4% 10% 24% cryogenic temperatures, there was concern regarding the Ethylene Glycol (v/v) Propylene Glycol (v/v) difficulties of removing the DMSO after thawing the cells Figure 1a. Viability from Permeating Cryoprotectants. The in microgravity. results show that cells cryopreserved in dimethyl sulfoxide and Our primary goal was to find an alternate ethylene glycol have a post-thaw viability of ~80%. cryoprotectant to DMSO to be used for future CBOSS Exploration Cell Science investigations. While 40 40 30 30 1ºC/min 1ºC/min systematically screening for potential permeating and 20 20 10ºC/min 10ºC/min non-permeating cryoprotectants, we used a human 10 10 Percent Viable Percent Viable 0 0 colorectal carcinoma cell line, MIP-101 (3) that has flown 1% 5% 10% 20% 1% 5% 10% 20% on several biotechnology payloads (4), including CBOSS- Raffinose (w/v) Trehalose (w/v)
01. We utilized data from immediate post-thaw viability, 40 40 culture recovery and clonogenic assay to determine a 30 30 1ºC/min 1ºC/min 20 20 candidate alternate cryoprotectant to DMSO. Once 10ºC/min 10ºC/min 10 10 alternates were identified, we explored the use of a binary Viable Percent 0 Viable Percent 0 system consisting of permeating and non-permeating 1% 5% 10% 20% 1% 5% 10% 20% Sucrose (w/v) Dextran (w/v) cryoprotectants (5) at concentrations suboptimal in single cryoprotectant systems in an effort to maximize Figure 1b. Viability from Non-Permeating Cryoprotectants. Among the cells cryopreserved in non-permeating agents, those cryoprotective effect at lower concentrations. cryopreserved in trehalose, raffinose and sucrose have post- MIP-101 cells were resuspended in cryopreservatives thaw viabilities of 30-40%. using RPMI culture media as a carrier solution. Afterwards, they were frozen according to either a two- o 15 15 step procedure involving initial cooling at -1 C/min 10 10 o 1ºC/min 1ºC/min 10ºC/min 10ºC/min overnight in a Nalgene Cryocooler at 4 C followed by 5 5
storage in liquid nitrogen (LN2) vapor, or by storing cells Increase Fold 0 Increase Fold 0 o 7.5% 1% 4% 10% 21% 1% 4% 10% 25% directly in the LN2 vapor phase at -10 C/min. The frozen cells were thawed by immersion and agitation in a 37oC Dimethyl Sulfoxide (v/v) Glycerol (v/v) water bath. A sample was taken immediately and assayed 15 25 20 ® ® 10 by Guava Viacount to determine the post thaw viability 1ºC/min 15 1ºC/min 10ºC/min 5 10 10ºC/min and cell density. Ability to preserve cellular function after 5 Fold Increase Fold 0 Increase Fold 0 cryopreservation was assessed by the recovery of viable 1% 3% 10% 17% 1% 4% 10% 24% cells in short- and long-term cell culture experiments. Ethylene Glycol (v/v) Propylene Glycol (v/v)
Culture Recovery: Cells were plated in 6-well plates Figure 2a. Recovery from Permeating Cryoprotectants. The and cultured. After 96 hours, cells were harvested and increase in cell growth of cells cryopreserved in dimethyl viability was determined by Guava Viacount. In the assay sulfoxide, glycerol, and ethylene glycol were ~12-fold over the to determine cell growth kinetics, cells were harvested at course of 96 hours, whereas the increase of cells cryopreserved 12, 24, 36, and 48 hours. in propylene glycol was ~18-fold.
Gravitational and Space Biology Bulletin 2005 83 T. Elliott et al. – Screening and Identification of Cryopreservative Agents 40 15 The performance of a cryoprotectant in either 30 10 1ºC/min 1ºC/min preserving post-thaw viability or culture recovery must be 20 10ºC/min 5 10ºC/min 10 weighed to determine a candidate alternate to DMSO. Fold Increase Fold Fold Increase 0 0 Post-thaw viability is most important because it describes 1% 5% 10% 20% 1% 5% 10% 20% Raffinose (w/v) Trehalose (w/v) not only the number of cells that survived cryopreservation, but the starting cell density for 15 15 subsequent culture. Culture recovery describes the 10 10 1ºC/min 1ºC/min 10ºC/min 10ºC/min growth potential of the cells; however, the overall 5 5
Fold Increase Fold Increase Fold cryoprotective action of a cryoprotectant hinges upon the 0 0 1% 5% 10% 20% 1% 5% 10% 20% post-thaw viability. We found that some cryoprotectants Sucrose (w/v) Dextran (w/v) preserve post-thaw viability and others preserve post-thaw Figure 2b. Recovery from Non-Permeating Cryoprotectants. culture recovery. Remarkably, the increase of cells cryopreserved in 20% As an alternate cryoprotectant, 10% ethylene glycol raffinose jumped up to ~30-fold, whereas the increase of cells performed best in terms of viability and the clonogenic cryopreserved in trehalose was ~10-fold, dextran ~7-fold, and assay. The second best candidate was 10% propylene sucrose ~7-fold. glycol, which did not preserve viability at the level of
DMSO, but cells cryopreserved in propylene glycol had 140% s 120% greater growth potential than DMSO and ethylene glycol. 100% It is interesting to note that while raffinose-preserved 80% cells had a relatively low post-thaw viability, they had the 60% highest protection in terms of post-thaw culture recovery 40%
20% after 96 hours. In addition, in the kinetics assay, Percent of control colonie control of Percent 0% raffinose-preserved cells are the only cells where the Control Dimethyl Glycerol Ethylene Propylene Raffinose Trehalose Dextran Sulfoxide Glycol Glycol initial population increased without first declining, except
Figure 3. Clonogenic Assay. Cells preserved in ethylene glycol for the control, unfrozen cells. demonstrate 100% colony formation ability. The efficiency of Trehalose did not perform well in terms of post-thaw preservation by dimethyl sulfoxide, propylene glycol, and viability, in relation to DMSO, and not as well as trehalose were in the range of ~80-90% of the colony formation raffinose in post-thaw recovery. ability of control cells. Non-permeating cryoprotectants do not show much potential as single system cryoprotectants, but as shown 4.50 in our binary cryoprotectant experiment, may be used in 4.00 conjunction with permeating cryoprotectants in order to 3.50 reduce cryoprotectant concentration, though more 3.00 12hr development of the binary system is required. 2.50 24hr 2.00 36hr 1.50 48hr REFERENCES Fold Increase Fold 1.00 0.50 Erdag, G., Eroglu, A., Morgan, J.R., and Toner, M. 2002. 0.00 Cryobiology. 44:218-228. DMSO Ethylene Propylene Raffinose Control Glycol Glycol Jessup, J.M., Frantz, M., Sonmez-alpan, E., Locker, J., Figure 4. Kinetics. After thawing, the viabilities of the cells drop as the cells recover and begin to grow after 24 hours. Skena, K., Waller, H., Battle, P., Nachman, A., Weber, M.E., Thomas, D.A., Curbeam, R.L., Jr., Baker, T.L., and Goodwin, T.J. 2000. In Vitro Cell. Dev. Biol—Animal. 7 36:367-373. 6 5 Lovelock, J.E., and Bishop, M.W.H. 1959. Nature. 183:1394-1395. 4
3 Niles, R.M., Wilhelm, S.A., Steele, G.D., Burke, B., Fold Increase 2 Christensen,T., Dexter, D., O'Brien, M.J., Thomas, P., and Zamcheck, N. 1987. Cancer Investigation. 5:545-552. 1
0 Polge, C., Smith, A.U., and Parkes, A.S. 1949. Nature. 1.4% Ethylene Glycol 2.5% Raffinose 1.4% Ethylene Glycol & 2.5% Raffinose 164:666.
Figure 5. Binary Cryoprotection. Preliminary data suggests that the combination of 1.4% (v/v) ethylene glycol and 2.5% (w/v) raffinose has at least an additive effect on the culture recovery after 96 hours.
84 Gravitational and Space Biology Bulletin 2005
PSEUDOMONAS AERUGINOSA GROWTH AND PRODUCTION OF EXOTOXIN A IN STATIC AND MODELED MICROGRAVITY ENVIRONMENTS. S. Guadarrama, E. deL. Pulcini, S.C. Broadaway and B.H. Pyle. Department of Microbiology, Montana State University–Bozeman, MT 59717 USA
Pseudomonas aeruginosa is a ubiquitous, water-borne to MSDM2 (Modified Simple Defined Media 2) at a ratio bacterium and opportunistic pathogen, which may thrive of 1:10 and used to inoculate 5 ml syringes each with 2 ml in water and other environments. It may form a biofilm of medium to simulate flight growth chamber of cells in an extracellular polymeric matrix, on the specifications. FZN inoculum growth cycle sampling mucous membranes of the lungs in cystic fibrosis patients times were time zero, 30 min, 3 h, 6 h, 9 h, 12 h, 15 h, 18 and on many other surfaces. An important virulence h, 21 h, and 24 h. For the REF inoculum, growth cycle protein of P. aeruginosa is Exotoxin A (ETA) (Campa et sampling times were time zero, 6 h, 12 h, 15 h, 18 h, 21 h, al., 1993). The microgravity environment of spaceflight and 24 h. For both inocula and every time point, syringes (10-4 to 10-6 x g) can provide important information as to were placed in a STAT control and in a MMG how the physiology of terrestrial organisms is affected by environment. VERT controls were included only for the gravity. Modeled Microgravity (MMG) systems are used REF inoculum. Time points differ between the FZN and to simulate the gravitational effects of spaceflight on REF inocula due to space constraints in the VERT rotator cells. Previous studies have shown changes in protein and relevance to ETA production. MMG and VERT expression in P. aeruginosa PA103 (ATCC 29260) samples were rotated at 15 RPM and all samples bacteria (Pulcini et al., 2004) and up-regulation of incubated at 37oC. Samples were drop plated on R2A virulence factors in Salmonella enterica serovar agar and incubated for 24 h at 30oC. Optical density (OD) Typhimurium and Escherichia coli (Nickerson et al., readings were taken at 540 nm. The remaining culture 2004) in response to MMG. Variations in the protein was fixed with formalin (2.0 % final concentration). ETA expression of P. aeruginosa in MMG reflect alterations in was quantitated by an ELISA assay developed for metabolic and physiological functions, and also in spaceflight experiment BACTER on STS-107. Anti- putative pathways responsible for the production of P. Pseudomonas Exotoxin A (Sigma) was used as the aeruginosa virulence factors (Pulcini et al., 2004). The primary antibody, Anti-Rabbit-HRP (Sigma) as secondary virulence of P. aeruginosa is suspected to be enhanced in antibody and orthophenylenediamine (OPD) as the HRP MMG. However, the growth and physiology of P. indicator substrate. A dilution series of standard ETA aeruginosa has not been well studied in MMG conditions. (CalBioChem) was included. Optical density was read at The goal of this study was to establish the growth of this 490 nm and sample concentrations were determined from microorganism in MMG systems, and to determine how the standard curve in ng/ml. Exotoxin A production is related to different stages of growth. It is anticipated that similar experiments will be performed in spaceflight. Fundamental questions concerning the effects of spaceflight on microorganisms in relation to crew health risks remain unanswered. 9 The MMG system used to study the influence of gravity on the growth cycle was the clinostat. Samples in a clinostat still experience unit gravity (g), however, the 8.5 constant rotation of the samples results in the g-vector being time-averaged to near-zero (Klaus et al., 2001). MMG is the term used to describe the resultant state of clinorotation. Vertical rotation (VERT) was utilized as a 8 rotational control to the clinostat. In the vertical system, Log CFU/ml the resultant force gravity vector is parallel to the gravity vector of the Earth. Static control (STAT) conditions 7.5 were achieved inside clinostat tubes in a stationary position. To assess the effects of MMG, the lag, log and 7 stationary phases of growth of the ETA producing strain, PA103, were studied. The experiment inoculum was 0 3 6 9 12 15 18 21 24 prepared in two different ways. One was suspended in Time (hours) glycerol (20 % final concentration) and frozen at -80oC (FZN) while the other was suspended in water and Figure 1. Growth cycle for frozen inoculum (n=3), refrigerated at 4oC (REF). Plate count results were used to ◊ Static, □ MMG, and refrigerated inoculum (n=3), adjust the inoculum suspension to ca. 5x108 CFU/ml ♦ Static, ■ MMG, X Vertical. before freezing or refrigeration. Both inocula were added
Gravitational and Space Biology 18(2) June 2005 85 S. Guadarrama et al. – Pseudomonas aeruginosa Growth and Production of Exotoxin A in Static and Modeled Microgravity Environments.
400 consistent and comparable results. Future work will focus on studying the effects of different speeds in the clinostat 350 and another MMG system such as the Synthecon HARV (High-Aspect Ratio Vessel), on the growth cycle of 300 PA103, including shear stress consequences on growth and virulence. In addition, proteomic analyses will be 250 performed at intervals over the growth cycle to determine variations in protein expression which may be related to 200 virulence.
ETA ng/ml ETA 150
100 REFERENCES
50 Campa, M., Bendinelli, M., and Friedman, H. 1993. Pseudomonas aeruginosa as an Opportunistic Pathogen. 0 Plenum Press, New York. pp. 107-113. 0 6 12 18 24 Time (hours) Klaus, D. M. 2001. Clinostats and Bioreactors. Gravitational and Space Biology Bulletin 14(2): 55-64.
Figure 2. ETA production for frozen inoculum (n=3), Nickerson, C., Ott, M., Wilson, J. W., Ramamurthy, R., ◊ Static, □ MMG, and refrigerated inoculum (n=3), and Pierson, D. L. 2004. Microbial Responses to ♦ Static, ■ MMG, X Vertical. Microgravity and other Low-Shear Environments. Microbiology and Molecular Biology Reviews. 68(2): 345-361. The growth cycle of Pseudomonas aeruginosa was apparently not affected by the MMG conditions of the Pulcini, E.deL., Broadaway, S.C., and Pyle, B.H. 2004. clinostat (Figure 1 and 2). The initial cell numbers in Pseudomonas aeruginosa Virulence and Proteomics in samples from the FZN inoculum diluted in MSDM2 were Simulated Weightlessness. American Society for consistently lower than those from the REF inoculum. Microbiology. Abstract I-033. The FZN inoculum cultures also reached lower numbers in the stationary phase, and there was a decrease in CFU/ml between 18 and 21 h that recovered by 24 h (Figure 1). This decline and recovery did not occur with the REF inoculum (Figure 2). The overall ETA production rate was fairly constant during the log phase with both inocula. For the FZN inoculum, the ETA concentration in the MMG was essentially equivalent to the STAT control (Figure 3). With the REF inoculum, the ETA concentration was somewhat higher in the vertical rotation system when compared to the MMG and STAT samples (Figure 4). The difference between the bacterial populations of the FZN vs. the REF inocula apparently affected the overall ETA production throughout the growth cycle (Figures 1, 2, 3 and 4). Mean ETA concentrations in samples from the REF inoculum were generally higher than from the FZN inoculum. Differences between the FZN and REF inocula may have been the result of selection of a particular population during freezing. As demonstrated here, due to the differences that can be induced in the experimental sample by pre-experiment storage conditions, it is crucial importance that all experiments, especially for spaceflight, are done with the inoculum prepared in the same way to obtain
86 Gravitational and Space Biology 18(2) June 2005
DEVELOPMENT AND TESTING OF AN EFFICIENT LED INTRACANOPY LIGHTING DESIGN FOR MINIMIZING EQUIVALENT SYSTEM MASS IN AN ADVANCED LIFE-SUPPORT SYSTEM. G. D. Massa1, J. C. Emmerich2, M. E. Mick1, R. J. Kennedy1, R. C. Morrow2, C. A. Mitchell1 1 Dept. of Horticulture & Landscape Architecture, Purdue Univ., West Lafayette, IN.2 Orbital Technologies Corp., Madison, WI. Orbitec had previously designed a 1-in-square “Chip- NASA has defined a metric to determine the on-Board” LED “light engine” for plant growth necessary launch capacity for an advanced life-support containing up to 132 LEDs with 5 possible colors, and 2 system (ALS), designed to support humans for an photodiodes that can detect a variety of wavelengths. For extended duration beyond low earth orbit. This metric this system, we utilized these light engines. However, allows the comparison of techniques and technologies due to our requirements, chips were populated only with over time and between mission scenarios, yielding the red and blue LEDs, with green LEDs and photodiodes optimum physical-chemical and bioregenerative array for also in place but not currently utilized. Future generations human life support. The metric in use is Equivalent could include different LEDs depending on individual System Mass (ESM), which equates power, volume, crop requirements. Each lightsicle strip has 20 light cooling and crew time into a mass-equivalency unit (Kg) engines which have 5 rows of LEDs, four rows of sixteen as shown by Equation 1 (Levri, 2003). 640-nm red LEDs and one row of sixteen 440-nm blue Equation 1 ESM = M + (V•Veq) + (P•Peq) + (C•Ceq) + LEDs giving a total of 80 LEDs per chip (Fig. 1 inset). (CT•D•CTeq) Heat from the LED chips is removed through a channel running behind the strip and a fan-driven airflow system Where M, V, P, C, CT and D are the mass, volume, mounted in the lightsicle enclosure (Fig. 1). This cooling power, cooling, crew time and mission duration, method allows plants to grow near or touch the strips respectively, for a given system, and the subscript without scorching. multipliers are the equivalency factors used to convert these into units of mass equivalents. Lighting for plant growth is estimated to account for 43% - 60% of the total ESM for biomass production, with much of this value due to the power requirements (Drysdale and Bugbee, 2003). Significant energy from overhead electrical lighting is lost by inaccurately targeting the light to all leaves in a crop canopy. When plants are young, light is lost by illuminating empty space around seedlings. If crops are planophiles (e.g. cowpea, soybean), which close their canopies as they age, then younger leaves shade out older leaves. This leads to a small percentage of leaves (approximately 10-15%) doing the photosynthetic work to support the entire crop stand. To avoid wasting light and to utilize more of a crop’s Figure 1. The components of an individual lightsicle. Inset is a photosynthetic capabilities, we envision an intracanopy light engine showing the LEDs and photodiodes. lighting system that allows switching of lights in response to plant growth. A reconfigurable lighting system is the Other components of the lighting system include a result of a jointly sponsored collaborative research control enclosure containing a power supply and circuitry agreement between Orbital Technologies Corp. (Madison, that control LED intensity via dimmer potentiometers, as WI) and the ALS NSCORT Crop Production group at well as incremental switching of light engines from the Purdue University. We have worked together to develop bottom to the top of lightsicles. Additionally, a timer a light-emitting diode (LED)-based system from earlier allows preprogramming of desired photoperiod. A proof-of-concept studies with fluorescent lighting (Frantz separate power and communications distribution et al., 2001). assembly allows integration of the 16 strips. Fig. 1 illustrates an individual unit or “lightsicle” of A mounting system was developed to suspend the the lighting array. The first array consists of sixteen such lightsicles within a crop-growth compartment. The strips, each mounted either independently or back to back. growth area consists of an EGC (Chagrin Falls, OH) Strips also can be mounted in a planar configuration, walk-in growth chamber with temperature, relative allowing an overhead control for intracanopy studies, and humidity, and CO2 control. A recirculating hydroponics eventually a separate close-canopy lighting system for system was constructed. The root-zone compartment is erectophile (e.g. wheat) and rosette (e.g. lettuce) crops, mounted on a table frame, and a framework of angle iron where illumination switching will track the plant was constructed around the hydroponic system. After silhouette from above. Initially, however, the array will lighting installation was complete, walls made from be used as an intracanopy lighting system for planophile reflective white poly film were hung between the top of crops. the framework and the table. Lightsicle mounts were suspended from struts placed horizontally over the framework at intervals. Brackets from the struts were Gravitational and Space Biology 18(2) June 2005 87 G. Massa et al. – LED Intracanopy Lighting Design for ALS attached to metal strips, and corresponding strips were fastened to the lightsicle mounting bracket (Fig. 1). Velcro was used to join the two sets of strips together. This mounting system allows ease of lightsicle removal for configuration changes, height adjustment, and maintenance. Lightsicles were mounted in a configuration to maximize light coverage uniformity within the growth compartment. The power and communications assembly was mounted to the framework adjacent to the growth compartment. Cables run from each individual lightsicle to this assembly, and separate cables extend from the distribution assembly to the control enclosure outside of the chamber. Photosynthetically active radiation (PAR) was measured at different positions within the canopy space. Measurements were taken while red and blue intensities Figure 3. Current versus PAR with 5, 10, 15, and 20 light were changed and incremental switching of light engines engines illuminated at each intensity for a 16-strip array. PAR occurred. Fig. 2 shows the increases in PAR with light was measured without plants with the light sensor centered in intensity at two different heights within the array. the array on the foam base plate using a Li-Cor LI-1776 cosine- corrected solar monitor.
We have developed a reconfigurable lighting array for plant growth in an ALS. This system is initially in an intracanopy arrangement for growth of planophile crops. Initial system tests indicate good performance with possible electronic modification in future systems to counteract component variations. Current drawn by the system strongly correlates with light output. The first cowpea crop grown in the system appeared healthy though somewhat elongated with weak stems. This test demonstrated the need for intense blue light early on to allow for de-etiolation and reduction in hypocotyl elongation. Other protocol modifications may include switching an extra light engine on to guide plant growth. Preliminary ESM calculations for an intracanopy lighting Figure 2. PAR at increasing light intensity within the 2 3 intracanopy lighting array. Measurements were taken array on a m and m basis are underway. These approximately 2.5 cm directly in front of either A. light engine calculations will allow us to define the critical sensitivity #5 or B. light engine # 15 of a lightsicle in the center of the points within the lighting subsystem that will yield a high array. When measurements were taken at engine # 5, only return on investment when improved, i.e. big reductions engines 1 through 5 were illuminated. When measurements in ESM. were taken at # 15, engines 1-15 were illuminated. ACKNOWLEDGEMENTS The differences between PAR values in Fig. 2A and The authors would like to thank Tom Crabb, Bruce B are due to radiation reflected off the poly film wall, Bugbee, and Ray Wheeler for helpful discussions. We radiation from surrounding engines, and variations in the gratefully thank Charles Barnes for his help and light engines and associated drivers. The maximum light encouragement. Thanks to the Orbitec technical staff for output measured within the system was approximately engineering. This work was supported in part by NASA: 900 µmols/m2/s (data not shown). NAG5-12686. In addition to PAR measurements, current draw was measured as light intensity was increased (Fig. 3). The correlation coefficient (R2 = 0.994) indicates good REFERENCES agreement between energy input to and PAR output from Drysdale, A., Bugbee, B. 2003 Optimizing a plant habitat the system. The y intercept indicates the baseline current for space: a novel approach to plant growth on the moon. value for an energized system with no lights on. We ICES 2003-01-2360, SAE International. measured this value at 2.6 Amps running with 24 volts DC. Frantz, J.M. Joly, R.J., Mitchell, C.A. 2001 Intracanopy lighting reduces electrical energy utilization by closed cowpea stands. Life Support Biosphere Sci. 7: 283-290.
Levri, J. 2003 Personal Communication. 88 Gravitational and Space Biology 18(2) June 2005
CELL BEHAVIOR IN SIMULATED MICROGRAVITY: A COMPARISON OF RESULTS OBTAINED WITH RWV AND RPM A. Villa1, S. Versari1, J.A.M. Maier2 and S. Bradamante1. 1CNR-ISTM, Via Golgi 19, Milan – Italy 2Dept. Preclinical Sciences-LITA Vialba, University of Milan, Via GB Grassi, Milan — Italy.
INTRODUCTION Some pathological conditions described in astronauts, Random Positioning Machine such as cardiovascular deconditioning, represent the adaptive response to the absence of gravity and are The RPM is essentially a 3-axis clinostat, which creates a partially due to the effects exerted by microgravity (µg) at condition in which the weight vector is continually the cellular level. Since endothelial cells are crucial in the reoriented as in traditional clinorotation, but with maintenance of the functional integrity of the vascular increased directional randomization (Klaus, 2001). The wall, it is noteworthy that we described endothelial instrument operates inside an incubator with controlled dysfunction in response to simulated µg. In particular, we temperature and atmosphere (CO2 percentage, relative have shown that cultured Human Umbilical Vein humidity...). Standard consumable experiment hardware Endothelial Cells (HUVEC) in the Rotating Wall Vessel can be used, such as 6 wells multidishes sealed with a gas (RWV) grow faster than controls, rapidly remodel their permeable membrane, in order to achieve a suitable gas cytoskeleton and, after a few days, markedly down- exchange. More sophisticated hardware, derived from regulate actin (Carlsson et al., 2002, Carlsson et al., spaceflight experience, will enable automatic medium 2003). The RWV system requires cells suspended or exchange, fixation, etc. Primary applications are cell and seeded on microcarriers. This is an obstacle to evaluate developmental biology and tissue engineering. Simulation other endothelial functions, among which cell migration, a of µg by the means of continuous random change of crucial event in vasculogenesis and angiogenesis. We orientation of objects relative to the gravity's vector can therefore decided to simulate µg using the Random generate effects comparable to those of true µg when the Positioning Machine (RPM) that allows to culture cells in changes are faster than the object's response time to standard plates. RPM has been demonstrated to be a good gravity. Thus, slow responsive living objects, are µg simulator for plants, osteoblasts and T lymphocytes. excellent candidates to be studied on RPM. Up to now, there are only few data about the behaviour of adherent cell cultures in RPM. MATHERIALS AND METHODS To optimize the RPM operative parameters, we chose as a HUVEC were obtained from the American Type Culture model human monocytoid U937 cells that have been Collection (ATCC) and cultured in M199 containing 10% studied in simulated and real µg (Hatton et al., 1999; fetal calf serum, Endothelial Cell Growth Factor (150 Maier 2004). On the basis of previous results, our aim mg/ml) and heparin (5 U/ml). The cell culture system was was: a) to evaluate the best operative experimental based on 2% gelatin-coated 6 wells multidishes. In order conditions of RPM, b) to compare the results with those to achieve a suitable gas exchange, every dish was sealed obtained in simulated (RWV) and real (spaceflight) µg with a gas permeable membrane (Breathe-Easy, Sigma) conditions; c) to use RPM to grow HUVEC in order to avoiding air bubbles formation. To be used in the RWV, confirm and, possibly, broaden the RWV results. HUVEC were seeded on beads (Cytodex 3, Sigma). Cells were subcultured using 0.05% trypsin 0.02% EDTA Rotating Wall Vessel solution. All culture reagents were from Sigma. U937 cells have been cultured in RPMI medium containing The RWV is a suspension culture vessel optimized to 10% calf serum. In order to assess the viability, cells have produce laminar flow and minimize mechanical stress on been counted every 48h after staining with Trypan Blue, cell aggregates in culture. It provides fluid dynamic using a Burker chamber. To simulate µg, we used the operating principles characterized by 1) solid body RWV bioreactor (Cellon) with 10 and 50 ml disposable rotation about a horizontal axis that is characterized by vessels and the RPM facility at the Dutch Experiment colocalization of cells and aggregates of different Support Center (DESC, Amsterdam, NL) accommodated sedimentation rates, optimally reduced fluid shear and in a dedicated temperature controlled incubator capable of turbulence, and three-dimensional spatial freedom; and 2) supplying a 5% CO2/air mixture. To observe the oxygenation by diffusion, excluding undissolved gases cytoskeleton, immuno-fluorescence/confocal microscopy from the bioreactor (Hammond et al., 2001). The of cells fixed with paraformaldehyde and stained with cylindrical culture vessel is filled with culture fluid and fluoresceine isothiocyanate (FITC)- phalloidine has been the cells or tissue particles are added. All air bubbles are performed. removed from the culture vessel. Oxygenation is achieved through a gas permeable silicone rubber membrane. Since RESULTS AND DISCUSSION the Rotary Cell Culture System™ has no impellers, airlifts, bubbles, or agitators, tissue damage from impact Cell proliferation and turbulence is decreased with shear stress and damage It is reported that µg affects cell growth. Here we essentially insignificant. evaluated the proliferation of U937 and HUVEC grown in
Gravitational and Space Biology 18(2) June 2005 89 A. Villa, et.al., Cell Behavior in Simulated Microgravity the RWV and RPM systems. Figure 1 reports the duplication time of U937 and HUVEC. A B As expected on the basis of in flight experiments performed on the Shuttle, U937 in the RWV and in the RPM grew 40% slower than controls. On the contrary, HUVEC proliferated 50% faster than controls. These effects are reversible upon return to normal growth conditions. We conclude that µg simulated either by RWV or RPM leads to comparable results.
C D U937
36
24 Time (h) 12
Figure 2. Confocal microscopy images of HUVEC grown in 0 RPM (B, D) and their ground controls (A, C) stained with control RWV RPM fluorescent phalloidin. 48 HUVEC ACKNOWLEDGMENTS
36 The research has been supported by ESA contract 14631/SH/NL/00. We are grateful to Jack Van Loon (DESC, Amsterdam, NL) for the expert technical help 24 and to Dr. Aviral Vatsa for its support with the confocal Time (h) microscope. 12
0 REFERENCES Control RWV RPM Carlsson SIM, Bertilaccio MTS, Ascari I, Bradamante S, Figure 1. Duplication time of U937 and HUVEC cultured in Maier JAM. Modulation of human endothelial cell RWV and RPM vs. static gravity controls. Standard Errors are behaviour in simulated microgravity. J Grav Physiol <10%. 2002, 9: P273-274.
Cytoskeleton reorganization Carlsson SIM, Bertilaccio MTS, Ballabio E. and Maier J.A.M. Endothelial stress by gravitational unloading: Different cell types cultured in µg show cytoskeleton effects on cell growth and cytoskeletal organization. reorganization. We therefore stained HUVEC grown in Biochim. Biophys. Acta - Molecular Cell Research (2003) simulated µg in the RPM and their ground controls with 1642: 173-179. fluorescent phalloidin to visualize the cytoskeleton.
Ground controls (A, C) show a well-organized Hammond T.G. and Hammond J.M. (2001) Optimized cytoskeleton with abundant stress fibers organized into suspension culture: the rotating-wall vessel. Am J Physiol bundles. HUVEC grown in RPM (B) for 96 h show major Renal Physiol 281, F12-F25. modifications of cell shape, as previously observed in
RWV experiments, with elongated extended podia (D), Hatton JP, Gaubert F, Lewis ML et al. The kinetics of disorganized actin fibers and clusters. Cytoskeleton translocation and cellular quantity of protein kinase C in modifications are reversible upon return to normal growth human leukocytes are modified during spaceflight. conditions. FASEB J 1999, 13 Suppl:S23-S33.
CONCLUSION Klaus D.M. (2001) Clinostats and bioreactors. Gravit The similar U937 behavior in space-flight (Hatton et al., Space Biol Bull 14, 55-64. 1999) and simulated µg conditions supports RWV and RPM as good tools to simulate µg on earth. In addition, Maier JAM. The adaptive response of cells to RWV and RPM exert similar effects on HUVEC gravitational unloading. 25th International Gravitational behaviour. Our results indicate that both the systems work Physiology Meeting (Abstract). June 6-11, 2004. at similar simulation levels, thus allowing a wider spread Moscow, Russia. of experiments.
90 Gravitational and Space Biology 18(2) June 2005
METHODS FOR THE CULTURE OF C. ELEGANS AND S. CEREVISIAE IN MICROGRAVITY Thomas Fahlen1, June Sunga1, Jon Rask1, Anna Herrera2, Kitty Lam2, Luke Sing1, Kevin Sato1, Ross A. Ramos1, Melissa Kirven-Brooks1, and Debra Reiss-Bubenheim3 1Lockheed Martin Space Operations, NASA Ames Research Center, Moffett Field, CA 94035 2 NASA/Ames Internship Program, NASA Ames Research Center, Moffett Field, CA 94035 3NASA Ames Research Center, Life Sciences Division, Moffett Field, CA 94035
To support the study of the effects of microgravity on biological systems, our group is developing and testing methods that allow the cultivation of C. elegans and S. cerevisiae in microgravity. Our aim is to develop the experimental means by which investigators may conduct peer reviewed biological experiments with C. elegans or S. cerevisiae in microgravity. Our protocols are aimed at enabling investigators to grow these organisms for extended periods during which samples may be sub- cultured, collected, preserved, frozen, and/or returned to earth for analysis. Data presented include characterization of the growth phenotype of these organisms in liquid medium in OptiCells™ (Biocrystal, LTD). Subculture and sampling activities of C. elegans and Figure 2. Growth of C. elegans in OptiCells™ with various S. cerevisiae in microgravity are more manageable when starting densities. Animals were inoculated to a density of 10, the organisms are grown in a closed liquid culture system. 100, or 1000 worms/ml 10 ml of CeMM in OptiCells™ and We have therefore examined the growth of C. elegans in incubated at 20 °C. Worms were periodically withdrawn and C. elegans minimal medium (CeMM) (Lu et al. 1993, counted. Error bars show the standard deviation from the Szewczyk et al. 2003) utilizing Opticells™ as growth mean. N = 3. chambers. The OptiCells™ are under investigation as a containment system for liquid C. elegans culture since they are amenable to flight subculture and sampling procedures. Our data show that C. elegans grow at a rate comparable to that in a standard culture flask, reach densities of up to 1 x 106 worms/ml in liquid medium in OptiCells™, and may be initiated with as few as 10 worms/ml (Figs 1 and 2). Similarly, S. cerevisiae cultured in standard YPD medium in OptiCells™ grow at a comparable rate to those in a static flask (Fig. 3). Note that the Opticells apparently mimic a static flask condition rather than a shaking flask condition. These results suggest that our culture conditions support the growth of C. elegans and S. cerevisiae in a manner comparable to Figure 3. Growth of S. cerevisiae in Opticells. Stationary phase conventional culturing methods. S. cerevisiae BY4743 were diluted 1:1000 in YPD broth, transferred to shaker flasks or Opticells™, and incubated at 30 °C. The flasks were incubated either with shaking or statically. The OptiCells™ were incubated statically with a 2mm space between replicate Opticells. Growth was monitored over time by measuring the optical density at 600 nm. Error bars depict the standard deviation from the mean. N = 3.
Characterization of the required conditioned transport and storage of CeMM is important as it affects the hardware, power, and space requirements for the experiment. CeMM is known to contain components that are light and temperature sensitive and is therefore typically stored at 4 ˚C in the dark. To characterize the Figure 1. Growth of C. elegans in OptiCells™ compared to effect of storage at elevated temperature, we grew worms flasks. Animals were inoculated 10 ml of CeMM in OptiCells™ in medium that had been stored at room temperature in or T75 vented flasks. Samples were incubated at 20 °C and worms were periodically counted. Error bars show the standard the dark for 10 months. As shown in Figure 4, this deviation from the mean. N = 3. severely affected ability of the medium to support C. elegans growth.
Gravitational and Space Biology 18(2) June 2005 91 T. Fahlen et al. – Methods for the Culture of C. elegans and S. cerevisiae in Microgravity
1.60E+06 S. cerevisiae cells must be held in stasis during launch 1.40E+06 Media Storage to allow controlled initiation of a flight experiment. We 4 C 1.20E+06 have examined methods that may allow us to prevent Room Temp 1.00E+06 yeast growth yet maintain viability prior to experiment
8.00E+05 initiation. It is common practice to freeze cells long term in YPD containing 15% (v/v) glycerol. Although 15% 6.00E+05 (v/v) glycerol affects the growth rate of the cells, we 4.00E+05 found that lower concentrations of glycerol, such as 5% 2.00E+05 (v/v) do not significantly affect the S. cerevisiae growth
0.00E+00 rate (data not shown). We assessed the use of the lower 0 5 10 15 20 25 30 glycerol concentration for preservation of S. cerevisiae at Time (days) –20 °C. As shown in Figure 6, we found that 5% glycerol Figure 4. Effect of CeMM storage temperature. Animals were in YPD medium was sufficient to significantly improve inoculated to OptiCells™ containing 10 ml of CeMM that had been stored at either room temperature or at 4 ˚C in the dark for the long term viability of S. cerevisiae cells at -20 °C. 10 months. Samples were incubated at 20 °C and worms were These data suggest that YPD supplemented with 5% periodically withdrawn and counted. Error bars show the glycerol may serve as an ideal freezing and growth standard deviation from the mean. N = 3. medium for S. cerevisiae during flight experiments.
As expected, during growth testing of S. cerevisiae in YPD medium in OptiCells™, we observed the formation of large bubbles, CO2, within the OptiCells™ (data not shown). Since bubbles are undesirable during flight experiments, and dextrose is a substrate converted to CO2, we sought to mitigate bubble formation by reducing the dextrose in the YPD medium, which typically contains 2.0% dextrose. To this end, we grew samples in OptiCells™ in YPD medium modified to contain 2.0%, 1.5%, 1.0%, or 0.5% dextrose. With lower dextrose concentrations, we found a slightly reduced growth rate and final cell density (data not shown), yet a dramatic reduction in bubble formation (Fig. 5). This suggests that modulation of the glucose concentration in YPD medium Figure 6. S. cerevisiae BY4743 survival in YPD + glycerol. 100 may be used to reduce bubble formation in OptiCells™ µl aliquots of stationary phase cells at 1 x 105 cells/ml in YPD, by yeast. or YPD + 5%, 10%, or 15% v/v glycerol were transferred to 1.5 ml microfuge tubes and placed at -20 °C. Samples were plated 0.7 for viability over time. N = 3. Error bars depict the standard
0.6 deviation from the mean.
0.5 The experiments described support the cultivation of C. 0.4 elegans and S. cerevisiae in microgravity utilizing OptiCells™. In each instance the OptiCell(tm) containers 0.3 provided growth rates similar to standard laboratory 0.2 vessels. Our data suggest that OptiCells, coupled with the
0.1 appropriate media, may provide a means to culture C. elegans and S. cerevisiae in a manner conducive to space 0.0 10 12 14 16 18 20 22 flight experiments. Time (Hours) REFERENCES Figure 5. Gas bubble formation in OptiCells. S. cerevisiae Lu, N.C. and K.M. Goetsch. (1993) Carbohydrate BY4743 was grown in OptiCells in YPD with 0.5%, 1.0%, 1.5%, requirement of Caenorhabditis elegans and the final or 2.0% w/v dextrose. The bubble size is expressed as the ratio development of a chemically defined medium. of distance to the bubble meniscus over the length of the Nematologica 39:303-331. OptiCell™ window. 0.5% dextrose did not produce measurable bubbles. Error bars depict the standard deviation from the mean. N = 3. Szewczyk N.J., E. Kozak, and C.A. Conley (2003) Chemically defined medium and Caenorhabditis elegans. BMC Biotechnology 3:19-27
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DEVELOPMENT OF THE EMCS HARDWARE FOR MULTIGENERATIONAL GROWTH OF DROSOPHILA MELANOGASTER IN SPACE M.E. Sanchez2, M. Shenasa1, A. Kakavand2, R.S. Stowers1, D. Leskovsky2, and S. Bhattacharya3 1Education Associates Program, 2Lockheed Martin Space Operations, 3NASA Ames Research Center, Moffett Field, CA
Understanding the changes that occur in living organisms to bring about adaptation to the space environment is essential to support future plans for long-term missions to the Moon and Mars. The European Modular Cultivation System (EMCS), a life science research facility developed by the European Space Agency (ESA), will serve as a habitat for culturing multiple generations of Drosophila melanogaster. Two different Prototype Containers (I and II) were designed and developed at Ames Research Center to test the feasibility of culturing Drosophila specimens using the EMCS (Figures 1 and 2).
Figure 3. Biocompatibility of PI, PII, and standard lab vials with Salad in a Bag or BreathEasy membranes, or standard cotton or buzz (cellulose acetate) plugs.
Neither Salad in a Bag nor BreathEasy membranes supported Drosophila growth but both cotton and buzz plugs did. Apparently, the diffusion rate of C02 and 02 through either membrane type is too low. Growth in PI Figure 1. EMCS Drosophila Prototype Containers and Food and PII compared favorably with control standard lab Compartments. A) Prototype I (PI) B) Prototype II (PII). PI vials with cotton or buzz plugs. We do not consider any has a fixed food reservoir while PII has a rotateable food differences in growth between cotton and buzz plugs cylinder. consequential as only single replicates of the experiment were conducted. The reason behind this is that in the testing phase only one of each custom-made prototype container is routinely built so that any problems encountered can be resolved through design changes in the next generation of the container. With the biocompatibility of PI and PII established, we next determined the effect of different starting population densities on progeny production in a single generation. The starting population size needs to strike something of a balance between generating a sufficient number of progeny that the results will be statistically significant, while at the same time not introducing the Figure 2. Fully assembled prototype containers with flies. variable of overcrowding. As shown in Figure 4, starting population sizes of 15 males/30 females (15M/30F) The objective of the study presented here was to produced greater numbers of progeny than 10M/20F. The optimize the biocompatibility of the prototype hardware. lower number of progeny production in PII relative to PI Fly behavior, humidity levels, and hardware mechanics is likely caused by first instar larval escape from PII. were assessed. Modifications to the hardware were made Based on our studies, 15M/30F is an appropriate number as required to resolve any suboptimal performance issues. of starting adults. Flies were grown in containers using standard techniques for fly handling and videotaped using the video capabilities of the EMCS ERM camera system. Since multigenerational growth is a requirement for space To determine the biocompatibility of PI and PII we experiments, we assessed the ability of PI and PII to tested both containers, as well as standard lab vial support growth over four generations. These data are controls, with the indicated membranes or plugs for their shown in Figure 5. ability to support Drosophila growth and survival (Figure 3). Wildtype flies were used in all experiments.
Gravitational and Space Biology 18(2) June 2005 93 M.E. Sanchez – Development for the EMCS Hardware EMCS video system was successful in imaging the behavioral parameters of the flies. Both analog video images and still digital images were captured. An example of a digital image is shown in Figure 6.
Figure 4. Comparison of parental density on progeny production in a single generation in PI and PII. Figure 6. Digital image of adult Drosophila in PI container acquired with the EMCS imaging camera system.
SUMMARY Two prototype containers (PI and PII) were developed for the purpose of supporting multigenerational growth of Drosophila on the International Space Station. Through population growth studies we determined that using cotton or buzz plugs allowed air circulation rates and humidity levels compatible with supporting growth of Drosophila. These containers support growth of 200-250 flies per generation. Population density studies showed that 30 females and 15 males is a good starting parental population size. The EMCS camera system successfully Figure 5. Ability of PI, PII, and control containers to support captures analog video images and still digital images. multigenerational Drosophila growth. Planned future studies include assessing the effects of different sterilization and packaging methods on food Fresh food reservoirs (PI) and food cylinders (PII) were integrity when stored for extended periods. The growth transferred to PI and PII containers after eclosion of each of mutant strains of Drosophila will also be analyzed as generation of adult Drosophila. Three days later, the food mutant growth rates will likely differ from wildtype flies. reservoirs and cylinders containing embryos and larvae In addition, the development of a sampling tool that will produced by the adults were transferred to new PI and PII enable fly extraction from the prototype containers is containers. planned
Over four generations, both PI and PII supported growth of larger fly populations than standard lab vials. This experiment demonstrates that PI and PII provide conditions sufficient for the growth of 200-250 Drosophila.
In the low gravity space environment where convection will not be occurring, we do not expect to observe significant changes in Drosophila growth rates or survivability because Drosophila has a relatively low metabolic rate that can be accommodated by exclusively diffusion-limited gas exchange. Also, the flight of adult Drosophila will promote air flow in the containers, and at least the adult Drosophila can move to regions where the 02 and C02 are favorable if major gradients were to develop.
For many experiments, video imaging of Drosophila in PI and PII containers will be an essential assay for understanding the effects of altered gravity on fly behavior. The EMCS camera system is designed for this purpose. The EMCS camera has 18X optical zoom and 4X digital zoom. Initial results demonstrate that the 94 Gravitational and Space Biology 18(2) June 2005
NEUROPHYSIOLOGICAL LONG-TERM RECORDINGS IN SPACE: EXPERIMENTS SCORPI AND SCORPI-T Michael Schmäh and Eberhard Horn University of Ulm, Gravitational Physiology, Albert-Einstein-Allee 11, 89081 Ulm, Germany The first challenge for SCORPI was the development The International Space Station ISS offers the opportunity of a proper immobilization technique. A 3-point immobi- for physiological long-term observations in microgravity lization seems to be the most suitable one (Fig. 1), but a in awake animals. The 3-month period of experimentation multi-point fixation at the base segment of each leg will favours heredity, development and physiological adapta- be tested during the precursor space flight experiment tion as scientific fields that can profit from those long- SCORPI-T on the Russian satellite FOTON-M2 in 2005. term exposures to microgravity. However, the most com- plicating problems are (i) the lack of proper animal habi- tats, and (ii) the available crew time. These facts dramati- cally decrease the types of experiments and, in particular, the animal species that can be used for long-term studies. From the methodological point of view, automatic working recording devices are the most suitable tech- niques. Experience with neurophysiological long-term recordings based on ground studies, in particular in in- sects (cf. Miller, 1979), favours the study of central inte- gration of neuronal, sensory and muscular activity of the organisms and, in particular, its adaptation to the micro- gravity environment. The lack of proper animal habitats on ISS favours the use of animals that are adapted to a life in extreme environmental conditions with rare food sup- ply. Only those animals can be used that are adapted to a life in extreme environmental conditions and that are able Figure 1. A 3-point immobilization of scorpions for neuro- to starve for long periods of their life due to limited ac- physiological long-term recordings in space. Pedipalps and cess to food and water. Desert animals such as beetles and opisthosoma base are fixed by aluminium clamps (C). The tail is scorpions are species of first choice. connected to a thread (TF) that restricts the extent of tail move- In 2001, an experiment with scorpions titled SCORPI ments protecting the electrodes from damage by the sting. Elec- trodes are inserted at eye, leg, opisthosoma, and brain. Despite was selected for flight on ISS mounted in the European of this immobilization, animals catch prey actively that is a nec- research facility BIOLAB. Its main purpose is to analyse essary condition for feeding. Animals survive in this harness for the adaptation of coordinating mechanisms between vege- months. tative and sensorimotor activities to microgravity by means of neurophysiological recordings. The second challenge was to develop the fully auto- Among the basic coordinating principles of physio- mated equipment that allow recordings of the visually logical mechanisms common in most organisms are bio- elicited activity (electroretinogram, ERG), the muscular logical clocks and their synchronization with external activity (electromyogram, EMG), the arousal of the brain Zeitgebers such as the daily light-dark rhythm and other (spontaneous cerebral electrical activity, SEA) and the geophysical fields. In humans, desynchronisation of these heart beat frequency (electrocardiogram, ECG) (Fig. 2). rhythmic events during space flights (Gundel et al., 1997; Dinges, 2001) or microgravity simulation such as bed rest head-down tilt (Samel et al. 1993) can cause physiologi- cal, behavioral and psychological disturbances. Studies in animals and lower organisms revealed that circadian rhythms may be altered, but do not disappear during space flights of rhesus macaques (Fuller et al., 1996), the beetle Trigonoscelis gigas (Alpatov et al., 1994), Chlamydomo- nas and Neurospora (Ferraro et al., 1995). The project SCORPI includes two important objec- tives, the hardware and the science objective. In particu- lar, SCORPI has to demonstrate the reliability of a con- tinuous neurophysiological multi-channel recording tech- nique in the restrained animals during a long-term space flight. Furthermore, it has to analyze the integration of motor, neuronal and sensory signals and its long-term Figure 2. Implantation sites of the electrodes for the neuro- adaptation to microgravity with specific consideration of physiological multi-channel long-term recordings. the biological clock system.
Gravitational and Space Biology 18(2) June 2005 95 M. Schmäh and E. Horn – Neurophysiological Long-term Recordings in Space: SCORPI and SCORPI-T The ERG is induced by short light pulses. Its basic Mounting of electrodes and the impact of animal shape is bipolar; in the case of rapid repetition of the movements on the reliability of the neurophysiological stimulus, adaptation occurs (Fig. 3). For SCORPI, this is recording will be studied during the precursor flight on not critical because the individual test light stimuli are FOTON-M2 that lasts 2 weeks. Thus, the specific goals of presented 10 to 20 min apart from each other. The pa- SCORPI-T are to study critical aspects of the experiment rameters EMG, SEA and ECG are recorded during spon- SCORPI such as effects of animal locomotion on the im- taneous activity. In general, during motor activity the mo- plantation of the electrodes, or the impact of stress to the tor signal (EMG) overwhelms all traces (Fig. 4); however, animals caused by launch vibrations, logistics and micro- high- and low-pass filtering facilitates easy detection of gravity at all. all other activities such as ECG (Fig. 5) and SEA (Fig. 6, upper trace). For the low frequent signals such as ERG and ECG, low pass filtering is necessary while for the SEA, a specific filtering window is used.
Figure 6. SEA after filtering from high-frequent noise. No mo- tor activity during the recording period. Arbitrary potential amplitude scale differs from those in Figs 3-5.
The techniques developed to date for immobilization and electrode implantation allow to record physiological
Figure 3. ERG from the median eyes and a lateral eye and its parameters for several months. Thus it will be possible to adaptation in case of repeated stimulation. Lowest trace: Stimu- study the stability of sensory, motor and neuronal integra- lus. Duration of the white light flash: 100 ms. tion in the long-term range in orbit and on ground. This technique can also be applied to long-term studies of eco- logical adaptation to extreme environmental conditions such as high temperatures, low humidity, or drought. Supported by DLR: grant 50WB0323 to Horn.
REFERENCES Alpatov, A.M., Rietveld, W.J., and Oryntaeva, L.B. 1994. Impact of microgravity on free- running circadian rhythm of the desert beetle Trigonoscelis gigas. Biol. Rhythm Res. 25:168-177. Dinges, D.F. 2001. Sleep in Space Flight - Breath Easy Sleep Less? Am. J. Respir. Crit. Care Med., 164: 337-338. Ferraro, J.S., Fuller, C.A., and Sulzman, F.M. 1995. The biological clock of Neurospora in a microgravity envi- ronment. Aviat. Space Environ. Med., 66:1079.
Figure 4. Simultaneous recordings of SEA, EMG and ERG. Fuller, C.A., Hoban-Higgins, T.M., Klimovitsky, V.Y., Ordinate in relative measures. Lowest trace: Light flash. Griffin, D.W., and Alpatov, A.M. 1996. Primate circadian rhythms during spaceflight: results from Cosmos 2044 and 2229. J. Appl. Physiol., 81:188-193. Gundel, A., Polyakov, V.V., and Zulley, J. 1997. The alteration of human sleep and circadian rhythms during space flight. J. Sleep Res., 6:1-8. Miller, T.A. (ed.) 1979. Insect Neurophysiological Tech- niques. Springer-Verlag, New York Heidelberg Berlin, pp308. Samel, A., Wegmann, H.M., and Vejvoda, M. 1993. Re- Figure 5. ECG from an awake scorpion after filtering. sponse of the circadian system to 6 degrees head-down tilt bed rest. Aviat. Space Environ. Med., 64:50-54.
96 Gravitational and Space Biology 18(2) June 2005
SPACE SHUTTLE FLIGHT ENVIRONMENT INDUCES DEGENERATION IN THE RETINA OF RAT NEONATES
J. Tombran-Tink 1,2 and C.J. Barnstable 2 1Pharmaceutical Sciences, UMKC, Kansas City, MO, and 2Yale University School of Medicine, New Haven, CT
Retinal degenerations can be promoted by many factors including ischemia, oxidative stress, and increased OS intraocular pressure. Damage to retinal neurons can be inhibited by neurotrophic factors such as PEDF, BDNF, ONL and CNTF (Tombran-Tink and Barnstable, 2003). We present new evidence that the environment encountered in INL
space shuttle flight can also disrupt normal retinal IPL development and mimic stimuli that induce retinal degenerations on earth. Experimental evidence linking anomalies in visual perception with space flights has accumulated since the Apollo missions as have data OS suggesting pathological stimuli that disrupt retinal structure and function on earth are encountered in the ONL space shuttle environment (Draeger 2000; Nicogossian et al., 1993). Orbital space flights cause physiological disturbances in humans including cephalad fluid shift, increased intraocular pressure, disruption of cardio- vascular function and stress on the musculoskeletal OS system (Drummer 2000; Wang et al 1996; LeBlanc et al. 2000). ONL
Understanding the negative effects of space flight to the central nervous system (CNS) is of particular importance because, unlike most organs, the CNS has very little Figure 1. RET-P1 (anti-rhodopsin) labeling showing length of regenerative capacity. In this paper, we report the photoreceptor outer segments. R: reference retinal section. A. physiological effects of space travel on the retina of Synchronous, B. Flight, C. Vivarium female Sprague Dawley rodents in an NIH.R3 experiment at various stages of rats. Upper: PN5 launch, dissected PN14: Unload weight (Wt): postnatal development during orbital flight on Mission A-10.9 g. B-10.1 g; C-10.7 g. Middle. PN8 launch, dissected STS-72, launched in 1996. Three groups of Sprague PN17. Wt: A-15.6 g. B-16.5 g; C-16.2 g. Lower. PN15 launch, Dawley rats were analyzed in this experiment: Flight dissected PN24. Wt: A-29.7 g. B-29.2 g; C-30 g. (housed in a modified Animal Enclosure Module [AEM]), Synchronous (housed identically to Flight litters) and the ganglion cell layer. There was a loss in the number of Vivarium (standard colony housing). The rats were in ganglion cells in the retina of flight animals as well, and flight for 9 days and sacrificed immediately after the those remaining appeared unhealthy. shuttle returned to earth. Eyes from age and weight- matched female pups launched at post-natal days 5 (PN5), Other abnormalities such as the detachment of the neural 8 (PN8), and 15 (PN15) were dissected, fixed in 10 % retina from the retinal pigment epithelium (RPE), loss of formalin, and retinal sections labeled with an antibody RPE cells or disruption of the RPE monolayer, and against rhodopsin or Hematoxylin and eosin for increased neovascularization were also associated with morphological and morphometric analysis of the retina. animals exposed to orbital space flight conditions (Fig 3). The most striking difference among the space flight retinas and controls at all time points of rat neonatal Experimental studies have linked the perception of “light development was the loss of outer segments of the rod flashes” and hallucinations by astronauts in flight to the photoreceptor neurons (Fig 1). The outer segments penetration of ionizing nuclei through the nervous tissue. contain the visual pigment rhodopsin, which is essential This study has not allowed us to determine whether the to the visual transduction cascade for light detection. In effects we observed in the neonatal rodent retina are addition, there was disruption of normal development of transient, reversible, or dependent on flight duration. Nor the inner plexiform layer (IPL) as evidenced by the does it answer the question as to whether these changes decrease in thickness of the width of this structure as are triggered by a warp in gravitational force, solar compared to ground controls (Fig 2). The IPL contains the radiation, the impact of launching or reentry into the synaptic connections that mediate information transfer earth’s atmosphere, or fluctuations in oxygen level. It is from amacrine and bipolar neurons in the outer retina to also possible that only neonatal retinas and not well- developed adult retinas are affected by the hazards
Gravitational and Space Biology 18(2) June 2005 97 J. Tombran-Tink and C.J. Barnstable – Space Flight Induces Retinal Degeneration
examined showed similar morphology, samples used were from animals that developed well, and there is no evidence that Vitamin A deficiency due to malnutrition causes thinning of the IPL, or promotes loss of ganglion cells and neovascularization in the retina. These changes are more likely related to earth-based degenerations caused by increased intraocular pressure and ischemia. The impact on the neural retina and visual transduction cascades, however, highlights the importance of developing rigorous research to study how the eyes adapt or respond to various space related assaults since the duration of manned space journeys will significantly increase in the near future and could have irreversible adverse effects on human health and performance. Knowledge of how this unusual environment affects molecular mechanisms and pathways of the CNS are key to accelerating development of appropriate physiological risk mitigation measures to remove biological barriers that could impede the astronauts’ ability to survive and function in future long-term human space exploration. Such studies could also lead to information that is Figure 2. Thickness of retinal layers in the three groups of pups important to similar earth-based retinal disorders. examined. There is a general shortening of outer segments and We thank Alison French, Paul Callahan (Ames Res thinning of the inner plexiform layer in space flight animals. Center) and Louis Ostrach (NASA, Washington, DC) for IPL, inner plexiform layer; IS, rod inner segments; OS, rod making this study possible. Supported by NIH and RPB outer segments. Inc.
REFERENCES Draeger, J., Michelson, G., Rumberger, E. 2000. Continuous assessment of intraocular pressure - telematic transmission, even under flight- or space mission conditions. Eur J Med Res. 5:2-4.
Drummer, C., Gerzer, R., Baisch, F., Heer, M. 2000. Body fluid regulation in micro-gravity differs from that on Earth: an overview. Pflugers Arch. 441:R66-72.
LeBlanc, A., Lin, C., Shackelford, L., Sinitsyn, V., Evans, Figure 3. Hematoxylin and Eosin staining of retinas of PN5- H., Belichenko, O., Schenkman, B., Kozlovskaya, I., PN14 pups. A:Synchronous, B:flight, C: Vivarium Note the Oganov, V., Bakulin, A., Hedrick, T., Feeback, D. 2000. disrupted choroid (Ch) and choroidal vessels, lack of distinct RPE layer (RPE), and thinner IPL in the flight samples Muscle volume, MRI relaxation times (T2), and body composition after spaceflight. J Appl Physiol. 89:2158-
2164. encountered in the space travel environment. Some of the
NIH.R3 pups were compromised, probably due to Nicogossian, A.E., Huntoon, C.L. & Pool, S.L. 1994. disrupted maternal care of pups, including nursing. Only Space Physiology and Medicine. Lea & Fibiger, Phila. 10% of the pups launched at PN5 survived and the Ronca, A.E. 2003. Mammalian Development in Space, In survivors weighed significantly less than controls. In Adv. Space Biology & Medicine, Vol. 9, H.J. Marthy contrast, 90% of the pups launched at PN8 survived, but (Ed.), Developmental Biology Research in Space, their body weights averaged 25% less than controls Amsterdam: Elsevier, pp. 217-252. (described in Ronca, 2003). Other factors leading to compromised physiological status of the pups such as Tombran-Tink, J., and Barnstable, C.J. 2003. PEDF: a malnutrition and hypothermia, related to poor maternal multifaceted neurotrophic factor. Nature Rev Neurosci. care, may also contribute to retinal anomalies. 4:629-636. Developmental differences in growth and body size can affect the emergence of certain milestones, such as eye- Wang, M., Hassebrook, L., Evans, J., and Varghese, T. opening, and might also affect other aspects of sensory 1996. An Optimized Index of Human Cardiovascular development as well. However, this is unlikely to account Adaptation to Simulated Weightlessness. Trans. for our results since the retinas of all flight animal Biomedical Engineering, 43:502-511. 98 Gravitational and Space Biology 18(2) June 2005
ANTIGENIC PROTEIN IN MICROGRAVITY-GROWN HUMAN MIXED MÜLLERIAN OVARIAN TUMOR (LN1) CELLS PRESERVED IN RNA STABILIZING AGENT 1 2,3 4 4 4 5 D.K. Hammond , J. Becker , T.F. Elliott , K. Holubec , T.L. Baker , J.E. Love 1Enterprise Advisory Services, Inc., Houston, TX; 2National Space Biomedical Research Institute; 3Baylor College of Medicine, Houston, TX; 4Wyle Life Sciences, Houston, TX; 5Biological Systems Office, NASA, Johnson Space Center, Houston, TX.
Cellular biotechnology experimentation on the time with mouse antibodies to vimentin and epithelial International Space Station (ISS) sometimes requires that membrane antigens, and rabbit antibody to the collection of cells be preserved for long periods of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). time under adverse conditions. During Expedition 3, four Triplicate blots were made for each antibody. The graphs different cell lines were grown using the Cellular (Figures 1-3) show the average of the scanned pixel Biotechnology Operations Support System (CBOSS), and volumes for the stained protein band in each lane. A were then preserved in either formalin or RNAlater™ control using untreated cells was run on each blot and the (Ambion) at refrigerator temperatures. RNAlater™ is pixel volumes normalized to the control value. Due to the marketed to preserve RNA in the refrigerator for 30 days. normal variability of blots, the numbers were calculated Cells treated with RNAlater™ have previously been as a percent of the control. Blots are shown below the shown to contain antigenic proteins that can be visualized corresponding bar on each graph. using Western blot analysis. These proteins appear to be stable for several months when stored in this RNA 140 stabilizer at 4ºC. Antigenic protein can also be recovered 120 from cells that have been processed using an 100 RNAqueous® kit (Ambion) to remove RNA (1). Prior 80 work demonstrated that mixed Müllerian ovarian tumor cells (LN1) (2, 3) were capable of being grown in rotating 60 cell cultures that are analogs for microgravity (4). These 40 Percent of control cells grew on the ISS in the static bioreactor, Biological 20
Specimen Temperature Controller (BSTC), and produced 0 cytokines, although in reduced amounts compared with G3 G9 G14 F3 F9 F14 C the ground controls (5). Experiment Day
In this set of experiments, LN1 cells grown on the ISS during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were grown in 15 mL of media on Cytodex™ 3 beads (Amersham) in Teflon bags, Figure 1. Average of three blots using mouse anti-vimentin stored for three months in 9mL of RNAlater™ in the antibody (Oncogene) with 5µg protein/lane compared to control refrigerator, and RNA was extracted using an (C) untreated cells (100%). Samples corrected for protein RNAqueous® kit. The RNA filtrate containing the protein loading using SYPRO Orange stained gel. was precipitated with a final volume of 5% trichloroacetic acid (TCA), washed in TCA, and suspended in buffer 140 containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein as determined 120
by the Bicinchoninic acid (BCA) method (6) were loaded 100 onto SDS-polyacrylamide gels (7). After electrophoresis, 80 gels stained with SYPRO Orange (Molecular Probes) were scanned for equal loading of protein. Further, gels 60
were transferred by Western blot procedures (8, 9) to 40 Percent of Control polyvinylidene fluoride membranes. Primary antibodies 20 were as noted below. A horse-radish peroxidase secondary antibody was used and the Western blots were 0 ® G3 G9 G14 F3 F9 F14 C stained with an enhanced chemiluminescent ECL Plus Experiment Day detection kit (Amersham) and scanned using a Storm™
840 gel image analyzer (Amersham). ImageQuant™ TL software (Amersham) was used to quantify the densities of the protein bands. Figure 2. Average of three blots using mouse antibody to The ground cell samples from day 3 (G3), day 9 (G9) and epithelial membrane antigen, EMA (DAKO) with 5µg day 14 (G14) and flight cell samples from the same days protein/lane. The highest molecular weight bands are the EMA (F3, F9 and F14) showed similar staining patterns over according to the manufacturer’s instructions.
Gravitational and Space Biology 18(2) June 2005 99 D. Hammond et al. – Antigenic Protein in LN1 Cells Grown in Space
These data demonstrate the presence of antigenic protein 120 in the RNA-stabilized LN1 cells, even after long periods 100 of time in refrigerator storage. The antigenic protein is
80 recoverable even after the RNA has been removed from the cells by the filtration method. All three proteins 60 examined here had similar profiles at different times in
40 the flight and ground samples. Previously, proteins from
Control of Percent human renal cortical epithelial (HRCE) cells had been 20 shown to exhibit similar characteristics (1). This work 0 further demonstrates that the technique can be generalized G3 G9 G14 F3 F9 F14 C to other cell lines and might be a good way to preserve Experiment Day proteins for long term storage. Since this preservative is even more effective in protecting RNA when stored frozen, it seems likely that protein protection would be similarly increased in freezing conditions. Further work Figure 3. Average of three blots using rabbit anti-GAPDH is needed to determine if freezing also protects the antibody (Abcam) with 4µg protein/lane. proteins for longer periods of time. In addition, a more comprehensive study should be undertaken to determine if this holds true over a large range of protein varieties 350 and sizes. Vimentin 300 EMA 250 (Grant support to J. Becker: NAG9-1341)
200 REFERENCES 150
100 Becker, JL, Finan, M, Widen RH (1992) Oncogene Percent of Control expression by a Müllerian tumor cell line. In Vitro Cell 50 Dev Biol 28II:149A 0 G3 G9 G14 F3 F9 F14 Becker, J, Papenhausen, PR and Widen, RH. (1997) Experiment Day Cytogenetic, morphologic and oncogene analysis of a cell line derived from a heterologous mixed Mullerian tumor of the ovary. In Vitro Cell Dev Biol, 33:325-331 Figure 4. Vimentin and EMA Westerns corrected for the GAPDH levels, since GAPDH is frequently used as a baseline Goodwin, TJ, Prewett,TL, Spaulding, GF, and Becker, JL for RNA and protein production. (1997) Three-dimensional culture of a mixed Mullerian tumor of the ovary: expression of in vivo characteristics. Although the BCA protein load was the same for all In Vitro Cell Dev Biol, 33:366-374. lanes, the ground samples and control appeared to have more protein in some lanes than the flight samples. When Hammond, DK, Becker, JL, Elliott, TF, Holubec, K, a scan was done of the SYPRO Orange-stained lanes, Baker, TL, and Love, JE (2004) Cell growth and cytokine considerably less band volume was present in the flight production of human mixed Műllerian tumor (LN1) cells samples. This could be due to breakdown of the proteins, grown on the International Space Station (ISS). Society which allowed the small peptides to run at the dye front for In Vitro Biology, San Francisco, CA and not be counted. Some loss of protein did occur for most of the samples. If one assumes that the general Love, JE, Hammond, DK, Elliott, TF, Holubec, K, Baker, breakdown would be equivalent for all samples, then the TL, Allen, PL and Hammond, TG (2003) Antigenic graph (Fig. 4), which demonstrates the amount of protein protein from RNALater™ treated cell cultures grown on per GAPDH protein would be one way of viewing the the International Space Station (ISS), using the Cellular data. In Figure 4, the antibodies for vimentin and EMA Biotechnology Operations Support System (CBOSS). show good agreement with each other when expressed in ASGSB, Huntsville, AL this way. They also show a good correlation of protein expressed per GAPDH for both flight and ground samples Bicinchoninic acid (BC) protein assay kit procedure over all times. #TPRO-562, Sigma7.Laemmli, UK
100 Gravitational and Space Biology 18(2) June 2005
COUNTERMEASURE FOR SPACE FLIGHT EFFECTS ON IMMUNE SYSTEM: NUTRITIONAL NUCLEOTIDES A. D. Kulkarni1, K. Yamauchi1, A. Sundaresan1,2, G.T. Ramesh3, N. R. Pellis4 1Department of Surgery, Medical School and GSBS, Univ. of Texas Health Science Center, 2USRA/NASA, 3Texas Southern University, 4JSC/NASA, Houston
Microgravity and its environment have adverse effects on 300 Control RNA Uracil the immune system. Abnormal immune responses 250 observed in microgravity may pose serious consequences, 200 especially for the recent directions of NASA for long- 150 term space missions to Moon, Mars and deep Space 100 response exploration. The study of space flight immunology is
% Increase in PLN 50 limited due to relative inaccessibility, difficulty of 0 performing experiments in space, and inadequate Gro up HU provisions in this area in the United States and Russian Housing space programs (Taylor 1993). Microgravity and stress Figure 1-A. Effect of dietary nucleotides and housing on in vivo experienced during space flights results in immune popliteal lymph node (PLN) proliferative response in mice. system aberration (Taylor 1993). In ground-based mouse RNA : RNA supplemented Control diet (0.25%) Uracil : uracil supplemented Diet diet (0.06%) models for some of the microgravity effects on the human body, hindlimb unloading (HU) has been reported to 1200 Control RNA Uracil cause abnormal cell proliferation and cytokine production 1000 (Armstrong et al., 1993, Chapes et al. 1993). In this report, we document that a nutritional nucleotide 800 supplementation as studied in ground-based microgravity 600 analogs, has potential to serve as a countermeasure for the response 400 immune dysfunction observed in space travel. % Increase in PHA 200 0 We employed two ground-based analogs, HU as an in Group HU vivo model and a clinostat bioreactor (BIO) as an in vitro Housing cell culture model. We examined the effects of supplemental nutritional nucleotides on immune function Figure 1-B. Effect of dietary nucleotides and housing on ex vivo mitogen (PHA) proliferative response in mouse splenocytes. in these analogs. BALB/c female mice (8-10 week old) RNA : RNA supplemented Control diet (0.25%) were divided into the following two groups: control non- Uracil : uracil supplemented Diet diet (0.06%) HU, and HU for 7 days. Mice were fed either control chow or chow supplemented with RNA or Uracil 400 Control RNA Uracil (referred as NT). Immune function was assessed by in 300 vivo popliteal lymph node proliferation (PLN), and in vitro phytohemagglutinin (PHA)-stimulated proliferation 200 of splenocytes and their cytokine production, (Kulkarni et 100 al, 2002). PLN response was calculated as weight gain in allogeneic challenged lymph node versus weight gain in 0 syngeneic challenged contralateral lymph node. Gro up HU Gro up HU % Increase in Cytokine levels Splenocytes were cultured in vitro in BIO with/without IL-2 INF-g PHA and nucleoside/nucleotide mixture (referred as
NS/NT). Cell proliferation was assessed by tritiated Figure 1-C. Effect of dietary nucleotides and housing on in vitro thymidine uptake assay by thymidine uptake in stimulated cytokine (IL-2 and INF-♦) production by splenocytes cultures versus unstimulated cultures. Cytokines released RNA : RNA supplemented Control diet (0.25%) Uracil : uracil supplemented Diet diet (0.06%) by cultured and stimulated cells were quantified by
ELIZA method (Cayman Co.). PLN response was 400 significantly suppressed in HU mice (P<0.05) and was Control NS/NT restored by NT supplemented diets (Figure 1). 300 Splenocytes from HU mice had decreased PHA stimulated proliferation and decreased IL-2 and IFN-γ 200 cytokine levels (P<0.05) as well (Figure 1-A,B,C). These 100 responses were restored by NT diets. In BIO cultures, % Increase in mitogen respo mitogen in Increase % PHA response was suppressed significantly, and NS/NT 0 restored the proliferation response (Figure 2). Results in control Flask BIO these figures are expressed as percent changes in groups. Figure 2. Effect of supplemental nucleotides on mitogen (PHA) In both models, we documented that dietary and stimulated proliferative response in mouse splenocytes. supplemental nucleotides (NT or NS/NT) restored several Control flask and BIO (Bioreactor) NS/NT : Nucleotide and nucleoside mixture supplemented medium (0Gravitational 012 M) and Space Biology 18(2) June 2005 101 A. D. Kulkarni et al. : Countermeasure For Space Flight Effects On Immune System: Nutritional Nucleotides
T-cell growth / REFERENCES: Infect ion f unction Wound Cyt okine IL-2 healing Release IL-3 IFN-g Armstrong JW, Nelson KA, Simske SJ et al., Skeletal unloading causes organ-specific changes in immune cell In vivo In vit ro Melanoma DTH funct ion NT Diet f unction NT Diet responses. J Appl Physiol. 75(6):2734-9, 1993.
Stem cell/ MLC/ T-cell Cell Dendrit ic Bowers RH, Cerra FB, Berdashadsky B, et al. Early Cell Growt h locomotion cells R Surf ace enteral administration of a formula (Impact ) markers Cort ico steroids supplemented with arginine, nucleotides, and fish oil in intensive care unit patients: Results of a multicenter, In 1 G In µG propsectiuve, randomized, clinical trail. Critical Medicine, 22(2):436-449, 1995. Figure 3: Left panel shows the summarized observations from 1 G study and right panel shows observations from ground-based microgravity analogs. Arrows indicate up or down modulation of Carver JD and Walker WA. The role of nucleotides in specific evaluations performed both in in vivo and in vitro models. Human nutrition. Nutr. Biochem, 6:58-72, 1995.
immunological functions in mice including cytokine Chapes SK, Mastro AM, Sonnenfeld G, et al. production, and decreased corticosteroid levels. Antiorthostatic suspension as a model for the effects of Many of the parameters of cellular functions are shown in space flight on the immune system. J. Leuk. Biology, 54 Figure 3. Similar effects are seen by NT supplementation (3):227-235. in 1 G (Kulkarni et al., 1990) and in µG analogs. These observations clearly document the advantages and Felix K, Wise K, Manna S, et al. Altered cytokine benefits of exogenous source of nucleotides in the form of expression in tissues of mice subjected to simulated nutritional NT supplementation. Observed decrease in microgravity. Molecular and Cellular Biochemistry, corticosteroid levels in HU mice by NT supplementation 266:79–85, 2004. may be one of the potential mechanisms for immune restoration observed in these mice. Hales NW, Yamauchi K, Martinez AA, et al. A countermeasure to ameliorate immune dysfunction in in Understanding of the mechanisms of immune restoration vitro simulated microgravity environment: Role of in microgravity analogs, including CNS mediated effects cellular nucleotide nutrition. In Vitro Cell Dev. Biol and their implications, is important in addressing the Animal, 38:213-217, 2002. NASA’s CRP and CRL maps. Nucleotide metabolism is very important for cell cycle. Perturbations in nucleotide Kulkarni AD, Rudolph FB, Van Buren CT. The role of metabolism that lead to a variety of diseases are primarily dietary sources of nucleotides in immune function: A in the de novo pathways. Supplemented nucleotides review. J. of Nutrition 124:1442s-1446s, 1994. participate and contribute primarily in the salvage pathway and they are utilized by rapidly proliferating Kulkarni AD, Yamauchi K, Hales NW, et al. Nutrition target cells (Kulkarni et al 1990). As compared with the beyond nutrition: plausibility of immunotrophic nutrition amounts of available nucleotides from the de novo for space travel. Clinical Nutrition, 21:231-238, 2002. synthesis and the endogenous salvage pathway, the levels of supplemental nucleotides in the experimental (Kulkarni Taylor GR, Overiview of spaceflight immunology. J. et al., 1990, 2002) and clinical studies are much smaller Leuk. Biology 54(3):179-188, 1993. (Bower et al. 1995, and Carver and Walker, 1995) and without any adverse or untoward effects. The amount of Yamauchi K, Sundaresan A., Hales NW, et al. Nutrition NT added was minimal in all the groups; however, the countermeasure to obviate immune dysfunction in beneficial effect was seen only in the microgravity microgravity. Proceeding of the ISTS, 2000-p-05p, 2002. analogs of HU or BIO and not in any control groups where there was no demand or stress involved. Thus, in Yamauchi K, Hales N.W, Robinson SM, et al. Nutrition conclusion our studies presented in this report document requirement of nucleotide for cellular immunity in the critical need of NT supplementation in physiological simulated microgravity. J. Appl. Physiol., 93:161-166, stress situations; such as space travel, where nutritional 2002. supplemental nucleotides have up-regulating and immunoprotective effects with potential as a countermeasure to the observed immune dysfunction in true microgravity.
Key words: nucleotide; nutrition; immunity; space travel
102 Gravitational and Space Biology 18(2) June 2005
CELL SURVIVAL AND PRESERVATION OF SIRNA-MEDIATED PROTEIN KNOCK-DOWN UPON SERUM-FREE CRYOPRESERVATION (-80°C) Charles A. Lambert, Christophe Deroanne, Sandrine Servotte, Pierre Mineur, Charles M. Lapière, Betty Nusgens Laboratory of Connective Tissues Biology. Tour de Pathologie B23/3, University of Liège, 4000 – Liège, Belgium
The small G proteins of the Rho family (Rho GTPases) (FCS)/ dimethyl sulfoxide (DMSO) (95:5)) was taken as are key operators in the signaling arising from integrins 100%. and cell-matrix focal adhesions. They function as binary In preliminary experiments, wild-type WI-26 cells molecular switches that cycle between an inactive GDP- were frozen in DMEM alone or supplemented with bound and an active GTP-bound form that in turn dextran, hydroxyethyl starch (HES), trehalose or DMSO activates downstream effectors and a variety of signaling as cryoprotective agents. After 11 days of storage at – pathways. The best known Rho GTPases, RhoA, Rac1 80°C in DMEM alone about 25% of cells attached to the and Cdc42 control the organization of the cytoskeleton support. The addition of dextran (2 and 8%) and HES (2.5 and the focal adhesions-mediated transduction of and 5 %) resulted in a lower survival than DMEM alone, exogenous and endogenous mechanical signals. Their while trehalose (200 mM and 1 M) did not affect the localization at the cross-road between signaling, cryoprotective effect of DMEM. As expected DMSO mechanical forces and the cytoskeleton along with added some cryoprotective effect when present at 5%, and reported effects of microgravity on cell shape and to a lesser extent at 10% (not illustrated). cytoskeletal arrangement led us to hypothesize that Rho In a second set of experiments, the cryoprotective GTPases might be involved in the reception and reaction potency of four mediums were tested on WI-26 (wild- of cells to gravity. To explore this hypothesis we created type, expressing Rho GTPase QL or transfected with stable cell lines, SV40-transformed fibroblasts (WI-26), siRNA). The selected mediums were: DMEM/DMSO expressing a constitutively active form (QL) of RhoA, (95/5, = Medium B), Medium B containing 5% BSA Rac1 or Cdc42 (manuscript in preparation). The reverse (w:v, = Medium C), DMEM/ProFreeze situation, i.e. suppression of the GTPases expression was (Cambrex)/DMSO (50/45/5, = Medium D) and Medium obtained by transfecting small interfering RNA (siRNA) D containing 5% BSA (w:v, = Medium E). After 50 days targeting each of these GTPases (Deroanne et al., 2003, at –80°C, cells were thawed and diluted (1/20) in DMEM 2005). containing 1% BSA before plating. Similar data Experiments using these cells have been selected for (illustrated for WI-26 expressing Rac1 QL in Fig. 1) were the 2nd batch of experiments to be conducted in the Biolab obtained with the wild-type WI-26 and the various facility on-board of the International Space Station. The derived cells. They indicate that the rank of deep impact of seric factors on the activity of Rho cryopreservative potency is Medium A = Medium C = GTPases together with practical constraints related to the Medium E > Medium D > Medium B. storage of frozen cells, their thawing and dilution on the ISS imposed a cryopreservation of the cells in serum-free conditions at -80°C. Here we describe the cryopreservative potential of various serum-free mediums that allow the persistence of the knock-down of the 100 GTPases by siRNA after freeze-thawing of the cells. WI-26 cells were transfected by siRNA targeting
RhoA, Rac1 and Cdc42 using calcium phosphate 50 precipitate. Naïve cells, cells treated with calcium phosphate alone or a with a scramble siRNA were used as control. After 16 hours the precipitate was washed-out and the cells kept for 48 hours in culture before 0 cryopreservation procedure. Cells were detached from A B C D E culture dish with trypsin, pelleted by centrifugation and suspended at 106 cells/ml in Dulbecco’s modified Eagle MEDIUM Medium (DMEM). Aliquots of cell suspension were pelleted, suspended in the indicated cryopreservative Fig. 1: Cryopreservation of WI-26 cells in various mediums. WI- mediums, left at -20°C for 40 minutes and stored at - 26 cells expressing constitutively active Rac1 were frozen at – 80°C. After the indicated periods of time, cells were 80°C in various cryopreservative mediums. After 50 days they thawed, diluted (1/20) in serum-free DMEM were thawed, plated onto collagen in serum-free DMEM, 1% complemented or not by 1% bovine serum albumin BSA for 4 hours and the relative number of attached cells was (BSA), plated on collagen-coated wells and transferred at measured. A: FCS/DMSO (95:5, = control medium); B: 37°C. After 4 hours the wells were washed twice with DMEM/DMSO (95:5); C: DMEM/DMSO (95:5) + BSA (5%, phosphate buffered saline and the number of attached w:v); D: DMEM/ProFreeze/DMSO (50:45:5); E: cells was measured by the content of DNA assayed by DMEM/ProFreeze/ DMSO (50:45:5) + BSA (5%, w:v). fluorimetry. The attachment of cells frozen in control cryopreservative medium (Medium A: fetal calf serum
Gravitational and Space Biology 18(2) June 2005 103 C.A. Lambert – Cell Survival and Preservation of Sirna-Mediated Protein The cryoprotection offered by Medium C and E was 1 2 3 4 5 6 further confronted to that of control medium for a variety of cell types (see Fig. 2). Data show that Medium E offered cryoprotection similar to or slightly better than Rac1 control medium for the various tested cells except NIH3T3. Medium C was significantly less efficient. Cdc42
ERK1,2
5 MO-1 JO-1 MG-63 1000
5 Fig. 3: Knock-down of Rho GTPases expression by siRNA is effective and retained after storage at –80°C. Cells were left 50 0 untreated (1), mock-transfected (2) or transfected with siRNA against RhoA (3), Rac1 (4) or Cdc42 (5) or a scramble siRNA 5 (6). After two days the cells were frozen in medium C for 50 days, thawed, plated and harvested after 24 hours. Total cell
0 0 extracts were run on SDS-polyacrylamide gels and blotted to A2058 ROS NIH3T3 membranes probed with anti-Rac1, anti-Cdc42 or anti-ERK1,2 100 antibodies.
Supported by Prodex PEA 90095 and PEA 90099 50 (ESA/NASA ILSRA-2001-074 & ESA-RA-LS-01- PREP/GB003)
0 REFERENCES C E C E C E MEDIUM Deroanne C.F., Vouret-Craviari V, Wang B, and Pouyssegur J. 2003 EphrinA1 Inactivates Integrin- mediated Vascular Smooth Muscle Cell Spreading via the Fig. 2. Survival of several cell types after cryopreservation at – Rac/PAK pathway. J Cell Sci. 116:1367-76. 80°C for 50 days in Medium C and E. Data are expressed as the percentage (mean + S.D) of attached cells as compared to control medium taken as 100%. MO-1, JO-1: human primary Deroanne C.F., Hamelryckx D., Ho T.T.G., Lambert fibroblasts; MG-63: human osteosarcoma cells; A2058: human Ch.A., Catroux P., Lapière Ch.M. and Nusgens B.V. 2005 melanoma cells; ROS; rat osteosarcoma cells; NIH3T3: mouse Cdc42 Downregulates MMP-1 Expression by Inhibiting transformed fibroblasts. the ERK1,2 Pathway. J.Cell Sci., in press.
Finally, the impact of freeze-thawing on the knock-down of the GTPases expression by siRNA was tested. WI-26 cells were transfected by siRNA as previously described (Deroanne et al., 2005) before freezing. After 50 days of storage, cells were thawed, harvested in DMEM containing 1% BSA, plated for 24 hours and then lyzed in Laemmli buffer. The expression of the GTPases was measured by Western blotting using antibodies specific for the three GTPases or against ERK1/2 as a control of protein loading. Transfection with the cognate siRNA reduced the expression of RhoA and Cdc42 by about 90 % and that of Rac1 by about 70 % , i.e. to levels identical to those observed in the cells before freezing, regardless of the cryopreservative medium used (illustrated for Rac1 and Cdc42 in cells frozen in medium C, Fig. 3). In conclusion these data demonstrate the feasibility to store cells frozen in serum-free cryopreservative medium at –80°C on Earth and to plate them after dilution with serum-free medium on ISS. Moreover, they show that siRNA-directed knock-down of proteins is retained in these conditions.
104 Gravitational and Space Biology 18(2) June 2005
TESTING OF SACCHAROMYCES CEREVISIAE MORPHOLOGICAL FIXATIVES AND FIXED SAMPLES STORED AT AMBIENT TEMPERATURE Diana Ly1, Diane Yu2, Beverly Girten3 and Jacob Cohen4 1Education Associates Program (University of San Francisco), 2Lockheed Martin Space Operations, 3NASA Ames Research Center, 4Science and Technology Corporation, Moffett Field, CA EMMYS-1, Effects of Microgravity on Model Yeast Specimen, is a yeast payload designed to investigate 2.50 samples grown on-orbit for morphological and molecular FPA A FPA B 2.00 analysis. Saccharomyces cerevisiae (yeast) is considered FPA Control a model eukaryotic microorganism for space studies in part due to its small size, fully sequenced genome, 1.50 availability of mutants covering most of the open reading =600 nm) λ frames and ability to be stored dry. Yeast is commonly 1.00 used to study radiation effects, protein-protein O.D. ( interactions and cell regulation pathways. 0.50 Due to the constraints set by current payload scenarios, EMMYS–1 is being optimized for storage and 0.00 operation at ambient temperature for a period of up to six 0 102030405060 months. Current testing includes determining the timeline Hours of the experiment (activation and termination at either the beginning or ending of the flight scenario) and fixative- Figure 1. Results of the FPA chamber containment test. FPA A hardware compatibility with the goal of obtaining samples (8 months) and FPA B (9 months) represent growth curves of useful to the scientific community. To recommend the yeast in extracted YPD stored in FPAs that contained fixative in the fixative chamber. FPA Control represents growth curve of most appropriate flight scenario for optimal science return yeast in extracted YPD stored in FPAs that did not contain this study investigates hardware compatibility, fixative fixative in the fixative chamber. There is no significant stability and staining capability. difference between yeast grown in YPD stored with fixative in S. cerevisiae, strain BY4743, were grown in Yeast- the FPA and the YPD stored alone in the FPA. Peptone-Dextrose (YPD) at 30ºC to an O.D. ( =600 nm) To analyze the fixative effectiveness over a period of of 1.5 to 1.7 for all experiments. Fixatives tested were time, fixative ability to kill yeast within one minute formaldehyde, paraformaldehyde, glutaraldehyde and exposure was tested (time determined empirically). acetic acid based fixatives at various concentrations in Fixatives were freshly prepared and stored at either 4ºC or either Phosphate Buffered Saline (PBS) or Cacodylic 25ºC. Aliquots were taken on a periodical basis, mixed Acid (CA). The hardware being optimized for the with yeast in a 1 to 2 ratio and washed with YPD. EMMYS-1 flight experiment is BioServe Space Samples were allowed to recover for 30 minutes then Technologies’ Group Activation Pack (GAP), which plated onto YPD agar plates. Plates were incubated for allows for simultaneous activation of 8 Fluid Processing two days and subsequently counted for colony forming Apparatus’ (FPA), while maintaining three levels of units. 4% fixatives stored for one month at 25ºC were not containment. able to completely kill the yeast within the one minute To determine materials compatibility, septa from the (data not shown). FPAs were submerged in 5 ml of fixative and stored in The samples stored up to one month, used for the the dark at ambient temperature (19 to 23ºC). fixative stability testing, were also used for staining Observations were made on the appearance of the septa at capability. In addition, fixed samples stored at ambient regular time intervals. All fixatives, except temperature for up to 8 months were also tested. DAPI, glutaraldehyde and fixatives with acetic acid, showed no Calcofluor, Mithramycin and Acridine Orange staining negative interactions with the septum after approximately methods were performed based on protocols assembled 8 months of storage in the dark at ambient temperature by Hasek and Streiblova (1996). In this report, only (data not shown). Septa stored in glutaraldehyde and DAPI results are presented; these results reflect those acetic acid based fixatives developed ridges on the obtained for the other nuclear stains (Mithramycin and surface. Acridine Orange). Percentage stained was determined by To test chemical containment ability of the septa in the number of properly stained yeast divided by the total the FPA, YPD was stored in one chamber with 12% number of yeast multiplied by 100. formaldehyde in 30% acetic acid stored in the adjacent Yeast fixed then stored at ambient temperature for chamber at ambient temperature for up to nine months. one month before staining showed a dramatic decrease in The YPD was then extracted and a S. cerevisiae growth the percent of DAPI stained yeast when compared with curve was obtained. There was no difference in growth those stained 5, 7 and 10 days after fixation (Figure 2). curves between yeast grown at 22ºC in YPD stored in No nuclear staining was observed in yeast fixed and FPAs with fixative when compared to those grown in stored at ambient for one month in 12% formaldehyde in YPD stored in FPAs without fixative (Figure 1). 0.6M CA. Gravitational and Space Biology 18(2) June 2005 105
D. Ly - Testing of Saccharomyces cerevisiae
120% 120% Control Day 5 8 months Day 7 100% 100% Day 10 1 month 80% 80% 60% 60% 40% 40%
Percent Yeast Stained Stained Yeast Percent 20%
Percent Yeast Stained Stained Yeast Percent 20% 0% 12% Formaldehyde/0.6M CA 12% Formaldehyde/PBS 0% 12% Formaldehyde/0.6M CA 12% Formaldehyde/PBS Figure 4. Percent of yeast stained with DAPI seven days post Figure 2. Percent of yeast stained with DAPI after being fixed fixation. Fixatives used were two week old fixatives stored at and stored at ambient for 5 days, 7 days, 10 days and 1 month. 4ºC (control) and those stored for eight months at ambient There was a dramatic decrease of approximately 70 percent in temperature. Yeast cells were fixed and stored at ambient for 7 yeast stored fixed for one month then stained. Data on yeast days prior to staining with DAPI. There is no significant fixed for one month in 12% formaldehyde in 0.6M CA showed difference between the control and the eight month old fixatives. no stained cells. Higher percentage fixatives stored at 25ºC for one In summary, the hardware compatibility tests month showed significant (p<0.001) increase in yeast determined that either formaldehyde or paraformaldehyde cells stained properly with DAPI compared to 4% based fixatives (in PBS or CA) can be used for up to a 6 fixatives (Figure 3). In general, yeast fixed with one month ambient temperature flight scenario. Furthermore, month old formaldehyde based fixatives had a higher the FPA can be stored in its inactivated form for the percentage of stained yeast compared to one month old expected flight scenario since the growth curves analysis paraformaldehyde based fixatives. of the YPD media stored in FPA containing fixative in the fixative chamber suggest that the septum provides an 120% adequate level of containment between the two chambers. Viability tests suggest that higher concentration 100% fixatives are more stable than lower concentrations. Yeast fixed in higher percentage fixative stained better 80% than at lower percentages. Although both the 6 and 12% formaldehyde and paraformaldehyde fixatives performed 60% * well after one month at 25ºC, it was observed that formaldehyde based fixatives have an overall higher 40% percent of stained yeast. The results from the staining and Percent Yeast Stained Stained Yeast Percent viability analysis of 12% formaldehyde based fixatives 20% * stored at ambient temperature for over eight months demonstrate that the fixative will be stable for the
0% expected six month ambient flight scenario and still allow for proper staining to occur. Since yeast fixed and stored CA CA CA A A A M M M M C M C M C /0.6 /0.6 /0.6 /0.6 /0.6 /0.6 at ambient show a decrease in the number of stained yeast yde yde yde yde yde yde ldeh ldeh ldeh deh deh deh rma rma rma mal mal mal Fo Fo Fo afor afor afor over time, the flight scenario where incubation and 12% 6% 4% Par Par Par 12% 6% 4% fixation occurs at the beginning is not recommended. Figure 3. Percent yeast stained with DAPI seven days post These results demonstrate that the optimal flight fixation with fixatives stored one month at 25ºC. There was a scenario for EMMYS-1 will consist of 1) incubation and significant difference between the 4% formaldehyde and 4% fixation just prior to return of payload and 2) 12% paraformaldehyde with the higher percentage fixatives. formaldehyde based fixative for morphological analysis. *symbol represents significant difference (p<0.001) compared However, our results could not rule out either 6% to other columns. formaldehyde or 6 and 12% paraformaldehyde for Yeast fixed with 12% formaldehyde based fixatives fixative purposes. Further studies will include testing of stored at ambient temperature over eight months showed fixatives for antibody staining, Green Fluorescence no significant difference compared to fixative stored at Protein staining and Electron Microscopy analysis. 4ºC for two weeks when stained with DAPI (Figure 4). In addition, viability tests using the fixatives stored at REFERENCE ambient temperature for eight months showed no viable Hasek J and Streiblova E. (1996). Fluorescence yeast after one minute exposure (data not shown). microscopy methods. Yeast protocols; methods in cell and molecular biology, Volume 53. Humana Press,
106 Gravitational and Space Biology 18(2) June 2005
POSSIBLE INVOLVEMENT OF FLOW DETECTION IN THE ACTIVATON OF OSTEOBLASTS 1 2 3 2 2 Muneo Takaoki , Hyekeyong Park , Naoko Murakami , Dai Shiba , and Jun-Ichiro Gyotoku 1 2 Space Environment Utilization Center, OSFO, JAXA; ISS Science Project Office, ISAS, JAXA, 3Advanced Engineering Services Co. Ltd.; Tsukuba 305-8505, Japan
One of the most potent mechanical stimulators for bone gation (Takaoki et al., 2004). Moreover, cooling of the cells may be the interstitial fluid flow. Fluid flow induces culture fluid, due to handling at room temperature, various events such as Ca2+ signaling, c-fos expression, induced similar response. To distinguish the effect of actin fiber reorganization, and appearances of bone rocking from the influence of cooling, we successfully markers in cultured cells (Chen et al., 2000). Fluid shear utilized small Styrofoam boxes and heat accumulators. stress may distort cellular membrane and other structures, With this simple system, it was shown that either fluid inducing cellular response. However, the nature of flow or cooling independently induced the responses molecules that convert the mechanical strain into biologi- (Figure 1). Although flow-cells are normally used for the cal signals remains unclear. purpose (Stevens and Frangos, 2003), this rocking The difficulty in identifying the mechanosensor mole- method was suitable for the parallel analyses of responses cules seems to lie, at least in part, in the lack of reliable in multiple flasks. methods for analysis. In contrast to regular cell culture experiments, that compare the effects of dilute sub- 3
stances, cellular responses to mechanical stimulations are n 2. 5
o
i s
often difficult to interpret. The mechanical stimulation s e
r 2
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may alter culture conditions drastically. It is extremely E
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o
f difficult to apply only mechanical stimulations, leaving
-
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e 1
all other conditions uniform. Excluding the effects of v
i
t a such environmental changes, induced secondarily by the l e 0. 5 operational procedure, is almost impossible. Thus, this R 0 Still 10 min 60 min 10 min 70 min study seeks to develop a simple method for applying at 37℃ after 1st after 1st after 2nd after 1st Rocking Rocking Rocking Rocking mechanical stimulation with minimum secondary at 60min influences, especially from temperature changes during Figure 2. C-fos expression response to the second stimulation. laboratory procedures. Culture flasks were rocked and then kept undisturbed for one hour before the second stimulation. Values are means and A B 6 3 standard errors for 3 flasks.
n 5 2. 5
o
i s
s The response reached a peak 10 to 15 min. after the e
r 4 2 p
x stimulation and settled to the background level within an
E
s 3 1. 5
o hour. The second stimulation given 60 min. later resulted
f
-
c
e 2 1 in a comparable response (Figure 2). In this study, we
v
i t
a kept all cultures inside the incubator without any distur- l
e 1 0. 5 R bance for at least 2 hours before rocking. 0 0 37℃ 37℃ 37℃ 4℃(1min) There was some possible mediation of the c-fos Still Rocked Still →37℃ St ill 2+ expression response by Ca and the ERK signaling Figure 1. C-fos expression induced either by rocking (A) or pathway. The addition of an ERK inhibitor, U0126 cooling (B) of the culture flask. MC3T3-E1 cells were pre- suppressed the response (Figure 3). cultured for 3 days at 37°C in T-25 flasks. Culture flasks were Fluid flow can be more efficiently detected when seen placed in small Styrofoam boxes with heat accumulators (small, as a drag force against a projected structure than as a refreezable pack equilibrated to 37°C). This minimized the shear stress to a smooth surface. One such projection, cooling during incubator door opening and rocking. Gently primary cilium, served as a flow meter in kidney epithe- replacing heat accumulators with cold (4°C) packs for 1 min. achieved cooling with the least possible fluid agitation (B). After lial cells. A transient receptor potential (TRP) channel replacing with warm packs again, all flasks were incubated at family protein, polycystin-2 (PC2) with polycystin-1, is 37°C for additional 10 min. Total RNA was extracted and c-fos one of the key molecules for this function. PC2 localized gene expression was evaluated with RT-PCR, and the values at the primary cilia and functioned as mechanosensitive were normalized to 18S rRNA as described previously (Takaoki Ca2+ ion channel. Ca2+ signalling initiated from primary et al., 2004). Values are means and standard errors for 3 flasks. cilium distorted by the drag of flowing fluid are sent to neighbouring cells through gap junctions (Nauli et al., We observed that conventional laboratory handling of 2003). Whitfield (2003) discussed the possible involve- culture flasks could stimulate the murine osteoblastic cell ment of similar mechanisms in bone cells. Interstitial fluid line MC3T3-E1. Fluid flow, generated either by manual flow could be caused by the slightest distortion of bone rocking or by unplanned agitation of culture flasks, tissue by a mechanical load. This flow could be easily possibly induced c-fos expression to a far greater extent detected by sensitive flow detecting primary cilia. than that caused by intended stimulation such as centrifu-
Gravitational and Space Biology 18(2) June 2005 107 M. Takaoki et al. –Flow detection by Osteoblasts Primary cilia were found in the osteoblastic cell line temperature sensing by PC2, or contributions of other MC3T3-E1. The cells also expressed PC2. These obser- bone-specific TRP channel superfamily proteins. vations suggested that bone cells and kidney cells The transient c-fos expression response could be in- detected fluid flow through similar mechanisms. How- duced by a variety of stimulations. These stimulations ever, the localization of PC2 to the primary cilia was not could be applied, either intentionally or unintentionally, as preferential as had been reported in kidney cells to the cell culture during laboratory procedures. How- (Figure 4). This weak localization of PC2 in the primary ever, the effects of these stimulations might be considered cilia and the difference in reported flow sensitivities trivial in regular experiments, where both reference (Chen et al., 2000, Nauli et al., 2003) suggested the controls and experimental groups undergo virtually involvement of molecules other than those in kidney identical environmental changes, and are treated side by cells. side in a similar manner. On the other hand, cultures must usually be placed in separate locations when applying 3 different mechanical stimulations. Therefore, it is
n 2. 5 extremely important to keep the environmental changes,
o
i
s s
e such as temperature, humidity, and CO2 concentration
r 2
p x
E uniform among the groups. This is particularly true in
s 1. 5
o
f space experiments, which must be precisely controlled to
-
c
e 1
v enable comparisons between flight and ground speci-
i
t
a l
e 0. 5 mens. R 0 Vehicle U0126 Vehicle U0126 Still Still Rocked Rocked REFERENCES
Figure 3. Suppression of c-fos response to fluid flow by U0126 Chen, N.X., Ryder, K.D., Pavalko, F.M., Turner, C.H., 37°C. Values are means and standard errors for 4 flasks. Burr, D.B., Qui, J., and Duncan, RL. 2000. Ca2+ regu- lates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts. Am. J. Physiol. Cell Physiol. 278:C989-C997.
Clapham, D.E. 2003. TRP channel as cellular sensors. Nature 426:517-524
Corey, D.E., Garcia-Anoveros, J.. Holt, J.R., Kwan, K.Y., Shuh-Yow, L., Vollrath, M.A., Amalfitano, A., Cheung, E.L.-M., Derfler, B., Duggan A., Geleoc, G.G., Gray P.A., Hoffman, M.P., Rehm, H.L., Tamasauskas, D., Zhang D.-S. 2004. TRPA1 is a candidate for a mech- anosensitive transduction channel vertebrate hair cells. Nature 432:723-730. Figure 4. Immunofluorescent staining of primary cilium (arrow) of an MC3T3-E1 cell. Immunostainings with anti- Nauli, SM, Alenghat, F.J., Luo, Y., Williams, E., Vas- acetylated α-tubulin (AcTub) or anti-PC2 antibodies (right) are silev, P., Li, X., Elia A.E.H., Lu, W., Brown, E.M., shown. Quinn, S.J., Ingbar D.E., and Zhou, J. 2003. Polycystins 1
and 2 mediate mechanosensation in the primary cililum of We observed that MC3T3-E1 cells expressed c-fos in kidney cells. Nature Genetics. 33:129-137. cooling as well as in rocking of the culture flasks. This
response to the cooling would not seem physiological, Stevens H.Y., and Frangos, J.A., 2003. Bone cell re- since we could find virtually no literature describing sponses to fluid flow. In Helfrich, M.H. and Ralston, S.H. temperature dependencies in bone metabolism. Neverthe- eds. “Bone Research Protocols”, Humana Press (Totowa, less, the response could be a factor observable only under NJ) pp381-398. culture conditions with a cell line. Detection of lowered
temperature by PC2 or other TRP channel superfamily proteins could also be possible. Some TRP channel superfamily proteins have been shown to sense either cold, cool or hot temperatures (Clapham, 2003), while another member was assumed to sense mechanical stimuli (Corey et al., 2004). Most of these proteins are expressed in the respective neurons in a highly specific manner, but not in bone or kidney cells. However, we have not entirely excluded the possibilities of mechanosensing and
108 Gravitational and Space Biology 18(2) June 2005
POLYAMINES PROTECT AGAINST RADIATION-INDUCED OXIDATIVE STRESS Albert W. von Deutsch1, Clarence D. Mitchell2, Chris E. Williams2, Kamla Dutt1,3, Natalia A. Silvestrov 4,5, Brenda J. Klement1,6, Imad K. Abukhalaf 7 and Daniel A. von Deutsch*,1,4,5. 1Space Medicine and Life Sciences Research Center, 2MBRS Program, MSM and Morehouse College, 3Department of Pathology, 4Department of Pharmacology and Toxicology, 5Clinical Research Center, 6Department of Neurobiology and Anatomy, 720 Westview Dr, SW, Atlanta, GA 30310. 7Department of Biotechnology and Genetic Engineering, Philadelphia University, Jordan. *Corresponding Author.
Astronauts and cosmonauts are exposed to a wide variety of Trays were placed on warmers (37°C) in the cell culture different hazards while in space that include radiation, which hood, uncovered and exposed to a UVC germicidal lamp for presents one of the most critical barriers to long-term 30 min (398.1 J/cm2, measured through the use of a missions. A major deleterious effect directly associated with thermopile - the distance to source was 71 cm). All other ionizing radiation is the production of reactive oxygen sources of light were off during UVC irradiation, and both species (ROS) such as peroxides and hydroxyl radicals. The medium and cells were exposed to UVC. Following free radicals generated by ultraviolet (UV) or ionizing exposure, cells were incubated for an additional 24 hours radiation can attack cellular lipids, proteins and DNA [1]. prior to analysis. Analysis: Cell viability was determined 24 Endogenous free radical scavengers such as glutathione and hours post exposure by the Trypan Blue dye exclusion the polyamines (e.g., spermidine and spermine) can inhibit method and cells counted on a hemacytometer. HO-1 content the action of ROS. In particular, heme oxygenase-1 (HO-1), was measured by ELISA (StressXpress). Calculations: Cell the enzyme involved in heme protein metabolism, can viability was calculated as either the percent of irradiated provide antioxidant protection through the production of the control [%UV Control= (Experimental/ Control)] or antioxidant bilirubin [2]. Furthermore, polyamines have been %Difference of Control [%Difference = (Experimental – UV shown to indirectly increase HO-1 content and antioxidant Control)/UV Control]. With % of Control, the control is at protection[2]. The ß2-adrenoceptor agonist clenbuterol has 100%, while with % Difference of Control, the control is at been shown to stimulate polyamine synthesis and by 0%. Thus, any value less than the control would be a negative extension, might provide a margin of antioxidant protection effect, meaning enhanced cell death, while any value greater through increasing HO-1 content. However, it is unclear than 0% would be protective. whether the polyamines are acting as a tertiary messengers Results. Cells were pre-treated with the ß2-adrenoceptor -7 for antioxidant protection in the ß2-adrenoceptor signal agonist clenbuterol (Cb, 10 M) and the antioxidant enzymes transduction pathway [3]. The purpose of this study was to catalase (CAT, 10 units/mL) and superoxide dismutase study the role of the polyamine pathway in attenuating free (SOD, 10 units/mL) and incubated for 24-hrs before exposure radical-induced damage. to UVC radiation (exposure time: 30 min). Treatment with Methods. Cells/Culture Conditions: Human fetal retinal Cb, CAT and SOD significantly increased the rate of cell cells (Ret 205, an SV-40T immortalized cell line) were a gift survival: Cb 2.13-fold (±0.34), CAT 2.61-fold (±0.26) and from Dr. Kamla Dutt [4]. Stock monolayer cultures were fed SOD 3.00-fold (±0.15), respectively, when compared to the DMEM/F12 (1:1) medium supplemented with: 10% FBS, UV control (1.0 ±0.05) (Figure 1). The combination of CAT NaHCO3, glutamine and gentamycin. Experiments were or SOD with Cb produced significantly higher (p< 0.05) carried out in 12-well trays (1.5 mL media/well) when cells levels of cell viability than in those treated with CAT or SOD were at 75% confluency, they were differentiated using 8-
bromo cAMP [4]. Treatment: All drugs were made up in 40 +3 35 phosphate buffered saline (PBS) and subsequently added to +2 p= 0.074 +2 *** *** *** their appropriate wells (100 µL/mL). The vehicle control 30 *** *** 25 wells received just PBS in the same volume as the treatment * groups. To stimulate ornithine decarboxylase (ODC) activity 20
Cell Viability Cell 15 and polyamine synthesis, cells were treated with clenbuterol Cells) Live (% (Cb, 10-7M), while the ODC inhibitor difluoromethyl- 10 ornithine (DFMO, 10-5M) was used to block polyamine 5 0 UV Ctrl Cb C S C+S C+Cb S+Cb synthesis. The ß2-adrenoceptor antagonist ICI-118551 (ICI, Treatment -6 10 M) was used to competitively inhibit Cb. When treating Figure-1. UVC and antioxidant protection. Comparison of antioxidant cells with both ICI and Cb, the cells were treated first with protection provided by Cb and the antioxidant enzymes. (+2) Compared to ICI (10-6 M) and one hour later the cells received Cb (10-7 Catalase and (+3) Compared to Superoxide Dismutase. Clenbuterol (Cb), M). To confirm that UVC was producing peroxides and catalase (C) and superoxide dismutase (S). N = 6. superoxides, cells were treated with the antioxidants catalase alone. Data expressed as mean ± S.D. (CAT, 10 units/mL) and superoxide dismutase (SOD, 10 To establish that Cb was acting through the ß2- units/mL). Cb (10-7M) served as a positive control with a adrenoceptor in providing a measure of protection against known response. In all experiments, each well received the UVC-induced oxidative damage, the ß2-adrenoceptor same volume of treatment and/or vehicle control. Exposure: antagonist ICI-118551 (ICI, 10-6 M) was used to Immediately before treatment (with vehicle, drugs or competitively inhibit Cb. When treating cells with both ICI enzymes), cells were fed fresh DMEM/F12 media [with pH and Cb, the cells were initially treated with ICI (10-6 M) and indicator] and incubated 24-hours prior to UVC exposure. one hour later the cells received Cb (10-7 M). All wells Gravitational and Space Biology 18(2) June 2005 109
A.W. von Deutsch et al. – Polyamines Protect against Radiation-Induced Oxidative Stress received the same final volume of treatment and/or vehicle treated controls, but HO-1 was significantly (p< 0.001) control (Figure 2). Cell viability, measured as the percent higher than the non-treated UV Controls. HO-1 content in the difference from the irradiated control (set to 0% difference, UV control group was significantly less than in the non- see methods). Treatment with ICI alone showed a small but exposed control group. In the Cb + ICI group, HO-1 levels significant (p< 0.05) agonist effect but ICI significantly (p< were not significantly different from the Cb-treated group. 0.01) attenuated Cb’s protective effect in the combination However, DFMO significantly inhibited Cb’s effect on HO-1
(*1a) p= 0.052 ***1 p< 0.001 Cb vs UV Control ++3 p< 0.01 ICI+Cb vs Cb 500 (***1b) 100 *2 p< 0.05 ICI vs UV Control *3 p< 0.05 ICI+Cb vs UVControl 400 75 ***1 300 (+p= 0.085) 50 200 ** (++1)
*2 *3, ++3 (ng/mg Protein) 25
Cell Viability Cell 100 CellularHeme Oxygenase-1 Content 0 0 Ctrl UV Ctrl Cb ICI Cb+ICI D Cb+D (% Difference in Live Cells) Live in Difference (% Figure 4. Heme Oxygenase-1 Content in Retinal Cells Exposed to -25 UV-Induced Oxidative Stress. Cells were treated with clenbuterol (Cb), Ctrl UV Cb ICI ICI+Cb ICI-118551 (ICI), DFMO (D) or their combination. Significance: Figure 2. ß2-Adrenoceptor Agonist Protection against UVC-Induced Compared to Control p= 0.052; Compared to UV Ctrl: (***1b) p< 0.001; Oxidative Stress. Cells were treated with the ß2-adrenoceptor agonist Compared to Cb (++1) p< 0.01. N= 5. -7 clenbuterol (Cb, 10 M), the ß2-specific adrenoceptor antagonist ICI- 118551 (ICI, 10-6 M) or their combination. N= 10. content, thereby showing that the polyamines were acting, in part, as tertiary messengers in Cb-induced antioxidant group [UV Ctrl 0.00 ±1.08%, Cb 58.28 ±9.54%, ICI 22.37 activity (Figure 4). ±10.02% and ICI+Cb 27.88 ±4.94%]. Discussion and Conclusion. Exposure of the media to To test whether polyamines are involved in Cb’s UVC radiation results in the production of ROS such as protective action, the ornithine decarboxylase (ODC) peroxides and hydroxyl radicals. Following exposure to UVC inhibitor difluoromethylornithine (DFMO, 10-5 M) was used radiation, cells showed significant levels of cell death. to inhibit polyamine synthesis, while the ODC substrate Treatment with the antioxidant enzymes CAT and SOD
***1 p< 0.001 DFMO vs UV Control, ***2 p< 0.001 Cb vs UV Control, significantly increased cell viability, suggesting ROS *3 p< 0.05 Cb+D vs UV Control, ***4 p< 0.001 Orn vs UV Control *5 p< 0.05 Orn+D vs UV Control. involvement. Likewise, treatment with the ß -adrenoceptor 75 2 ++5 p< 0.01 Orn vs Orn+D, +++4 p< 0.001 Orn vs D, ++ 3 p< 0.01 C b+D vs D , + + 2 p< 0.01 C b+ D vs D agonist Cb or the ODC substrate ornithine significantly +++2 p< 0.001 Cb vs Cb+D. 50 increased cell viability in exposed cells. In contrast, the ODC ***2, +++2 inhibitor DFMO increased the level of retinal cell death and 25 when combined with Cb or ornithine, it attenuated their *3, ++3 ***4, +++4 ***1 Cell Viability Cell *5, ++5 protection. This suggests that polyamines mediate Cb/Orn- 0 induced antioxidant protection. Previous studies conducted in
(% Difference in Live Cells) in Difference (% our laboratory showed that Cb increased skeletal muscle -25 polyamine concentrations while reducing atrophy, thus Ctrl UV D Cb Cb+D Orn Orn+D Figure-3. Clenbuterol and Ornithine Protection Against UVC- further supporting the protective role of polyamines [3]. In Induced Damage. Comparison of antioxidant protection provided by addition, the ability of Cb to act through the polyamine clenbuterol (Cb, 10-7M), ornithine (Orn, 10-7M) and the ODC inhibitor -5 spermine (as an NO donor) in stimulating HO-1 activity and DFMO (D, 10 M). Control = Ctrl. Data expressed as mean ± S.D. N=10. the subsequent production of bilirubin results in increased ornithine (Orn, 10-7 M) was used to stimulate polyamine antioxidant protection and cell viability [2]. Thus, with synthesis without ß2-adrenoceptor involvement (Figure 3). increased protection provided by both elevated polyamine Twenty-four hours post-treatment, cells were exposed to and bilirubin levels, the magnitude of UV-induced cell death UVC for 30 minutes. Results suggest that DFMO is reduced. This study confirms that Cb acts through significantly (p< 0.001) decreased cell viability while Cb and polyamines and the antioxidant enzyme HO-1 to protect ornithine both significantly (p< 0.001 each) increased cell against UV-induced oxidative damage. This work was viability. Treatment with DFMO significantly attenuated the supported by NASA Grant NCC9-112 and NIH Grants protective effects of both Cb (p< 0.01) and Orn (p< 0.01), 1R25RR17694 and 5P20RR011104. suggesting that polyamines are involved in the cells ability to References. protect against UVC-induced oxidative damage. 1. Fang YZ, Yang S, Wu G. Free radicals, antioxidants, and To determine whether the polyamines serve as tertiary nutrition. Nutrition. 2002; 18:872-879. messengers in the ß2-adrenoceptor agonists signal 2. Hartsfield CL, Alam J, Cook JL, Choi AM. Regulation of transduction pathway for antioxidant protection, changes in heme oxygenase-1 gene expression in vascular smooth muscle the antioxidant enzyme heme oxygenase-1 (HO-1) content cells by nitric oxide. Am J Physiol. 1997; 273(5 Pt 1): L980- was measured. Cells were treated with Cb (10-7M), ICI (10- L988. 6M), DFMO (D, 10-5M) or their combination. As in the 3. von Deutsch DA, Abukhalaf IK, Wineski LE, Silvestrov NA, previous experiments, cells were exposed to UVC for 30 min. Bayorh M, and Potter DE. Changes in muscle proteins and In Cb-treated cells, HO-1 content only approached being spermidine content in response to unloading and clenbuterol treatment. C.J. Physiol. Pharmacol. 81:28-39, 2003. significantly higher (p= 0.052) than the non-exposed/non-
110 Gravitational and Space Biology 18(2) June 2005
ERYTHROPOIETIN AND IL-3 RECEPTOR CELL SURFACE EXPRESSION IS DECREASED UNDER CONDITIONS THAT MODEL SOME ASPECTS OF MICROGRAVITY Kun Xu, Laurie Feldman, Kerry L. Davis, and Arthur J. Sytkowski Laboratory for Cell and Molecular Biology, Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, MA 02215, USA
Human and experimental animals in space develop the To determine the specificity of the RCCS effect on EpoR, anemia of space flight (Tavassoli, M., 1982; Udden et al., the abundance of IL-3 receptor was also studied. 1995). Studies have shown that erythropoietin (Epo) is Similarly, 125I-IL-3 binding to RCCS-cultured cells was implicated in this microgravity-induced abnormality significantly reduced compared to that to flask-cultured (Udden et al., 1995). The status of the Epo receptor cells (p<0.01) (Figure 2). This result suggests that culture (EpoR) in microgravity, however, is unknown. The in the modeled (simulated) microgravity environment of Rotary Cell Culture System (RCCS), based on NASA the RCCS has a generalized effect on cell surface growth rotating wall vessel (RWV) technology and manufactured factor receptors. by Synthecon Inc., is an in vitro culture system. Within this system individual cells, to a certain extent, experience an environment with similarities to true microgravity 2.5 (Gao et al., 1997; Battle et al., 1999). BaF3 cells stably expressing the transfected human Epo receptor (BaF3- 2.0 EpoR cells) were cultured in RPMI-1640 supplemented -3 with 5% fetal bovine serum and either 1 unit/mL of 1.5 recombinant human Epo (rhEpo) or 5 ng of recombinant 1.0 mouse interleukin-3/mL (rmIL-3) in either the RCCS or
2 o 10 x CPM, in 175 cm standard tissue culture flasks (control) at 37 C 0.5 o in a humidified atmosphere of 95% air/5% CO2, 37 C for 48 hours. Cells were then harvested by centrifugation, 0.0 incubated with 125I-labeled rhEpo or 125I-labeled rmIL-3, 0.00 0.25 0.50 0.75 and the bound/free ligands were separated by centrifuging 125I-IL-3, nM the cells through a serum cushion according to published methods (Yonekura, 1991). The radioactivity of the cell 125 pellet was quantified by gamma scintillation Figure 2. The binding curves of I-IL-3 to BaF3-EpoR cells spectrometry. grown in flasks () and RCCS ( ). Each point shown Starting from 0.1 nM of 125I-Epo (the lowest represents the mean of triplicates ± S.E. concentration tested) upward, the specific binding of radiolabeled ligand to flask-cultured cells () was Cell viability from both flask- and RCCS-cultured significantly greater than that to RCCS-cultured cells ( ) cells was assayed by trypan blue dye exclusion. Both (p<0.01) (Figure 1). At 3.2 nM (the highest concentration exhibited cell viabilities of greater than 90% (p>0.05) tested), the bound radioactivity (surface EpoR density) of (Figure 3). This result excludes the possibility that the RCCS-cultured cells was only 56.6% of that of the flask- effect of RCCS culture on cell surface receptors is due to cultured counterpart. The non-specific binding of mechanical damage to the cells or other intrinsic factors, radiolabeled Epo to the flask- (S) and RCCS (U)- such as toxicity. cultured cells were similar.
100
80 20
-3 60 15
10 40 Survival, % CPM x 10 5 20
0 0 0 1 2 3 Flask RCCS 125I-Epo, nM
Figure 3. Histogram of survival rates of BaF3-EpoR cells Figure 1. The binding curves of 125I-Epo to BaF3-EpoR cells grown in RCCS and flasks. Each bar shown represents the mean grown in flasks () and RCCS ( ). Each point shown of triplicates ± S.E. represents the mean of triplicates ± standard errors (S.E.).
Gravitational and Space Biology 18(2) June 2005 111 K. Xu et al. – Erythropoietin and IL-3 Receptor Cell Surface Expression Is Decreased in Modeled Microgravity
The possibility of nutrient depletion as a factor contributing to the reduction of cell surface Epo receptors was excluded by the following study. Cells were cultured in RCCS with either 1 ( ) or 2 ( ) mg/mL of glucose in the cell culture medium. No changes were observed in both specific binding (SB) and non-specific binding (NSB) between the two glucose levels (Figure 4).
14 12 10 -3 8 6 CPM x 10 CPM 4 2 0 SB NSB
Figure 4. The histograms of 125I-Epo binding to Ba/F3-EpoR cells grown in Low ( ) and high ( ) glucose in RCCS. Each bar shown represents the mean of triplicates ± S.E.
This is the first study showing possible microgravity effects on the EpoR and the potential involvement of the EpoR in the anemia of space flight. This study also suggests that the expression of other cell surface growth factor receptors may be affected by gravity.
REFERENCES
Battle, T., Maguire, T., Moulsdale, H., and Doyle, A. 1999. Progressive maturation resistance to microcystin-LR cytotoxicity in two different hepatospheroidal models. Cell Biol Toxicol 15: 3-12.
Gao, H., Ayyaswamy, P. S., and Ducheyne, P. 1997. Dynamics of a microcarrier particle in the simulated microgravity environment of a rotating-wall vessel. Microgravity Sci Technol 10: 154-165.
Tavassoli, M. 1982. Anemia of spaceflight. Blood. 60: 1059- 1067.
Udden, M. M., Driscoll, T. B., Gibson, L. A., Patton, C. S., Pickett, M. H., Jones, J. B., Nachtman, R., Allebban, Z., Ichiki, A. T., Lange, R. D., and et al. 1995. Blood volume and erythropoiesis in the rat during spaceflight. Aviat Space Environ Med 66: 557-561.
Udden, M. M., Driscoll, T. B., Pickett, M. H., Leach-Huntoon, C. S., and Alfrey, C. P. 1995. Decreased production of red blood cells in human subjects exposed to microgravity. J Lab Clin Med 125: 442-449.
Yonekura, S., Chern, Y., Donahue, K. A., Feldman, L., Vanasse, G. J., and Sytkowski, A. J. 1991. Erythropoietin receptors induced by dimethyl sulfoxide exhibit positive cooperativity associated with an amplified biologic response Proc Natl Acad Sci U S A 88: 2535-2539.
112 Gravitational and Space Biology 18(2) June 2005
SECRETION AS A KEY COMPONENT OF GRAVITROPIC GROWTH: IMPLICATIONS FOR ANNEXIN INVOLVEMENT IN DIFFERENTIAL GROWTH Greg Clark, Araceli Cantero-Garcia, Tim Butterfield, Marianne Dauwalder, and Stanley J. Roux Molecular Cell & Developmental Biology, University of Texas, Austin, Texas 78712 USA
Materials for cell wall growth are delivered to the wall by secretory vesicles, and recent studies have highlighted the role of secretion in plant growth generally and plant gravitropic growth specifically. Funke and Edelmann (2000) show that the growth of graviresponding nodes of Tradescantia is controlled by auxin-induced secretion and subsequent infiltration of wall materials that promotes wall extension. Similarly, Zhang and Hasenstein (2000) demonstrate that Brefeldin A, a potent inhibitor of the delivery of secretory vesicles from the trans-Golgi to the plasma membrane in plant cells (Driouich et al, 1993), Figure 1. Autoradiography of 3H–galactose incorporation and blocks the asymmetric delivery of expansins to the cell annexin immunostaining in young Arabidopsis shoots that have wall of graviresponding maize roots. They further show been gravistimulated, showing (A) asymmetric deposition of that this effect is correlated with a delay in the labeled polysaccharides in mainly epidermal cells of the hypocotyl (arrows), and (B) asymmetric anti-AnnAt2 staining of graviresponse. The data of Morris and Robinson (1998) the hypocotyl epidermis that corresponds to the faster growing reveal that Brefeldin A also blocks actin-dependent side (arrows). cycling of PIN proteins, which serve as important auxin efflux facilitators, to the plasma membrane, and they strongly suggest that other auxin transport inhibitors may work by interfering with membrane-trafficking processes. Annexins are a multi-gene, multifunctional family of calcium-dependent membrane-binding proteins found in most eukaryotic cells. Annexins appear to be important targets of Ca2+ action in plant cells and are proposed to be key participants in the process of secretion of newly synthesized plasma membrane and wall materials during growth and development. We have identified eight different members of the annexin gene family in Arabidopsis. Using in situ RNA localization, we found that two of these annexins, AnnAt1 and AnnAt2, exhibited mostly distinct localization patterns with highest expression levels in secretory cell types in young Arabidopsis seedlings (Clark et al., 2001). More recently, we have obtained immunolocalization Figure 2. Autoradiography of 3H–galactose incorporation and data for these two individual annexins in conjunction with annexin immunostaining in young Arabidopsis roots that have autoradiography data of 3H-galactose incorporation in been gravistimulated, showing (A) asymmetric deposition of young Arabidopsis seedlings (Clark et al., 2004). For the labeled polysaccharides in mainly epidermal cells of the root (arrows), and (B) asymmetric anti-AnnAt1 staining of the root immunolocalization experiments we used monospecific epidermis that corresponds to the faster growing side (arrows). anti-annexin antibodies raised against divergent 31-mer peptides from AnnAt1 and AnnAt2. These peptides show Figure 1 shows asymmetric polysaccharide secretion only 35% amino acid identity. As a marker for as demonstrated by 3H-galactose incorporation and polysaccharide secretion, we used 3H-galactose, which is asymmetric localization of AnnAt2 in epidermal cells of taken up by seedlings, converted into more complex gravistimulated hypocotyls. In gravistimulated roots, we polysaccharides and detected by immunocytochemistry. also observed asymmetric polysaccharide secretion as We observed strong similarities between this pattern of demonstrated by 3H-galactose incorporation and secretion and the individual protein localization patterns for correlated this secretion asymmetry with an asymmetric these two annexins in most cases. localization of AnnAt1 in epidermal cells (Figure 2). In In the present study, we applied these same both cases, we observed an asymmetric distribution techniques to determine the localization pattern for these pattern for an individual annexin and for labeled two individual annexins and surveyed secretion activity in polysaccharides which were almost identical for the faster graviresponding hypocotyls and roots in young growing side. The asymmetric 3H-galactose incorporation Arabidopsis seedlings. We also examined hypocotyl and and annexin immunostain shown in gravistimulated root growth for several T-DNA mutants of these two seedlings was repeated several times to insure that annexins in the dark. sectional differences were not the source of the
asymmetries. Additionally, control non-stimulated
Gravitational and Space Biology 18(2) June 2005 113 Clark et al. – Annexin Involvement in Differential Growth seedlings were run in parallel and showed an even mutant of AnnAt2 exhibits inhibited growth in distribution of 3H-galactose incorporation and annexin hypocotyls. Taken together these results suggest that these immunostain in the epidermal cells on both sides of two annexins may play a role in differential growth hypocotyls and roots, respectively. during gravitropism.
ACKNOWLEDGEMENT
The work presented here was supported by NASA grant NAG2-1586 to SJR and GBC.
REFERENCES
Clark G.B., Sessions A., Eastburn, D.J., and Roux S.J. 2001. Differential expression of members of the annexin multigene family in Arabidopsis. Plant Physiol. 126: 1072-1084.
Clark, G.B., Lee, D., Dauwalder, M., and Roux S.J. 2004. Immunolocalization and histochemical evidence for the association of two different Arabidopsis annexins with secretion during early seedling growth and development. Planta (in press).
Driouich, A., Zhang, G.F., and Staehelin A.L. 1993. Effect of Brefeldin A on the structure of the Golgi apparatus and on the synthesis and secretion of proteins and polysaccharides in sycamore maple (Acer pseudoplatanus) suspension-cultured cells. Plant Physiol. 101: 1363-1373.
Funke, M., and Edelmann, H.G. 2000. Auxin-dependent cell wall depositions in the epidermal periplasmic space of graviresponding nodes of Tradescantia fluminensis. J. Exp. Bot. 51: 579-586.
Figure 3. (A) Hypocotyl lengths in 3.5-day-old etiolated wild- Morris, D.A., Robinson, J.S. 1998. Targeting of auxin type plants and annexin knockout mutant annAt2-1, and (B) root carriers to the plasma membrane: differential effects of lengths in 7-day-old etiolated wild-type plants and annexin Brefeldin A on the traffic of auxin uptake and efflux knockout mutants annAt1-1 and annAt1-2. Asterisk above the bar indicates a significant difference (p<0.05) between the carriers. Planta 205: 606-612. length of that mutant and the wild-type plants. Zhang, N. and Hasenstein, K.H. 2000. Distribution of
expansins in graviresponding maize roots. Plant Cell In order to clarify the function of individual Physiol. 41: 1305-1312. Arabidopsis annexins we have obtained T-DNA knockouts for these two annexins. In initial experiments, an annexin T-DNA knockout mutant for AnnAt2, annA2- 1, shows inhibited hypocotyl growth in 3.5-day-old dark grown seedlings (Figure 3a) and annexin T-DNA knockout mutants for AnnAt1, annAt1-1 and annAt1-2, show inhibited root growth in 7-day old dark grown seedlings (Figure 3b). We have presented autoradiographic and immunolocalization data linking two different annexins with the enhanced secretion that accompanies asymmetric growth during gravitropism in Arabidopsis. Specifically, there is a correlation with asymmetric secretion and differential localization of AnnAt1 in gravistimulated roots and AnnAt2 in gravistimulated hypocotyls. Additionally, we have shown that T-DNA mutants for AnnAt1 exhibit inhibited growth in roots and a T-DNA
114 Gravitational and Space Biology 18(2) June 2005
PHOTOTAXIS AND AEROTAXIS IN A CALCIFYING ALGA P.Jackie Duke1 ,Wendy Callejas2, Lan Doan1, and Mary Marsh3 1Dept. of Orthodontics, Dental Branch, University of Texas Health Science Center at Houston, 2University of Houston- Downtown, 3Dept. of Biochemistry and Molecular Biology, Medical School, University of Texas Health Science Center at Houston, Houston, TX 77225, USA.
INTRODUCTION reached, concomitant with the start of bioconvection The coccolithophore, Pleurochrysis carterae, is a (convection due to movement of microorganisms.) The unicellular marine alga, with an outer covering of scales bioconvective process begins with the accumulation of known as coccoliths, instead of a cell wall (Figure 1; 1). cells at the surface of the medium, which in turn requires Coccoliths are composed of calcium carbonate (CaCO3) that the cells swim up (8). The objective of the current in the form of calcite, and are formed intracellularly in the study was to determine if taxes other than gravi- or Golgi cisternae in a very precise and well defined process gyrotaxis, specifically photo- and aerotaxis, were the before being positioned outside the cell membrane (1). cause of surface accumulation of cells. Because our Because this is an excellent model of calcification in a deselection was due to lack of videomicroscopy, single cell, we proposed P. carterae for flight on the macroscopic methods were used in these experiments. International Space Station, and spent several years in experiment development prior to having the experiment MATERIALS AND METHODS deselected from flight. In addition to being a model for calcification, P. carterae was to have been used to Cells from a stock cell culture of P.carterae, Plymouth address questions of reproduction, cellular polarity, strain 136, a swimming strain, were expanded in F/2 secretion, and gravitaxis in the microgravity environment medium (18oC), then divided into 4 t-flasks. After 24 (2-4). Requiring only low levels of light and capable of hours, cultures were observed for accumulations of cells being stored for over 70 days in the cold and dark before in the region of the air bubble. For phototaxis, box use, P. carterae remains an excellent choice for space covers, with only a small opening at the bottom of one flight studies (5,6). Additional descriptions of the side, were placed over the t-flasks as an obstruction of the proposed experiment and various educational links light source. Cells in these cultures did not accumulate in regarding this alga can be found at (7). any region of the culture, and cultures did not grow due to the lack of light. The t-flasks were then placed horizontally in the incubator with the boxes covering all but the top 25% of the flask. The air bubble was located in the dark region of the flask. Flasks were incubated overnight, and observed for accumulation of cells in particular regions of the flask.
Figure 2: T-flask from phototaxis experiment. Cells are visible as a cloud in the medium, and are located in the left hand side of the flask which was exposed to light during culture in the incubator.
Figure 1: Scanning electron micrograph showing coccooiths on RESULTS AND DISCUSSION the surface of P. carterae. Photo by Dr. Mary Marsh. During the initial culture of the cells to expand cell In preparation for the gravitaxis portion of the spaceflight numbers, a green halo of cells surrounded the air bubble experiment, we spent considerable time assessing in each flask, demonstrating aerotaxis. The first gravitaxis in P carterae at 1G, using the Wintrack 2000 phototaxis experiment used boxes that completely software. In these studies, we determined that cells of covered the flask, with only a small opening for light. Pleurochrysis carterae do not start to move in a This arrangement did not support cell growth as after the measurably oriented manner until a certain cell density is incubation period, no accumulations of cells were seen. This agrees with previous experiments in which we Gravitational and Space Biology 18(2) June 2005 115 P. Jackie Duke – Phototaxis and Aerotaxis in a Calcifying Alga demonstrated that no increase in cell number is seen in cultures of P carterae kept in the dark. REFERENCES In the partially covered t-flasks, cells accumulated in the portion of the flask that was not covered, Algae and Dentistry:http://www.db.uth.tmc.edu/ demonstrating phototaxis (Figure 2). The bubbles in orthodont/Duke%20NASA/index.html Bees, M. A. and these flasks were carefully sequestered in the dark region, Hill, N.A. 1997. Wavelengths of bioconvection patterns. so that the aerotaxic response would not interfere with the Journal of Experimental Biology 200: 1515-1526. phototaxic response. The lack of accumulations around the air bubble in these cultures demonstrates that the Duke, P.J., and Montufar-Solis, D. 2000. “Pleurochrysis cell’s response to light is stronger than the response to the carterae: a model to study biologically directed presence of air. Similar results were seen in experiments mineralization in space.” First International Symposium with Euglena gracilis, a fresh water photosynthetic alga on Microgravity Research and Applications in Physical (9). Sciences and Biotechnology, ESA Publications Division, These simple experiments demonstrate that ESA SP-454:337-348. macroscopic methods can be used to observe movement of populations of algal cells, thus obviating the need for Marsh, M. 1999. Biomineralization in Coccolithophores. microscopic observations to see overall cell Gravitational and Space Biol Bulletin. 12: 5-15. accumulations. These observations, which can be captured automatically on videotape in several of the Montufar-Solis, D., Duke P.J., and Marsh, M.E. 2003. current cell culture systems developed for ISS, can be Bioconvection in cultures of the calcifying unicellular used to determine if bioconvection occurs in space. alga Pleurochrysis carterae. J Gravitational Physiology. Although bioconvection, according to theory, should not 10: 270-272. occur in a microgravity environment, there may be enough g disturbances on ISS to begin the process, which Montufar-Solis D., Marsh M.E., and Duke, P.J. 2002. might be then continued by swimming action of the Cell density affects cell motility in P. carterae cultures. In: organisms themselves. Proceedings of the Eight European Symposium on Life The results in these studies were definitely density Sciences Research in Space, Stockholm, Sweden, June 2- dependent—at lower densities the cloud of cells would 7, , ESA SP-501, pp293-294. not be visible to the naked eye. At higher densities, bioconvection would occur. Montufar-Solis, D., Marsh, M.E., and Duke, P.J. 2002. Finally, such results must be taken into account when Cell density affects cell motility in P. carterae cultures. designing space flight experiments—for example, the Journal of Gravitational Physiology 9:P265-266. light source should be perpendicular to the gravity vector in the culture system to ensure that the cells are Montufar-Solis, D., and Duke, P.J. 2001. Pleurochrysis responding to gravity and not to light. Alternatively, carterae is an excellent model to study biomineralization during g-response experiments, an infared source of light and gravitaxis on the ISS. American Institute of can be used. Aeronautics and Astronautics Bulletin. AIAA (4989):478- 483. CONCLUSIONS Porterfield, D.M. 1997. Orientation of motile unicellular From this experiment we conclude that P.carterae is algae to oxygen: oxytaxis in Euglena. Biol. Bull. 193: aerotaxic and phototaxic, and that the phototaxic response 229-230. is stronger than the aerotaxis response. In the ocean, P. carterae is found near the surface of the water, due probably to phototaxis and aerotaxis, not gravitaxis or gyrotaxis. Future experiments will determine the response to air during the nonphotosynthetic period of the light:dark cycle.
ACKNOWLEDGEMENTS
Supported by UTHSC Summer Research Program for High School Students 2004 (WC) and NASA NAG2- 1498 (MM).
116 Gravitational and Space Biology 18(2) June 2005
CO-EXPRESSION AND HORMONAL REGULATION OF GENES IN RESPONSE TO GRAVITY AND MECHANICAL STIMULATION IN THE ARABIDOPSIS ROOT APEX. Jeffery M. Kimbrough1*, Christopher S. Brown1,2 and Heike Winter Sederoff1 Plant Gravitational Genomics Group, 1Dept. Botany and 2Kenan Institute, North Carolina State University, Raleigh, NC, USA *corresponding author [email protected]
Plant roots direct their growth in response to gravity, light, and mechanical stimuli. Spatial changes in the rates of cell elongation and division are responsible for the directional growth. Local changes in hormone concentration and/or sensitivity have been shown to be part of the signal transduction and response mechanisms that result in those tropic growth resonses. Part of most hormone regulated mechanisms are regulation of transcription and transcript stability. We have focussed our analysis of whole genome microarrays on the differential regulation of transcript abundance changes during the first hour after gravity and mechanical stimulation with respect to the involvement of hormones – especially auxins and brassinosteroids. Root apices from 7-day-old etiolated Arabidopsis seedlings were harvested and analyzed for relative changes in transcript levels in response to gravistimulation and mechanical stimulation using the Arabidopsis ATH1 GeneChip (Affymetrix). In a time Figure 1: Genes regulated (up or down) by both stimuli were course experiment, approximately 150 root tips were classified into functional groups. The number represents the harvested before (0 time point) and 2, 5, 15, 30, 60 percentage of genes in each functional group from all genes regulated under both stimuli. min after 135◦ reorientation by pouring RNAlater (Ambion) onto the plates and cutting off the root apex (~7.5 mm). Mechanical stress control seedlings (0, 2, 5, 15, 30, 60 min) were moved horizontally for 5 sec without changing their orientation towards the vector of gravity and were processed in an identical manner. Total RNA was extracted and purified (RNeasy column, Qiagen), amplified and hybridized to microarrays using standard protocols (Kimbrough et al. 2004). From 24,000 transcripts analyzed, we found 1730 with significant changes in abundance at two consecutive time points after reorientation (gravity) and 1691 transcripts regulated after mechanical stimulation (mechanical). While both stresses had 1641 regulated transcripts in common (Figure 1), 65 genes were gravity-specific up regulated and 26 were specifically regulated by the mechanical stimulus. Of those regulated by mechanical stimulus, 16 showed an Figure 2: Intensities of red and green reflect degree of up or increase in abundance and 10 transcripts showed a down regulation, respectively, compared to vertical control (0). decrease compared to the vertical control. Of the transcripts regulated by both stimuli, 897 showed an increase in abundance and 744 a decrease (Kimbrough et al. 2004). Many of the stimuli specific genes exhibited co-regulation throughout the time course (Figure 2).
Gravitational and Space Biology 18(2) June 2005 117 J. Kimbrough – Response to Gravity and Mechanical Stimulation in the Arabidopsis Root Apex similar fashion. In some cases, transcripts showed (A) distinct differences in their temporal profiles. A specific example of this is the cytokinin regulated gene for a disease resistance protein (At4g11190, see Figure 3C). This gene showed up-regulation from 2 through 15 minutes during gravity stimulation but was not up- regulated until 30 minutes in mechanical stimulation. This suggests the regulation of this gene is controlled through a similar pathway but at different times in gravity or mechanical stimulation. The xyloglucan endoglycosyl transferase gene (TCH4) was observed to be transiently up-regulated throughout both mechanical and gravity stimulation and was previously shown to be up-regulated upon treatment of cytokinins
(B) and the combined treatment of BR and auxin. This analysis can reveal which genes are regulated by gravity/mechanical stimulation through certain hormones but it cannot determine local or spatial effects of hormones. It is probable that these hormones affect gene expression through gravity and mechanical regulated genes in specific cell types in the root or preferentially on the upper or lower side of the root to produce gravitropic curvature. Currently, we are analyzing the expression profile of certain genes in mutants affected in hormonal response. The results will allow us to identify whether genes regulated by (C) gravity/mecahincal stimulation and hormones are regulated through parallel pathways or if they are controlled in a single linear pathway.
REFERENCES
Kimbrough JM, Salinas-Mondragon R, Boss WF, Brown CS, Sederoff HW (2004) The Fast and Transient Transcriptional Network of Gravity and Mechanical Stimulation in the Arabidopsis Root Apex. Plant Physiol. 136: 2790-2805
Goda H, Shimada Y, Asami T, Fujioka S, Yoshida S (2002) Microarray Analysis of Brassinosteroid-Regulated Genes in Arabidopsis. Plant Physiol. 130: 1319-1334 Figure 3: Cluster analysis of hormone-regulated genes showing changes in transcript abundance throughout 1 hour of gravity (left) and mechanical stimulation (right). (A) IAA or BR responsive Rashotte AM, Carson SDB, To JPC, Kieber JJ (2003) genes; (B) IAA and BR responsive genes; (C) cytokinin (zeatin) Expression Profiling of Cytokinin Action in Arabidopsis. regulated genes. Plant Physiol. 132: 1998-2011
Hormones including, but not limited to auxin, cytokinins and brassinosteroids are known to be mediators regulating gene expression changes in plants. Previously identified genes whose expression levels change upon treatment with cytokinins (Rashotte et al., 2003), brassinosteroids and/or auxin (Goda et al., 2002) were analyzed to determine their transcript abundance profile throughout gravity and mechanical stimulation (Figure 3). We observed many hormone regulated genes that were also regulated throughout gravity and mechanical stimulation in a 118 Gravitational and Space Biology 18(2) June 2005
ALTERATION OF GROWTH AND GRAVITROPIC RESPONSE OF MAIZE ROOTS BY LITHIUM Timothy J. Mulkey Department of Life Sciences, Indiana State University, Terre Haute, IN 47809 Measurement of Elongation: A computer-based root The alkali metal lithium is known to produce a number auxanometer system (Mulkey et al, 1982b) was used to of effects in plants and animals. Lithium salts have been determine the effects of lithium on elongation of intact an important tool in dissecting the intracellular pathways primary roots of maize. Roots were placed into the of signal transduction systems. Lithium induces an auxanometer chamber containing half-strength Meyer's increase in diacylglycerol (DAG) in animal cell culture; solution. the mechanism of this effect is not fully understood. Isolation of Membrane and Cytoplasmic Fractions. Brami et al. (1993) suggest that lithium may alter the One-cm root tip segments (at least 3 g of roots) were degradation of phospholipids by acting on phospholipase ground on ice with homogenization buffer containing 50 C or D. In the second messenger signal transduction mM MES-NaOH, pH 7.0, 5mM MgCl2, 0.5 mM DTT, system phosphatidylinositol-4, 5-disphosphate (PIP2) is 0.25 M Sucrose, 5 mM EDTA, and 0.5 mM PMSF using converted into phopholipase C (PLC). a mortar and pestle (1:2 w/v). The homogenate was Lithium can alter the second messenger signal filtered through Miracloth and centrifuged for 10 min at transduction system by altering enzyme activity. Myo- 7,000 g to discard nuclei and cell walls. The supernatant inositol-1-phosphatase (I-1-P) converts D-glucose-6- was collected, centrifuged for 30 min at 100,000 g for 30 phosphate to myo-inositol via the intermediate L-myo- min; and the supernatant was used for procedures inositol-1-phosphate. This enzyme can hydrolyze both requiring cytoplasmic fractions. The pellet was the D- and L-enantiomers of I-1-P. Lithium inhibits myo- suspended in homogenization buffer at the same volume inositol-1-phosphatase activity in both plants and animals and centrifuged again at 100,000 g for 30 min. The final although plant tissue is less sensitive to the effects. In pellet was suspended in homogenization buffer (1 ml/5 g pollen of lily, 50 mM of lithium is required to produce initial fresh weight of root), and used for procedures 50% inhibition of the enzyme (Gumber et al., 1984). requiring membrane fractions. The protein content of It has been reported that lithium salts increase inositol fractions was determined by BCA Protein Assay kit. phosphate levels in animal tissue but that lithium has no Protein Phosphorylation. In vitro protein effect on inositol phosphate levels in plants. Morse et al. phosphorylation assays were conducted at 30C in a total (1987) found no evidence that lithium enhances the reaction volume of 100 µl containing 50 mM MES-NaOH recovery of inositol phosphates in the leaf pulvina of the (pH 7.0), 0.5 mM DTT, 5 mM MgCl2, 0.2 mM EGTA and legume Samanea saman during the night movement of the 200 ug of membranous or cytoplasmic proteins. Ten µl of leaves. Additionally they found that inositol is not the 1% Triton X100 was added 15 min before rate-limiting factor in the biosynthesis of phosphorylation of the membrane fractions. The reaction phophatidylinositol in Samanea saman. was initiated by adding 0.037 Bq [gamma-32P]-ATP. The Lithium salts alter ethylene biosynthesis in mung reaction was terminated after 1 min by adding the same beans (Vigna radiata) (Lee and Kang, 1987). Ethylene volume (100 µl) of electrophoresis sample buffer and by inhibits plant growth and is produced through the boiling the reaction mixture for 5 min. The reaction conversion of methionine to s-adenylsylmethionine to mixture of membrane protein was centrifuged at 10,000g aminocyclopropane carboxylic acid to ethylene. Lithium for 5 min and the supernatant was used for analyses by ions may alter ethylene biosynthesis by effecting the SDS-PAGE. The reaction mixture of cytoplasmic protein conversion of ACC to ethylene. was used without further centrifugation. Lithium inhibits thigmomorphogenesis in Bryonia Results: Figure 1 illustrates the effect of 1 mM dioica (Boyer et al., 1983) and Bidens pilosus (Desbiez et lithium chloride on response of roots to 0.1 nM auxin. al., 1981). Rubbing the internodes of Bryonia decreased The solid line indicates that 1 mM lithium is added at 60 the height of the plants by inhibiting cell division and elongation. Pricking one cotyledon on a Bidens pilosus plant inhibits the growth of the hypocotyl within one hour. Lithium treatment of the plants reduced the amount of inhibition which was observed. Thus it was concluded that lithium inhibition of thigmomorphogenetic responses resulted from lithium inhibition of the effect of ethylene formed in the mechanical stimulation of the plant tissues. Plant Material: Grains of maize (Zea mays L., Pioneer 3343) were soaked overnight in running tapwater and placed between wet paper towels on opaque plastic trays. The trays were placed in a vertical position with the grains aligned along the vertical axis. The seeds were germinated at 30 C and used when the primary root is 1- Figure 1. Pretreatment of Maize Roots with 1 mM Lithium 1.5 cm in length. Chloride and exposure to 0.1 nM Auxin. (n=16)
Gravitational and Space Biology 18(2) June 2005 119 T. Mulkey – Lithium in Growth and Gravitropic Response
Figure 4. In vitro Protein Phosphorylation of Cytoplasmic Figure 2. Pretreatment of Maize Roots with with 1 mM Lithium Fractions from Corn Roots. This gel illustrates the effect of the Chloride and exposure to 1.0 µM Auxin. (n=16) interaction of calcium, EGTA, auxin and lithium on protein phosphorylation patterns of the cytoplasmic fraction isolated min and 0.1 nM auxin is added at 120 min. Note the 30- from maize root. Description of the treatments for each lane 35% increase in elongation rate after the addition of can be found in the caption for Figure 3. auxin. The dash line illustrates the effect of adding IAA to roots at 120 min without previous exposure to lithium. Differences in the protein phosphorylation pattern in Figure 2 illustrates the effect of 1 mM lithium chloride on both fractions are evident. Phosphorylation of membrane response of roots to 1.0 µM auxin. The solid line polypeptides in the 14 to 22 kDa range increased under indicates that 1 mM lithium is added at 60 min and 1.0 conditions that promote elongation and were suppressed µM auxin is added at 120 min. The dash line illustrates when elongation was inhibited. These effects are not due the effect of adding IAA to roots at 120 min without to an ion effect but appear to be a lithium specific previous exposure to lithium. This treatment produces a response. This preliminary data suggests that at least part very strong inhibition (90-95% of the control rate) of of the effects observed with lithium and auxin treatment elongation. These results are similar to the interactions may be mediated through a second messenger system. observed between ethylene biosynthesis inhibitors and REFERENCES auxin previously reported Mulkey et al. (1982a, 1982b). Boyer, N. M.O. Desbiez, M. Hofinger, and T. Gaspar. 1983. Figure 3 and 4 show preliminary data on the effects Effect of lithium on thigmomorphogenesis in Bryonia dioica. of lithium and auxin on protein phosphorylation pattern. Ethylene production and sensitivity. Plant Physiol. 72:522-525. In the first lane of each panel is the control, the fourth lane is 1 mM Lithium, lane G is 1 mM lithium and 1 µM Brami, B.A., U. Leli and G. Hauser. 1993. Elevated phosphatidyl-CMP is not the source of diacylglycerol auxin, and lane K is 1 mM lithium and 0.1 nM auxin. accumulation induced by lithium in NG108-15 cells. Jour. Neurochemistry 60:1137-1142.
Desbiez, M.O., N. Boyer and T. Gaspar. 1981. Hypocotyl growth and peroxidases of Bidens pilosus. Effect of cotyledonary prickings and lithium pretreatment. Plant Physiol. 68:41-43.
Gumber, S.C., M.W. Loewus, and F.A. Loewus. 1984. Further studies on myo-inositol-1-phosphatase from the pollen of Lillium longiflorum Thunb. Plant Physiol. 76:40-44.
Lee, M.S. and B.G. Kang. 1987. Action of lithium ions on ethylene biosynthesis in hypocotyl segments of Vigna radiata. Korean Biochem J. 20:117-121.
Figure 3. In vitro Protein Phosphorylation of Membrane Morse, M.J., R.C. Crain, R.L. Satter. 1987. Fractions from Corn Roots. This gel illustrates the effect of the Phosphatidylinositol cycle metabolites in Samanea saman interaction of calcium, EGTA, auxin and lithium on protein pulvini. Plant Physiol. 83:640-644. phosphorylation patterns of the membrane fraction isolated from maize root tips treated with: A = Control; B = EGTA (5 Mulkey, T.J., K.M. Kuzmanoff and M.L. Evans. 1982a. mM); C = Calcium (1 mM); D = Lithium (1mM) + Calcium (1 Promotion of growth and hydrogen ion efflux by auxin in roots mM); E = Lithium (1mM) + EGTA (5 mM); F = Lithium (1mM) of maize pretreated with ethylene biosynthesis inhibitors. Plant + Calcium (1 mM) + IAA 10-6 M; G = Lithium (1mM) + IAA 10- Physiol. 70:186-188.
6 M; H = Lithium (1mM) + EGTA (5 mM) + IAA 10-6 M; I = -10 Mulkey, T.J., K.M. Kuzmanoff and M.L. Evans. 1982b. Lithium (1mM) + Calcium (1 mM) + IAA 10 M; J = Lithium Promotion of growth and shift in the auxin dose/response (1mM) + EGTA (5 mM) + IAA 10-10 M; K = Lithium (1 mM) + -10 relationship in maize roots treated with the ethylene biosynthesis IAA 10 M; L = Lithium (1 mM) + Calcium (1 mM) inhibitors aminoethyoxyvinylglycine and cobalt. Plant Science Letters 25:43-48. 120 Gravitational and Space Biology 18(2) June 2005
GRAVITY AND LIGHT: INTEGRATING TRANSCRIPTIONAL REGULATION IN ROOTS 1* 1 1 1 1 1,2 Raul Salinas-Mondragón , Anna Brogan , Nicholas Ward , Imara Perera , Wendy Boss , Christopher S. Brown 1 Heike Winter Sederoff 1 2 Plant Gravitational Genomic Group, Dept. Botany, Kenan Institute for Engineering, Technology & Science, North Carolina State University, Raleigh NC 27695 USA * corresponding author: [email protected]
Light and gravity give clues about time and space which combined stimulation: Dark-grown plants which were plants use to direct their growth. Roots grow towards the gravity stimulated either in the dark or after 15min or 1h vector of gravity (positive gravitropism) to access water of light exposure and compared to vertical controls (Fig and nutrients in the soil and to provide stability. Their 1). Plants were harvested by adding RNAlater (Ambion) response to light depends on its wavelength and intensity. and the root apices (ca. 7 mm) separated. RNA extraction Roots grow towards red light (positive phototropism) and (Qiagen), cDNA synthesis (Ambion) and Real Time PCR away from blue and white light (negative phototropism) (Sybr Green) were carried out as described by Kimbrough (Kiss et al. 2003). Plants respond to these signals via et al. 2004. differential cell elongation which results in directional growth. Light and gravity are simultaneous stimuli so Our first experiment was to perform Real Time PCR to plants must respond to both at the same time and integrate confirm our microarray data (Kimbrough et al. 2004), them into a single response. Our hypothesis is that comparing the wild type and our transgenic (t2-8). We integration of light and gravity responses in roots can be found that gravity induces fast and transient changes in observed at the level of gene expression. One of the transcript abundance of specific genes as shown by fastest known signal transduction elements described for microarray experiments of Arabidopsis root apices in gravity and light responses is inositol- 1,4,5 triphosphate comparison to mechanical stress (Kimbrough et al. 2004). (InsP3). Cellular levels of InsP3 increase within 15s to 30s For some nuclear genes, these transcriptional changes in response to stimulation by gravity or light, respectively occur within one minute as shown by real-time PCR (Morse et al. 1987; Perera et al. 1999). We therefore analysis. Interestingly the transcripts that were fast and analyzed light induced changes in transcript abundances transiently up regulated by gravity stimulation in the dark, of genes known to respond specifically to gravity in roots did not show any significant changes in the transgenic of wild type and transgenic plants dampened in their plants with dampened InsP3-signaling (Fig. 2). InsP3-signaling (Kimbrough et al. 2004; Perera et al., in preparation.).
Using whole genome Arabidopsis GeneChips (Affymetrix), we monitored the time course of gravity- induced transcript abundance changes of 22,744 genes in the root apices of wild type and transgenic Arabidopsis seedlings expressing inositol-polyphosphate-5- phosphatase (T2-8) (Kimbrough et al. 2004; Salinas- Mondragon et al. in preparation). Inositol polyphosphate 5-phosphatase specifically hydrolyzes the 5’-phosphate of
InsP3 and leads to substantially reduced levels of InsP3 in the expressing transgenic plants (Perera et al., in Figure 1. Experimental set-up to study the combined stimulation preparation). Several of the genes that exhibited gravity of gravity and light. C0 is the vertical control pre-adapted to specific transcript abundance changes in wild type plants green light (18 h). C1 and C2 are the controls after 15 minutes were not up-regulated in response to gravity stimulation or 1 hour of incandescent light stimulation respectively, prior to in the transgenic plants (Salinas-Mondragon et al. in gravistimulation. preparation). This indicates that the gravity-induced regulation of these genes is mediated by and dependent on InsP3. This makes the transcript abundance of these genes This suggests that gravity induced changes in transcript a quantifiable marker for InsP3-regulation and integration abundance of at least some genes are InsP3-mediated. of other environmental stimuli. Five of the fastest gravity specific genes, that we previously selected from the microarray data (At5g48010, In all the experiments we used 7-day-old dark-grown wild AtPEN1; At2g16005, expressed protein; At4g11310, type and transgenic Arabidopsis seedlings (t2-8). Light cysteine protease; At5g38020, SAMt-homologue; regulation of the transcript abundance changes for At4g23670, major latex related protein), also show a fast specific genes was analyzed using exposure to directional and transient up regulation in the wild type, under (seedlings were approximately 50 cm from the source of directional incandescent light. The levels of these light) incandescent light (2 µmol s-1m-2 or approximately transcripts in the transgenic (t2-8) plants were not up- 100 lux). The interaction of light and gravity induced regulated in response to light stimulation. (Fig. 3). transcriptional regulation was investigated using Gravitational and Space Biology 18(2) June 2005 121 R. Salinas-Mondragón et al. – Gravity and Light: Integrating Transcriptional Regulation in Roots A sequential combination of gravity and light stimulation phosphatase, we were able to show that the integration of and the subsequent analysis of transcript transcriptional regulation by light and gravity stimulation abundances showed that a short period of light (15 min) is dependent on InsP3-mediated signal transduction. has a synergistic effect on gravity induced transcriptional Furthermore we show that light has a synergistic and regulation (Fig. 4). This effect is transient and mediated transient effect on the transcript abundance of very early by InsP3 because no transcriptional changes for those gravity induced genes. It has been shown that genes were detected in the transgenic plants (t2-8). phytochrome mediated light signal transduction enhances root gravitropism (Feldman and Briggs, 1987). Overall, the data presented here may suggest that InsP3 and the increase of those very early and transient transcripts may function in gravi-and photofacilitation, a concept recently proposed by LaMotte and Pickard (2004).
Figure 2. Transcript abundance of At2g16005 increases more than 3-fold in gravity stimulated root apices in the dark within 1 Figure 4. Transcript abundance of At5g38020 increases rapidly min. after reorientation but showed no up-regulation in the same and transiently after gravity stimulation in the dark. A short time-span in gravity stimulated transgenic (t2-8) plants. light exposure (15 min.) enhances its response to gravity, while a 1h light exposure prior to gravity stimulation abolishes its up- regulation.
REFERENCES
Feldman, L. J. and W. R. Briggs (1987). "Light-regulated gravitropism in seedling roots of maize." Plant Physiol. 83: 241-3.
Kimbrough, J., R. Salinas-Mondragon, W. F. Boss, C. S. Brown and H. Winter Sederoff. "The fast and transient transcriptional network of gravity and mechanical stimulation in the Arabidopsis root apex.“. Plant Physiol. 2004 Sep; 136 (1): 2790 – 805.
Kiss, J. Z,. Correll, M. J., Mullen, J. L., Hangarter, R. P. and Edelmann, R. E. “Root phototropism: how light and gravity interact in shaping plant form”. Gravitational and Space Biology Bulletin. 2003 16 (2): 55 – 60.
LaMotte, C. E. and B. G. Pickard (2004). "Control of gravitropic orientation. II. Dual receptor model for gravitropism." Functional Plant Biology 31(2): 109-120.
Figure 3. Light induces changes in transcript abundance in the Morse, M. J., Crain, R.C., Satter, R.L. (1987). "Light-stimulated inositol same genes that were identified as fast and transiently up- phospholipid turnover in Samanea saman leaf pulvini." Proc Natl Acad Sci U S A 84: 7075-7078. regulated genes after gravity stimulation in wild type plants.
Perera, I. Y., I. Heilmann and W. F. Boss (1999). "Transient and CONCLUSION sustained increases in inositol 1,4,5-trisphosphate precede the differential growth response in gravistimulated maize pulvini." Proc Natl Acad Sci U S A 96 (10): 5838-5843. Gravity stimulation induces fast and transient increases in transcript abundance in specific genes in Arabidopsis root apices in the dark. When light-grown plants were gravity stimulated, the abundance of these transcripts did not change. However, a short light stimulus transiently enhanced gravity induced transcriptional changes. Using transgenic plants dampened in their InsP3-mediated signaling, by over-expression of inositol polyphosphate 5- 122 Gravitational and Space Biology Bulletin 18(2) June 2005
Author Index
Abukhalaf, I.K., 109 Horn, E., 95
Baillie, D., 11 Jackie Duke, P., 115 Baker, D.A., 17 Johnsen, R., 11 Baker, T.L., 83, 99 Jones, L.B., 83 Barnstable, C.J., 97 Joseph, J., 71 Becker, J., 99 Beckingham, K.M., 17 Kakavand, A., 93 Bhattacharya, S., 93 Kennedy, R.J., 87 Blustein, D., 81 Kimbrough, J.M., 117 Boss, W., 121 Kirven-Brooks, M., 91 Bradamante, S., 89 Klement, B.J., 109 Broadaway, S.C., 85 Kulkarni, A.D., 101 Brogan, A., 121 Brown, C.S., 117, 121 Lam, K., 91 Butterfield, T., 113 Lambert, C.A., 103 Lapière, C.M., 103 Callejas, W., 115 Leskovsky, D., 93 Cantero-Garcia, A., 113 Licata, A.A., 39 Cavanagh, P.R., 39 Love, J.E., 83, 99 Clark, G., 113 Ly, D., 105 Cohen, J., 105 Convertino, V.A., 59 Maier, J.A.M., 89 Marsh, M., 115 Das, G.C., 83 Massa, G.D., 87 Dauwalder, M., 113 Mick, M.E., 87 Davis, K.L., 111 Mineur, P., 103 Deroanne, C., 103 Mitchell, C.A., 87 Doan, L., 115 Mitchell, C.D., 109 Douglas Armstrong, K., 17 Morrow, R.C., 87 Dutt, K., 109 Mulkey, T.J., 119 Munjaal, R., 17 Elliott, T.F., 83, 99 Murakami, N., 107 Emmerich, J.C., 87 Nusgens, B., 103 Fahlen, T., 91 Feldman, L., 111 Park, H., 107 Forsburg, S.L., 3 Pellis, N.R., 101 Perera, I., 121 Girten, B., 105 Pulcini, E. deL., 85 Guadarrama, S., 85 Pyle, B.H., 85 Gyotoku, J-I., 107 Rabin, B.M., 71 Hammond, D.K., 83, 99 Ramesh, G.T., 101 Herrera, A., 91 Ramos, R.A., 91 Hinkle, N., 81 Rask, J., 91 Holubec, K., 99 Reiss-Bubenheim, D., 91 Gravitational and Space Biology 18(2) June 2005 A - 1 Author Index
Rice, A.J., 39 Takaoki, M., 107 Rose, A., 11 Texada, M.J., 17 Roux, S.J., 113 Todd, P., 71 Tombran-Tink, J., 97 Salinas-Mondragón, R., 121 Sanchez, M.E., 93 Versari, S., 89 Sato, K., 91 Villa, A., 89 Schmäh, M., 95 von Deutsch, A.W., 109 Schwarzkopf, R.J., 83 von Deutsch, D.A., 109 Servotte, S., 103 Shenasa, M., 93 Ward, N., 121 Shiba, D., 107 Williams, C.E., 109 Shukitt-Hale, B., 71 Winter Sederoff, H., 117, 121 Silvestrov, N.A., 109 Sing, L., 91 Xu, K., 111 Smith, A., 81 Sonnenfeld, G., 31 Yamauchi, K., 101 Stowers, R.S., 93 Yu, D., 105 Sundaresan, A., 101 Sunga, J., 91 Zhao, Y., 11 Sytkowski, A.J., 111
A - 2 Gravitational and Space Biology 18(2) June 2005