The NIH Catalyst from the Deputy Director for Intramural Research

Fostering Communication and Collaboration

The nihCatalyst A Publication for NIH Intramural Scientists

National Institutes of Health a Office of the Director Volume 3, Issue 5 September-October 1995

Tenure Panel Intersecting Orbits: Gets First-Year NIH and NASA’s Joint Explorations Report Card

by Celia Hooper by Rebecca Kolberg

IH’s new Central Tenure Com- uter space may remain far Currently, two NIH labs —one at mittee recently completed its beyond NIH’s scientific reach NICHD and one at NEI—are weighing N first year of life—and the first O for quite some time, but some the potential applications of NASA- data on tenure decisions are in, along intramural researchers are taking advan- developed technologies in basic with mixed reactions from the scien- tage of the next best research. >• biomedical tists that the group evaluates and thing—a chance to | Not only do the two serves. explore the earth- projects tackle quite “We haven’t lived with the new | bound applications of 3 different scientific tenuring system long enough to see if technology designed problems, but they it produces a better product” than the for the final frontier. also diverge in the old system, says Mark Boguski, a NLM “The nice thing nature of the devices investigator recently tenured by the about working with being tested and in NIH is that the rest of the formality of their the scientific commu- arrangements with the nity looks to NIH for federal space agency. guidance, and if use- NICHD’s evaluation of ful results are reported NASA’s bioreactor for

by NIH researchers, three-dimensional tis-

the technology starts sue culture is a five- to spread,” says year, $4.8 million Stephen Davison, a interagency agree- biotech program man- ment. On the other ager with NASA’s NICHD's Joshua Zimmerberg, hand, NEI and NASA Microgravity Science foreground, and Leonid Margolis scientists' fledgling and Applications Divi- assemble a NASA bioreactor. collaboration to ex- in sion Washington. continued on page 18 . Central Tenure Committee (CTC). “The NIDCD Director James B. Snow Jr., real test will be not just whether the who was appointed NIH’s representa- system is fair at the front end but CONTENTS tive to NASA’s Life Sciences and Micro- whether at the back end it produces a 2 10-11 gravity Advisory Committee last spring, scientist with a better post-tenure track From the DDIR Commentary: record than before.” agrees that NIH and NASA make com- New Genetic Clues 4 To Hearing Impairment Actually, the idea for the committee patible partners in many areas of bio- Science Ethics Forum: was hatched for rather different rea- medical research. “Research on Earth New Intramural Panel 12-13 sons in the 1980s, under NIH Director could benefit from the application or Hot Methods Clinic: 5 More Than James Wyngaarden, says CTC’s execu- transfer of technologies specifically Research Grapevine & Just the FACS tive secretary, Richard G. Wyatt. The developed for space-related purposes, Interest Group Gazette goal of forming the new tenure com- in 14 and research space or space-like 6-7 mittee, according to Wyatt, was to Lab Behind the Leader environments could improve knowl- Klausner at NCI, bring the expertise of senior scientists edge of the normal function of human A Whale of a Job 22 into major decisions at NIH. As is the Awards biologic systems on Earth,” Snow told 8-9 case at universities, tenure at NIH this year’s American Institute of Aero- Scientific Cybernauts: 23 accords a scientist permanence and Speeding Up Searches nautics and Astronautics’ Life Sciences Cartoons independent responsibility for labora- and Space Medicine Conference in 24 continuecl on page 15 . Houston. Catalytic Reactions The NIH Catalyst From the Deputy Director for Intramural Research

Safety and Security at NIH

he recent contamination of a water cooler in vidual scientist if a few thousand counts of hydrogen-3, Building 37 with phosphorus-32 has raised phosphorus-32 (P-32), or carbon-14 are left contaminat- important issues about how we protect staff. T NIH ing a lab bench, or if a researcher wants to mouth-pipet Although we are a diverse community, spanning multi- Escherichia coli carrying a recombinant plasmid encod- ple campuses and a wide range of professions, we ing human cDNA sequences? First, such activities repre- share a desire to minimize threats to our safety and sent bad laboratory practice, which could lead to slop- security. The tough question is, how can we create a py handling of more hazardous materials or organisms. safe, secure work environment without destroying the Second, perception and acceptance of risk is very per- open intellectual atmosphere essential to biomedical sonal. Because we work in a crowded environment, research? one person’s carelessness invariably results in the expo-

In my view, there are two general categories of safe- sure of others, and it is inappropriate for one researcher ty and security risks. First, there are risks such as fires to decide whether others should be exposed to ques- and chemical spills that pose an immediate danger to tionable material—no matter how small the risk. Finally, the health and well-being of NIH staff. There is little deliberate or careless violation of rules and regulations controversy about the importance of minimizing or regarding “low-level” risk subjects all of NIH to the pos- eliminating such risks. The second set of risks such as sibility of public censure and harsh regulatory sanctions

recombinant DNA activities and exposure to low-level that could make it far more difficult to conduct our dai- radioactivity—risks that do not constitute an immediate ly work. threat to health or safety—is more problematic. The recent, apparently deliberate, P-32 contamina- In the first category, the danger of exposures to fire, tion of a water cooler and perhaps of a scientist’s food toxic chemicals, pathogenic organisms, and high-volt- or drink in Building 37 illustrates some of these points. age equipment is a fact of life in the modern laboratory. Although the exposures should not pose a health risk to Fortunately, scrupulous use of appropriate safety equip- any of the staff involved, the attendant negative publici- ment and precautions, proper training, and mainte- ty led to questions about our security and handling of nance of clear corridors can greatly reduce the chance radioisotopes and demonstrates the potential price of

of lab accidents and facilitate emergency response problems in this arena. I cannot overemphasize the when accidents do occur. emotional distress experienced by the affected individu- A different sort of concern in the first category is the als, the exacerbation of mistrust within our local com- threat of criminal acts such as theft, personal assaults, munity, and the intensity of demands that NIH “do and violent action by malicious individuals or groups. something” to prevent such an event from recurring. Although rare, such events do occur at NIH. To guard Last year, NIH suggested to the Nuclear Regulatory against crime, NIH police patrol the campus and we all Commission (NRC) that security governing the storage take precautions such as locking unoccupied labs, limit- and use of certain radioisotopes, including P-32, be ing access to NIH buildings after normal working hours, relaxed to facilitate their use within labs. When the P-32 and controlling access to buildings that house nonhu- contamination occurred on June 28, NIH was undergo- man primates. Importantly, any steps to tighten security ing an NRC inspection to determine, at least in part, the are taken only after the risks are weighed against the effectiveness of our security arrangements for radioiso- possibility that tougher security measures will interfere topes and whether our request for less stringent restric- with normal research activities. Currently, there is no tions should be granted. Although our request was

plan to lock all NIH buildings during working hours based in good faith on known risks, it did not take into because the need for such action has not been shown account the dramatic nature of any contamination with to outweigh the high cost of hiring enough security radioactive materials and the emotional reaction to such

guards to provide “true” security and because it would contamination. I believe that our decision to withdraw significantly interfere with the normal flow of people that request, to strictly enforce current security regula- and research materials. Our best defense against crime tions, and to search scrupulously for other possible con- is for everyone to be vigilant, for example, by question- tamination—with only negative results so far—has been ing strangers about the nature of their business in NIH a reasonable response to the P-32 case. In fact, the NRC buildings and reporting suspicious or criminal activity to recently gave us high marks for the overall quality of police immediately. our radiation-safety program. The second category of potential risks at NIH Although we may wish to govern ourselves by safe includes factors that do not appear to pose an immedi- and appropriate research practices, the reality is that

ate danger but that, over a period of time, may result in NIH is governed by oversight bodies such as the NRC, a statistically detectable hazard. In some cases, the the Occupational Safety and Health Administration, and long-term health risks are unknown or indeterminable, the Environmental Protection Agency. One of my but a reasonable person might perceive such a risk, or responsibilities is to enforce safety regulations, but

there might be public concern about the possibility of another is to explain to regulators the special circum- such risk. Activities that fall into this category are exper- stances that affect NIH research activities. The good iments involving recombinant DNA research on non- working relationship that we have with regulatory agen- pathogenic organisms and gene products and the use of cies today is due in no small part to the reputation that low-level radioisotopes. Many researchers question NIH has developed as an institution that is responsive

rules and regulations in these risk areas that they think to public concerns. I hope that everyone will work with serve no useful purpose. me to maintain and foster this good reputation. However, recent events underscore the importance of observing all guidelines and regulations for this risk Michael Gottesman

category. You may ask, what does it matter to an indi- Deputy Directorfor Intramural Research

2 ?

September-October 19 9 5

Catalytic Reactions

Below are comments we received for topics raised in the July- reduced exposure to second-hand smoke. The cost for smokers

August issue, along with some general reactions. is some inconvenience, personal responsibility to minimize lit- tering, and exposure to the elements in inclement weather. Discussion of further smoking restrictions on the NIH cam- On ethics column on “Authorship and Ownership’’ pus leads to a more general policy question. Should NIH, a Thank you for writing a clear article on authorship and owner- renowned center for health-related medical research, be a per- ship. It was the first time that I can recall hearing about the missive partner with smokers? Or should NIH enforce a strict Guidelines for the Conduct of Research in the Intramural interpretation of the federal policy to provide a smoke-free, Research Program. Is this available in the NIH Library, or do I drug-free work environment and seek to reduce and eventually need to look elsewhere? eliminate smoking on campus? The state of Maryland and —Bill Bennett, NCI the American Medical Association have taken activist roles to reduce smoking, particularly in public places, and FDA is currently examining the classification of tobacco as a drug- I’ll be happy to send you a copy of the research conduct gu idelines. delivery system. NIH could also establish an active leadership Other intramural researchers who are interested in obtaining a role by reducing exposure of employees to toxic materials (e.g., copy of the guidelines should contact the Office of Intramural tobacco smoke) and by helping its employees eliminate habits, Affairs (phone: 496-3561). such as smoking, that are linked to debilitating disease. NIH —-Joan P. Schwartz, NINDS could achieve these goals in a manner that does not infringe on personal choice or privacy, perhaps, for example, by estab- On a “burning issue” at NIH lishing programs to help interested people break these habits, Federal regulations forbid smoking inside NIH buildings. Smok- and also by offering reduced health- and life-insurance rates ing is, however, permitted on the NIH campus, and employees for nonsmokers. taking a “smoke break” tend to cluster around the entrances of —Gerry Dienel, NIMH the buildings, thereby exposing everyone who enters and -leaves the building to second-hand smoke. Smoking around the On the National Institutes of Dent

Clinical Center entrances was prohibited as of July 1, and there Dent is a genius. The toiling, the drudgery, the cynicism—our is further discussion about whether the no-smoking zones world, captured in detail. Dare I say that his cartoon strip is the should be extended to include all entrances to all buildings on best thing in The Catalyst campus. The direct benefit to all NIH employees and visitors is —Daniel Fierer, NIAID

Calendar Convenience Catalyst Mailing List

It’s happened again. You’ve misplaced your Yellow Sheet, We are working toward improving our distribution system and that interesting seminar you planned on attending this for The NIH Catalyst. Over the past .year, many labs and afternoon turns out to have taken place yesterday. Maybe offices have moved to other locations at NIH, and we are

it’s finally time to enter the computer age and sign up for an doing our best to keep the mailing list up-to-date. If you electronic subscription to NIH’s weekly Calendar of Events. have recently moved and want to remain on our mailing

To receive the calendar via e-mail, send an e-mail message list, let us know. Intramural researchers arriving at or leav- to listserv@list. nih.gov with the message “SUBSCRIBE CAL- ing NIH should also contact us to be added to or deleted ENDAR Your Name”. from the mailing list (phone: 496-0450; fax: 402-4303; e-mail: [email protected]). n

Safety and Security at a Glance

If you have questions about course work on laboratory Correction safety" or other concerns about the safe use of chemicals or biological materials in the lab, contact Deborah Wilson at The Interinstitute Interest Group Directory, pages 12-13 of the Occupational Safety and Health Branch (phone: 496- the July-August issue, contained an incorrect e-mail address 2960). For nonemergency questions about fire hazards or for Janet Yancey-Wrona, who is the contact for the Nucleic regulations, contact the NIH Fire Department (phone: 496- Acid Biochemistry Interest Group. Yancey-Wrona’s correct 2372) or John McCabe with Fire Prevention (phone: 496- e-mail address is [email protected] 0487). For nonemergency questions about crime risks or other security issues, contact Patrick Coajou with the NIH Police (phone: 496-5685).

3 The NIH Catalyst

Science Ethics Forum

New Ethics Panel byjoan p. Schwartz, ph.o., ninds Seeks Scientists’ Input

n an effort to encourage NIH to develop and refine guidelines for the NIH Committee on Scientific researchers to adhere to high ethical conduct of research, including proce- Conduct and Ethics Istandards and to ensure that allega- dures to protect both whistle-blowers Chair tions of scientific misconduct are handled and scientists accused of scientific mis- Joan Schwartz, NINDS Members impartially and expeditiously, the Office conduct, to develop a model for binding Richard Asofsky, NIAID of Intramural Research (OIR) has estab- arbitration, and to determine areas in Bruce Baum, NIDR lished an Committee additional NIH which guide- Peter Blumberg, NCI on Scientific Conduct and lines may be needed Jane Cheng, NCI Ethics, which is com- The OIR feels (e.g., mentorship), Sue Cheng, NINDS of scientists from to Ted Colburn, NIAAA posed STRONGLY THAT THIS develop effec- most of the institutes, tive mechanisms for Robert Desimone, NIMH Andrew Dwyer, CC centers, and divisions. COMMITTEE CAN MAKE ethics training in Victor Ferrans, NHLBI The OIR feels strongly the NIH scientific com- AN IMPORTANT CONTRI- James Fozard, NIA that this committee can munity—including this BUTION TO THE NIH David Gorelick, NIDA make an important contri- ethics column, and Christine Grady, NINR bution to the NIH scientif- SCIENTIFIC COMMUNITY. to develop mecha- Betty Graham, NCHGR ic community. nisms to deal rapidly Victoria Hampshire, NCRR As chairman of this and fairly with allega- Christy Ludlow, NIDCD Ron Mason, NIEHS committee, I welcome all suggestions tions of scientific misconduct and with Ralph Nossal, DCRT from NIH staff for topics and issues you disputes related to authorship, sharing of John O'Shea, NIAMS would like to see addressed by our panel. data and reagents, mentoring, supervi- Alan Schechter, NIDDK The committee, which held its first meet- sion, and other conflicts in the scientific John Wilbur, NLM ing on Sept. 14, has three basic charges: workplace. Peggy Zelenka, NEI n

Wednesday Afternoon Lectures: Back by Popular Demand

he popular Wednesday After- top lectures by scheduling the talks in Center’s Office of Special Events noon Lecture Series returned for an easily accessible location and in a (phone: 594-5595). a second season on Sept. with T 13 standard time block that scientists can In a new development, all lecture a presentation by famed epidemiologist set aside on their calendars. As in the hosts wall try to set aside some of the Charles Hennekens of Harvard Medical 1994-95 series, most of this season's speakers’ time to meet with interested School in Boston. And the rest of the lectures will again be held on Wednes- students, fellows, and postdocs. Young lineup promises to rival the 1995-96 days from 3 to 4 p.m. in Masur Auditori- scientists who want to participate in inaugural series, which attracted some um, Bldg. 10. The Office of Education these meetings, please contact the head of the world’s most fascinating grants continuing medical education of the host group as soon as possible to researchers to the Bethesda campus. (CME) credits to lecture attendees. For reserve a spot. Interest-group contacts The series was started last year in an more information on the lecture series, are listed in the July-August issue of effort to improve attendance at NIH’s contact Hilda Madine of the Clinical The NIH Catalyst.

Oct. 18 Peter Kim Nov. 15 Elaine Fuchs Whitehead Institute for Biomedical Research, University of Chicago Cambridge, Mass. “Of Mice and Men: Cytoskeleton and Disease” ''Design of Proteins and Drugs” Host: Cell Biology Interest Group Host: NIGMS Nov. 20 Phillip Sharp Oct. 25 Carlos Bustamante Massachusetts Institue of Technology, Cambridge University of Oregon, Eugene “RNA Splicing and Biology” “Imaging Protein-Nucleic Acid Complexes with the Hosts: NIAID and the NIH Fellows Committee Scanning Force Microscope” Nov. 22 John Robbins Host: Structural Biology Interest Group NICHD Nov. 1 Thomas Kunkel “Something Old and Something New; Something NIEHS Borrowed and Some Things Yet to Do” "DNA Replication Fidelity, Mismatch Repair, and NIH Dyer Lecture (OD) Genome Stability” Nov. 29 Richard Anderson NIH Mider Lecture (OD) University of Texas Southwestern Medical Center, Nov. 8. Christopher Walsh Dallas Dana Farber Cancer Institute, Boston “Compartmentalization of Signal Transduction in “Molecular Mechanisms for Bacterial Resistance to Caveolae” the Antibiotic Vancomycin” Hosts: Cell Biology and Signal Transduction Host: Molecular Biology Interest Group Interest Groups

4 . .

September-October 19 9 5

Research Grapevine

We welcome your contributions to On the clinical front, James Cook of the of the amino acid homocysteine in the this new feature, which is intended University of Kansas Medical Center in blood to an elevated risk of coronary to provide the intramural research Kansas City discussed the application of artery disease by increasing thrombogenic commu nity with the latest news from the transferrin-receptor assay for assessing tendencies. He noted that the prevalence scientific meetings in a wide range of a patient’s iron status. Traditionally, the of artery narrowing among patients in the fields. For information on submit- serum-transferrin-receptor assay has been Framingham Heart Study was found to ting a brief update on a meeting that used to gauge erythropoiesis. But Cook correlate directly with serum homocys-

you have attended , contact The reported that this test may also be used to teine levels, with patients who had the Catalyst (phone: 402-1449; fax: 402- distinguish iron-deficiency anemia from highest homocysteine levels being twice 4303; e-mail: catalyst@od 1 em 1 the anemia produced by chronic disease. as likely to have advanced arteriosclerosis od.nih.gov). The assay found that iron-deficiency-ane- as those with average levels. Other stud- mia patients had serum transferrin-recep- ies indicate that excessive levels of homo- American Association for tor levels that were three times higher cysteine may dismpt the anti-coagulation Clinical Chemistry than normal, while patients with chronic- process, thereby predisposing individuals disease anemia had normal levels of to thrombotic heart disease. In addition, olecular diagnostics, automa- transferrin receptors. These results indi- Jacob says there’s experimental evidence tion, and point-of-care testing cate that the serum transferrin-receptor that B vitamins, particularly folic acid, M were the themes of the Ameri- assay may be a valuable substitute for may help to lower homocysteine levels. can Association for Clinical Chemistry’s bone-marrow examination, which has In addition to their implications for the annual meeting July 16-20 in Anaheim, been the standard method of distinguish- treatment and prevention of thrombotic Calif. In a fitting tribute to the theme ing between these two types of anemia. heart disease, the homocysteine findings of molecular diagnostics, NCHGR The role of homocysteine in coronary help to underscore tire often overlooked Director Francis Collins received the artery disease was the focus of a presenta- fact that coronaiy artery disease is a multi-

AACC's National Lectureship Award tion by Robert Jacob of USDA’s Western factorial disorder and is not solely deter- and delivered a superb talk entitled Human Nutrition Research Center in San mined by cholesterol concentrations, a “The Human Genome Project and the Francisco. Jacob provided an update on Future of Medicine.” recent studies linking high concentrations -Ronald Elin, CC

The Interest Group Gazette

IH’s family of interinstitute inter- expertise. Members are informed by epidemiological manifestations, and its est groups has grown consider- fax and/or e-mail of all official group prevention. Umberto Saffiotti, the group’s Nably over the past year, and more activities and other useful news. For more organizer from NCI’s Laboratory of new members continue to be added to information, contact Vincent Hearing Experimental Pathology, plans to have the fold. Here are some details about (phone: 496-1564; fax: 402-8787; e-mail: three to four meetings per year at NIH’s four of the most recent arrivals, three of [email protected]). Bethesda campus and, possibly, some at which are in the early, planning stages The Lymphoma and Leukemia NIEHS at Research Triangle Park. N.C. that rely heavily on the involvement of Interest Group, organized by Ivan For more information, contact Saffiotti rank-and-file scientists. Horak of NCI’s Metabolism Branch, held (phone: 496-2818; fax: 402-1829; e-mail:

Since it was established at the begin- its first meeting in September and plans saffiotu@ dce4 1 . nci . nih gov) . ning of this year, the Pigment Cell to meet from 2 to 3 p.m. on the second The first organizational meeting of the Research Interest Group has attracted Monday of each month in the Bunim Virology Research Interest Group will about 50 active members. The group, Conference Room, 9S-235, Bldg. 10. This be held on Nov. 9 from 2 to 3 p.m. in which serves as a forum for scientists group is devoted to the biology and ther- Bldg. 4, Rm. 433/437. All NIH scientists from a wide variety of disciplines interest- apy of lymphoma and leukemia. The in the field of virology are invited to

ed in the study of pigment cells, holds Nov. .13 meeting will feature a presenta- attend. The planning of future meetings infonnal and interactive seminars on the tion by NCI’s Jonathan Ashwell on the and seminars will take place during the third Monday of each month from 3:00 to regulation of normal and pathological first meeting, and there will also be a dis- 4:30 p.m. in Bldg. 37, Rm. 6B23. Interests apoptosis of T cells. Organizers plan to cussion of additional activities aimed at include the development, growth, differ- notify members of group activities via enhancing the scientific and social entiation. and function of melanocytes, as the NIH Calendar of Events and e-mail. interactions among virologists within well as what causes some melanocytes to For more information, contact Horak and around NIH. Researchers who are be transformed and grow into primary (phone: 594-1127; fax: 402-3647; e-mail: unable to attend but would like to malignant melanoma tumors and, eventu- [email protected]). Also in the for- convey their interests and ideas should ally, to metastasize. Typically, group mem- mative stage, the Carcinogenesis Inter- contact Bernie Moss of NIAID’s Laborato-

bers make the presentations, but occasion- est Group intends to hold its first infor- ry of Viral Diseases (fax: 480-1147; ally, outside speakers are invited to partic- mal meeting in November. The group’s e-mail: [email protected]).

ipate. The goal of the group is to foster purpose is to discuss and understand the

synergistic interactions among researchers process of carcinogenesis, as well as its -Lorna Heartley

who have complementary interests and/or causes and mechanisms, its clinical and

5 — —. The NIH Catalyst

Rousing the Sleeping Leviathan, NCI’s New Leader Gets Down to Business

hat’s huge, innately magnifi- importance of service and the essential instability and the cell cycle, and the rela- cent, and could use a strong perspective that scientists bring to scientif- tionship between genetic instability and Wshove to get back on course? ic leadership. the fundamental decision between life and The man on the street might answer, “A death that cells are capable of making. . . beached whale.” Around NIH, the WPat do you think is the toughest I expect that we will be developing a very response might very well be, “NCI.” In an task currently confronting NIH? vigorous and active cancer genetics pro- interview with Tloe NIH Catalyst shortly Klausner: There’s no question that the gram in terms of basic, clinical, and epi- after being sworn in as NCI’s new director, toughest task is the challenge of these demiological studies. I think the possibility

Richard Klausner made it clear that his incredible diseases [cancers] that present of really integrating clinical, basic and leadership team will waste no time in flex- such a daunting problem. Cancer will be population-based studies here provides ing its collective muscle to push NCI’s the number one killer of Americans by the many exciting opportunities. And this is once proud intramural research program end of this century. ... We need to find reflected in the new division structure, off the shoals and into exciting new ways [both to] maintain the spectacular which basically divides the intramural pro- waters of scientific discovery. For starters, progress in basic science [but], as impor- gram into three areas based upon the the 43-year-old cell biologist, who has tantly, to somehow bridge the huge gap three fundamental mechanisms of spent most of his past 16 years at NICHD, between this spectacular progress and the approaching the acquisition of knowledge has already streamlined NCI’s division very poor progress that we have made in and information—basic, clinical, and pop- structure and established an advisory the cure and prevention of most cancers ulation-based. board of intramural scientists. He’s also over the past 25 to 30 years. I also see great opportunities in injecting some new blood into the scientif- immunology. That’s one of the areas in ic community with the recruitment of a What do you see as the greatest which NCI has always been strong. I want noted molecular epidemiologist, Alfred strengths and weaknesses of NCI’s to continue to see that supported and

Knudson of the Fox Chase Cancer Center intramural program? enhanced. ... I hope we can help stimulate in Philadelphia, and two world-class yeast Klausner: I think the greatest strength of a real renaissance in the development of a geneticists from Seattle, Leland Hartwell of NCI's intramural program is the greatest the University of Washington and Steve strength of the NIH intramural program,

Friend of the Fred Hutchinson Cancer that is, the available resources and the Research Center. institutional opportunities to be a real community of scholars, the freedom to What was your perception ofNCI conceptualize research programs with very

before you came here? few constraints ... . Such freedom places

Klausner: I saw it as a somewhat trou- upon us a compelling demand to respond bled institute that had a very top-down by making sure ... that this is not only a leadership style. It tended to be an institu- great place to be, but that this is a place tion that ran by fiat and fear—fear of where great science is done. problems, fear of crises. It’s no secret that I think it is an institution that needs a it’s not been a place where people have variety of both structural and cultural

uniformly loved to work ... . That does changes so that it functions as a true meri- not mean that there haven’t been pro- tocracy and that it has real mechanisms [in grams and individuals who have thrived place] to select for and reward excellence. modern tumor immunology in much the and done well. My own feeling is that it is It needs a culture in which the indepen- same way that there’s been a renaissance in an intramural program that does not have dent development of the careers of people the immunology of autoimmune disease. the feel of the type of the intramural pro- can thrive. And it must become an institu- take to gram that I want to be associated with tion where all aspects of its administrative What steps do you plan to but I think that it will. function are structured to serve the scien- enhance the interactions between

tist and not the other way around. basic and clinical science?

Given your accomplishments as a Klausner: It is abundantly clear from the bench researcher and the current What do you see as the most fruit- exciting advances in human cancer biolo- administrative turmoil at NCI, why ful and interesting avenues for gy over the past few years that many of did you decide to take the job as basic and clinical cancer research them have come out of the ability of the institute director? at the institute right now? basic science to inform us about the dis- to Klausner: I really decided to do it both Klausner: The major areas that I think the ease, and, importantly, of the disease

out of a sense of the challenge that it rep- NCI needs to think about strengthening inform the basic science. ... For example, resented and, frankly, out of a sense of relate to cancer genetics and to the many we are going to want to develop molecu- responsibility as a member of the commu- fundamental aspects of the biology of the lar pathology, molecular diagnostics, and devel- nity .... I felt if asked, I would be willing cell that we now know are directly related cancer genetics and integrate such

to serve. I think Harold [Varmus] has pro- to cancer, including genetic and genomic opment among these three divisions

vided a fantastic model to scientists for the instability, the relationship between genetic basic, clinical and population-based. The

6 September-October 19 9 5

by Celia Hooper and Rebecca Kolberg

three division directors, George Vande those expectations will be reviewed. And what’s wrong—of all of the NCI’s pro- Woude, Philip Pizzo, and Joseph Frau- if a person is doing spectacularly as a lab grams, not just developmental therapeutics. meni, are deep in conversation about how chief and wants to remain as lab chief, I we actually set up working structures so don’t really see how enforcing retirement How can intramural researchers that the level of communication within helps anyone. help you shape the “new” NCI? this institute skyrockets. And there’s room Klausner: Part of the new governance for skyrocketing. What does the future hold for structure is something called an IAB, an NCI’s Frederick Cancer Research intramural advisory board that Claude Klee The Bisbop-Calabresi report made Center? will chair. This will have about 15 mem- dozens of recommendations on Klausner: We are actively discussing bers, intramural scientists from all the intra- ways of improving NCI’s intramur- Frederick. What I can say is that although mural divisions and people at all different al program, and we’d like to get there have been concerns about closing levels from tenure track to lab chief. This your reactions on a few of them. Frederick down, I see the Frederick cam- body will be very, very important in How do you feel about the recom- pus as a fantastic resource for the NIH reviewing the functioning of the intramural mendation to reduce the current and the NCI. I think it would be very program on an ongoing basis. The first percentage of the budget devoted to short-sighted to close it down, so we will thing they are going to need to do is intramural research? not be doing that. review the rules, the regulations, the

Klausner: I don’t know what the exact administrative processes, and the communi- percentage is. It’s actually not a number Will NCI be doing less AIDS cations pathways. This body will provide a that’s easy to get at ... . While I don’t research? filter mechanism so that decisions about the know exactly the right number, I do actu- Klausner: I have no commitment to functions of the intramural program will be ally think that it’s probably too high ... doing less AIDS [research], but I think we discussed by active intramural scientists. ... and we will be developing a plan that, will be calling less things AIDS. . . . We are At the same time this committee will set its over time, brings that percentage down. looking at the programs very carefully to own agenda to interact with NCI leadership and those responsible for the administrative structures in order to constantly look at how we can improve things and to address

the ongoing and changing needs of the sci-

entists. ... This will be a very well-publi-

cized, very accessible group. It will serve as

a line of communication between all intra-

mural scientists and the division and institute leadership, without worrying about going through chains of command.

What impact will the changes in NCI’s intramural program have on intramural research at other institutes? What do you think about the sug- make sure that we are accurately describ- Klausner: We are very interested in

gestion to establish an open ing the research we do ... . But as to developing things that I believe will be of grants competition? whether the cancer institute does AIDS real interest to all intramural programs, Klausner: Well, in the “Klausner” report research [or other] noncancer research, such as a good, user-friendly information- [issued in 1992 by the Task Force on the one of the lessons of the past 20 years is management system, so that we can, to Intramural Research Program, which was the hubris of deciding what exactly is can- the fullest extent possible, delegate headed by Klausner], we talked about the cer research and what isn’t. authorities to the laboratories and out of NIH creating special fellowships that people separate administrative branches. would compete for. I really like that idea. What about drug-development activities at NCI? Now that we’ve spent a lot of time What about appointing lab and Klausner: We will be reviewing the discussing NCI’s future, what branch chiefs and scientific direc- Developmental Therapeutics Program. I about the future ofyour own sci- tors for renewable five-year want the NCI to remain committed to entific career? terms? developmental therapeutics, but I think it’s Klausner: I’m going to keep my lab. I

Klausner: I see pros and cons of that. I’m a good time to look at that program. FIow- love being an intramural scientist at perfectly happy if in a lab there’s a group ever, I'm not singling out that program. NICHD ... and I hope they will continue of people who would like to rotate being Under this new administration, we are to love having me there. I’m absolutely lab chief. But the reality is that we have going to have real reviews—conceptual committed to maintaining my lab and certain expectations of lab chiefs, and reviews, not critique reviews pointing out maintaining myself as an active scientist. ®

7 The NIH Catalyst Scientific Cybernauts

Scientists, Start Your Engines! Finding Research Information on the Internet

mong the reasons that the As a recent “Hot Methods Clinic” erence Information. What this is:

research community has so helps to illustrate, the ability to smooth- This reference information is intended Aenthusiastically embraced the ly navigate the World Wide Web is to provide the reader with general Internet is the access it provides to vast among the most useful computing skills information regarding the process repositories of scientific information that a scientist can have [see March- known as PCR, or the Polymerase and to a wealth of databases for scien- April issue, page 12], To provide an Chain Reaction, and ... tific analysis. That’s all fine and good, idea of how search results vary depend- http://www.promega.com/pcrref/pcr- but how, in the Net’s ocean of informa- ing upon the engine chosen, I conduct- ref.html (3K) tion, can an individual scientist quickly ed a simple “experiment.” Using the 4) A Decade of PCR locate those sites that will be of greatest term “PCR,” I performed a search on Cold Spring Harbor Laboratory and The use in his or her own research? each of the three of the most-used Perkin-Elmer Corporation celebrate 10 One way to find search engines, InfoSeek, years of amplification with a videotape fruitful sites is to Lycos, and WebCrawler. library in which Nobel prize winners wander around the YOU MAY FIND ONE The results follow. Note Kary Mullis and James Watson and 19 Internet, simply using the difference in the other distinguished scientists review your mouse or key- ENGINE SUPERIOR amount of detail each the applications ...

board to roam through engine provides about http ://www . cshl . org/books/decade . html “tunnels” on Gopher TO ANOTHER FOR each site, as well as the (3K) servers or to surf fact that although they 5) MGD: PCR Primers Query Form YOUR PURPOSES. though the “links” were all given the same [MGI I User Support I Documentation

between sites on the search word, the engines I MGD I Citations I Markers I Probes

World Wide Web ranked some of the sites I PCR I Homology I Mapping I Map-

(WWW). Although serendipitous search- in different order. In addition, some ping Tools I Other Resources], PCR ing may uncover some wonderful engines returned more “hits" than oth- Primers Query Form. Search PCR resources, most scientists prefer a more ers, reflecting both the incidence of Primer Data Using the No Forms Inter- efficient mode of exploration. “Search such sites in the engine’s index and the face. Pre-generated lists ... engines”—computer resources that can method used to determine what infor- http ://www. informatics, jax. org/per. html be accessed free of charge through any mation is present at a particular site. (3K)

WWW browsing program [see box, page 91 —are what you need if you really want jr tm to soup up your research performance. These engines enable you to search for InfoSeek Lycos + any word or combination of words in the text of a wide range of Internet sites. A list of 10 sites was returned, and the The first 10 of 1,523 documents that After the words selected for a search are top five are listed below. contained the word “PCR” were printed, entered, a list of sites will appear. Some and the first three of those 10 are listed picking and choosing might be neces- 1) PCR Primer: A Laboratory below. sary at this point. As in any computer Manual search, if the terms are too broad, you Edited by Carl Dieffenbach, National 1) http://www.panvera.com/ may get a huge—and thus probably use- Institute of Allergy and Infectious Dis- catalog/pcrkits.html less— list of sites. Alternatively, if you eases, Gabriela Dveksler, Uniformed last fetched: 02-Jul-95 make your terms too specific, you might Services University of the Health Sci- bytes: 11933 wind up with a “list” with nothing on it. ences. From its first-published account links: 10 Try to use only a couple of relatively in 1985, the polymerase chain reaction title: PanVera Catalog, PCR Kits distinctive, but not too arcane, terms to has become a ... http://www.cshl.org/ and Primer Sets design a search of appropriate scope. books/pcr_primer.html (3K) outline: PCR Kits and Primer Sets LA You may also find one engine supe- 2) PCR Methods & Applications PCR Kit Version 1*, 50 reactions Product rior to another for your purposes. For A New Interdisciplinary Journal of Number: TAK RR011 PCR in vitro Single example, two major search engines, Research, Methods, Reviews, and Com- Site Amplification and Cloning (SSAC) InfoSeek and Lycos, return some infor- ment. Scientists have seized vigorously Kit*, 20 reactions Product Number: TAK mation about the site other than its on the power and flexibility of the R015 name, while another, WebCrawler, just polymerase chain reaction (PCR), and excerpt: PanVera Catalog, PCR Kits and returns a list of names. Also, the criteria this enthusiasm is generating a host Primer Sets PCR Kits and Primer Sets LA resulting in rankings may vary from of PCR-based and other ... PCR Kit Version 1*, 50 reactions Product search engine to search engine. Finally, http://www.cshl.org/journals/pcr/ (9K) Number: TAK RROl l Application Ampli- some Internet sites may be part of one 3) PCR Reference Information fication of large DNA templates (up to engine’s index and not another’s. PCR (Polymerase Chain Reaction) Ref- 40 kb) Amplification of cloned inserts

8 . .

September-October 19 9 5

by Dale Graham Ph D., DCRT ,

(e-mail: degraham@helix. n ih .gov)

and genomic DNA Description PCR enables you to access the site simply 9) Cookie, http://wsinti05.win.tue. technology has been widely used in by clicking on highlighted text. nl:4243/4 molecular genetics research, especially 10) MGD Home Page, http ://www. for genome analysis and sequencing 1) BioGuide, http://bioinformatics. informatics.jax.org/mgd.html

studies. However, efficient amplification weizmann . ac . il : 70/ 1 s/bioguide 11) Implications for Molecular Biol- of DNA fragments greater than 5 kb has 2) PanVera Catalog, TaKaRa PCR ogy in Hypertension Research,

been problematic. The Takara LA PCR Products and Molecular Biology http ://www. pitt . edu/~racst 1 2/thesis . html Kit is designed to overcome this limita- Kits, http://www.panvera.com/cata- 12) List of Journals from CSHL

tion. The LA PCR Kit includes all the log/pcrmb.html Press, http://www.cshl . org/journals/ reagents necessary for amplification of 7)3) MGD: PCR Primers Query Form,

large DNA templates; routine extension http://www.informatics.jax.org/per . html The information in this article deals only

to 20 kb, with . . 4) Long PCR Protocol, http://twod. with searching WWW, or Hypertext 2) http://twod.med.harvard.edu/ med.harvard.edu/labgc/estep/long- Transfer Protocol (HTTP), sites and not labgc/estep/longPCR_protocol. html PCR_protocol.html with other useful Internet sites such as last fetched: 19-Jul-95 5) RegFonn: PCR, http://www.vnu. Gopher or File Transfer Protocol (FTP) file date: 02-Jun-95 co.uk/eol/pcr/PCreg.htm servers. For information on locating bytes: 6270 6) College Nobel Laureate Lecture, search engines for other kinds of links: 5 http://www.physics.csulb.edu/WWW- Internet sites, use your WWW browser

title: Long PCR Protocol pages/nobel . html to access DCRT’s Information Sheet outline: Long PCR Reagents and Guide- http://bio-stockrooml .tamu. on Internet Resources. The address, lines General Guidelines for Long PCR edu/catalog/enzym.txt, or URL, for the Information Sheet

Conditions and Enzyme Mixtures Effi- http://bio-stockrooml.tamu.edu/cata- is http ://www. nih. gov/dert/expo/infos/ cient Long PCR results from the use of log/enzym.txt resources.html n two polymerases: a non-proofreading 8) PanVera Catalog Product Index,

polymerase is the main polymerase, http://www.panvera.com/catalog/index. excerpt: Long PCR Protocol Long PCR html Reagents and Guidelines (Modified

from Cheng et al. (1) ) General Guide- lines for Long PCR Conditions and In Search ofSearch Engines Enzyme Mixtures Efficient Long PCR To reach a search engine program, fire up a WWW browser program, such as

results from the use of two polym . . Netscape or Mosaic. If you’re using Netscape, clicking on the Net Search button will

3) gopher://bioinformatics.weiz- take you to a page with search engine sites. Another option is to select the Open mann.ac.il:70/l ls/bioguide Location in Netscape or the Open URL command in Mosaic and other browsers, and last fetched: 31-Jul-95 then type in the Uniform Resource Locator (URL) of the search engine you want to bytes: 1567 use. Bear in mind that URLs never contain returns, tabs, or spaces. Also, remember links: 7 that capital and lower case letters usually must be copied exactly. * excerpt: Select one of: * What is PCR? Search Engine URLs What are some good reference books InfoSeek Search, http://www.infoseek.com * for PCR? How should I select a set of The Lycos Home Page: Hunting WWW Information, http://lycos.cs.cmu.edu primers to use for PCR? * Programs Webcrawler Searching, http://webcrawler.com/ for designing PCR primers? * What is Sites with Lists of Search Engines “Hot-start” PCR? * What is AP-PCR or

RAPD PCR? * What is “Touchdown” W3 Search Engines

PCR? * Is there http :// cuiwww .unige. ch/meta-index. html This site is provided through the University of Geneva, and the search engine sites found here range from greatly useful to helpful only for searches of niche items, such as fonts. CUSI (Configurable Unified Search Interface)

http ://Web . nexor. co.uk/susi/cusi . html The query “per” found 200 docu- This site is maintained by Nexor UK. By filling out a single form, you can search several engines. ments and returned 25. The first 12 WWW are shown below. Uniform Resource Experimental Meta-Index Locators (URLs), which normally are http://www.ncsa. uiuc.edu/SDG/Software/Mosaic/Demo/medtaindex.html not included in WebCrawler results, This site not only provides access to some WWW search engines, but enables are included here. When used on- you to search Gopher seivers, Wide Area Information Servers (WAIS), and other line, WebCrawler returns a list with useful sites. the site name as a live link that

9 1 The NIH Catalyst Commentary A Locus for Dominant Nonsyndromic Hearing Impairment Maps Near the Huntington’s Disease Gene

nvestigators in the Section on Linkage Studies and Molecular plagued by inherited hearing loss in which the gene responsible is Cloning, Laboratory of Molecular Genetics, NIDCD—and our still unknown. I collaborators at the Department of Otolaryngology at Vander- bilt University in Nashville—have discovered a novel locus for Mouse Models nonsyndromic hereditary hearing impairment on chromosome 4p Mouse strains that exhibit the NSHHI phenotype may help in in the region of the Huntington’s disease gene. This locus causes identifying novel genes important in human NSHHI. The deafness,

a dominant, progressive low-frequency hearing loss (LFHL) in a or dn, mouse is among the most intriguing of a couple dozen large U.S. family. The region of linkage spans a distance of such models. The responsible dn gene maps to mouse chromo-

approximately 1.7 megabases (Mb), and we are hoping to narrow some 19 (D19MIT14, 60, and 41), which is syntenic with human this region as additional polymorphic markers are examined. chromosome 9q21 and therefore represents an additional potential There is a possibility that mutations in known sequences, mapped location for a human NSHHI-related gene (10). Tilted mice, during the efforts to clone the Huntington’s disease locus, are named for their characteristic tilted heads and discovered at Jack- responsible for this phenotype. son Laboratoiy in Bar Harbor, Maine, are another possible model. Genetic factors account for most cases of hearing impairment The defective gene in tilted mice maps to the syntenic mouse in young children. chromosome 5 and Among individuals may be the mouse with genetic hearing version of the human loss, approximately dominant NSHHI 74% have an autoso- gene that our lab mal-recessive mode recently localized to of inheritance, about a region on chromo- 25% have an autoso- some 4p that is mal-dominant type, bounded by the and the remaining Huntington's disease 1% having X-linked locus. The tilted- or mitochondrial mouse mutant has a types of hereditary recessive cochleo- hearing impairment. vestibular loss recog- One-third of individ- nizable by the uals with hereditary mouse’s tilted head hearing impairment and inability to swim have other associated (11). Although in

symptoms recogniz- mice it is not easy to able as a syndrome. detect loss of low-fre- The other two-thirds quency hearing that have no known asso- may be associated ciated findings and with the tilted mutant are classified as hav- gene, this promises to ing “nonsyndromic” Haplotype analysis offamily with nonsyndromic hereditary hearing be a valuable model. hereditary hearing impairment. Parentheses indicate inferred genotype Question marks indicate unknown Mutations in genes . impairment (NSHHI) genotype. Markers genotyped are displayed vertically, from top to bottom: D4S43, that are homologous (1,2). In many types D4S127, D4S412, D4S126, and D4S432. Boxes indicate inheritance of the in mice and humans share a of inherited hearing chromosome linked to the disease. Dashed lines indicate that the affected parent sometimes loss, morphologic or similar but not identi- is uninformativefor that marker. neuroepithelial cal phenotype. This

defects are observed in inner ear structures (3). At the present point is well illustrated by the relationship between the shaker-

time, however, little is known about the molecular mechanisms mouse-in which mutations in the myosin VIIA gene result in hear- involved in the development and homeostasis of the inner ear. ing loss and a vestibular abnormality—and human Usher syn- Linkage studies have identified seven dominant gene loci and drome type IB—in which mutations in the same gene result in seven recessive loci for autosomal NSHHI. One of these loci is retinal degeneration, in addition to hearing loss and vestibular

also the cause of Usher syndrome type IB and is linked to chro- abnormality. Several other mouse strains could serve as potential mosome llql3-5 (4,5). Mutations in this gene, a novel myosin models for NSHHI, even though these mice also have altered

VIIA gene (6), are apparently able to cause Usher’s syndrome in vestibular function in addition to hearing loss (12-15). some families and recessive NSHLII with vestibular malfunction in others. Human Studies Two NSHHI loci map to the X chromosome (7,8). Mutations in In our current study of an extended family in the United States the Pou3F4 gene have been shown to cause X-linked fixation of with more than 100 members, the majority of patients have a bilat-

the stapes with perilymphatic gusher (9), a rare congenital defect eral and symmetric hearing loss involving frequencies of 250, 500, leading to mixed hearing loss. Despite this progress in our under- and 1000 Hz at the onset of symptoms. The progression of the standing of the genetic causes of hearing loss, many families are hearing loss follows one of three patterns: 1) confined to the low

10 September-October 19 9 5

by Edward Wilcox Ph D., NICHD, Lesperance, , and Marci M.D.,

Children 's National Medical Center, Washington, D C.

frequencies, 2) involving all frequencies and producing a flat References audiogram, or 3) involving low and high frequencies while sparing 1. M.L. Marazita, L.M. Ploughman, B. Rawlings, E. Remington, K.S. Arnos, and W.E. Nance. “Genetic epidemiological studies of early-onset deafness in the U.S. school-age population." Am. the middle frequencies (2000 Hz). As a result of this comparative- ]. Med. Genet. 46, 486-91 (1993). ly mild pattern of hearing loss, few of the family members use 2. C.W. Cremers, H.A. Marres, and P.M. van Rijn. “Nonsyndromal profound genetic deafness in hearing aids, and none have cochlear implants. The age of onset is childhood.” Ann. N. Y. Acad. Sci. 630, 191-96 (1991). 3. S.D.M. Brown and K.P. Steel. “Genetic deafness-progress with mouse models.” Hum. generally in the second decade of life; no family members showed Mol. Genet. 3, 1453-36 (1994). hearing loss before age 5, but all affected members had developed 4. P. Guilford, H. Ayadi, S. Blanchard, H. Chaib, D. Le Paslier, J. Weissenbach, et al. “A human

hearing loss by age 15 (16). gene responsible for neurosensory, nonsyndromic recessive deafness is a candidate homologue of The family was genotyped for D4S432, D4S412, D4S127, D4S43, the mouse sh-1 gene.” Hum. Mol. Genet. 3, 989-93 (1994).

5. W.J. Kimberling, C.G. Moller, S. Davenport, I. A. Priluck, P.H. Beighton, Greenberg, et al. and D4S126 markers on human chromosome 4p. These five mark- J.H. “Linkage of Usher syndrome type I gene (USHlB) to the long arm of chromosome 11.” ers span a genetic distance of approximately 5 centimorgans (17). Genomics 14, 988-94 (1992).

6. The region has a much higher recombination rate than would be D. Weil, S. Blanchard, J. Kaplan, P. Guilford, F. Gibson, J. Walsh, et al. “Defective myosin VILA gene responsible for Usher syndrome type IB.” Nature 60-61 expected for its physical size (less than 3 Mb) (18). 374, (1995). 7. W. Reardon, H.R. Middleton-Price, L. Sandkuijl, P.D. Phelps, S. Bellman, L. Luxon, et al. “A Four recombinants were identified by haplotype analysis (see fig- multipedigree linkage study ofX-linked deafness: linkage to Xq 13-q21 and evidence for genetic 111-21 ure). Individual has a recombination between D4S127 and heterogeneity.” Genomics 1 1, 885-94 (1991).

8. D4S412. Unaffected individuals 111-17 and 111-19 A.K. Lalwani, J.R. Brister, J. Fex, K.M. Grundfast, A.T. Pikus, B. Plo- have recombinations between D4S126 and plis, et al. “A new nonsyndromic X-linked sensorineural hearing impairment linked to Xp21.2.” Am. J. Hum. Genet. 55, 685-94 D4S432. Since 111-11 is an affected individual with To OUR KNOWLEDGE, (1994). the same recombinant haplotype, we postulate 9. Y.J.M. Dekok, S.M. Vandermaarel, M. Bitnerglindzicz, I. Huber, A.P. that the breakpoints for 111-11 vs. 111-17 and 111- Monaco, S. Malcom, et al. “Association between X-linked mixed deafness and mutations in the POU domain gene POU3F4.” Science 267, 19 lie on opposite sides of the gene for hearing THERE HAS BEEN NO 685-88 (1995). loss. is fully informative D4S126 not a marker; the 10. B.J. Keats, N. Nouri, J.M. Huang, M. Money, D.B. Webster, and C.l. affected parents of the recombinants are homozy- STUDY THAT SPECIFI- Berlin. “The deafness locus (dn) maps to mouse chromosome 19.” Mam. Genome 6, 8-10. gous at this locus, preventing detection of recom- (1995). 11. M.F. Lyon and A.G. Searle. Genetic Variants and Strains ofthe Labora- binants in their children. Thus the most likely CALLY SCREENS FOR tory Mouse, 2nd ed. Oxford, England: Oxford University Press (1989).

location for the gene is between D4S412 and 12. M.S. Deol. “The inner ear in Bronx waltzer mice.” Acta Otolaiyngol. D4S432, a distance of 1.7 Mb (19). The maximum MORE SUBTLE FORMS OF 92, 331-36 (1981). 13. F. Gibson, Walsh, P. Mburu, A. Varela, K.A. Brown, M. Anotonio, LOD score was 5-05 at q = 0.05 for D4S412. Given J. et al. “A type VII myosin encoded by the mouse deafness gene shaker- the - - marker order of D4S412 D4S126 D4S432, HEARING LOSS IN 1.” Nature 374, 62-64 (1995). the multipoint mapping yielded a maximum LOD 14. M.S. Deol and M.C. Green. “Snell’s waltzer, a new mutation affecting behaviour and the inner ear in the mouse.” Genet. Res. 8, 339—45 score of 6.5 when the disease gene was placed in PATIENTS WITH HUNT- (1966). the interval between D4S126 and D4S432. 15. K.B. Avraham, D.M. Kingsley, L.B. Russell, N.G. Copeland, and N.A.

This region on chromosome 4 has been well “Molecular characterization of the deaf Snell's INGTON S DISEASE. Jenkins. genetic waltzer mouse. ” Am. Hum. Genet. suppl.), A210 (abstr.). mapped. More than 20 genes were identified in J. 55 (3, (1994) 16. The Vanderbilt University Hereditary Deafness Study Group. “Domi- the course of the lengthy search for the Hunting- nantly inherited low-frequency hearing loss.” Arch. Otolaryngol. 88, ton’s disease gene, HD or IT15 (20), which was 40-48 (1968).

finally located at 4pl6.3 in 1993 by the Huntington’s Disease Col- 17. S. Baxendale, M.E. MacDonald, R. Mott, F. Francis, C. Lin, S.F. Kirby, et al. “A cosmid contig laborative Research Group. Expression of IT15 has been detected and high resolution restriction map of the 2 megabase region containing the Huntington’s disease gene.” Nat. Genet. 4, 181-86 (1993). in all areas of the cerebral cortex, predominantly in neurons (21 ). 18. B.A. Allitto, M.E. MacDonald, M. Bucan, J. Richards, D. Romano, W.L. Whaley, et al. Hearing loss has not been described as a clinical feature of Hunt- "Increased recombination adjacent to the Huntington disease linked D4S10 marker.” Genomics ington’s disease; however, to our knowledge, there has been no 9, 104-12 (1991). Fan, DeYoung, R. Lagace, R.A. Lina, Z. Xu, Murray, et al. “Isolation of yeast artifi- study that specifically screens for more subtle forms of hearing loss 19. J.B. J. J.C. cial chromosome clones from 54 polymorphic loci mapped with high odds on human chromo- in patients with Huntington’s disease. multipoint data for our The some 4.” Hum. Mol. Genet. 3, 243-46 (1994) NSSHI locus suggest a location proximal to IT15 and D4S126, a 20. The Huntington’s Disease Collaborative Research Group. “A novel gene containing a trinu- region that includes the gene for the a-2C-adrenergic receptor cleotide repeat that is expanded and unstable on Huntington’s disease chromosomes.” Cell 72, 971-83(1993). (ADRA2C) (22). Alpha-2-adrenergic receptors are widely expressed 21. G.B. Landwehrmeyer, S.M. McNeil, L.S. Dure, P. Ge, H. Aizawa, Q. Huang, et al. “Huntin-

in the brain, especially in regions with high dopamine content. gon’s disease gene: regional and cellular expression in brain of normal and affected individuals.” Another candidate gene in this region is the a-2-macroglobulin Ann. Neurol. 37, 218-30 (1995).

22. O. Riess, U. Thies, 1. Siedlaczck, S. Potisek, R. Graham, Theilmann, et al. “Precise mapping of receptor-associated protein (A2MRAP) also known as the low-den- J. the brain a2-adrenergic receptor gene within chromosome 4pl6.” Genomics 19, 298-302 sity-lipoprotein protein receptor-related, protein-associated (1994). (LRPAP1) (23,24). Although the function of this protein is 23. Y.S. Jou, R.D. Goold, and R.M. Myers. “Localization of the 0c2macroglobulin receptor-associat-

ed protein gene (LRPAP1) and other gene fragments to human chromosome 1 6.3 by direct unknown, its corresponding receptor is important in proteinase 1 4p cDNA selection.” Genomics 24, 492-500 (1994). inhibition and lipoprotein metabolism. 24. F. Van Leuven, C. Hilliker, L. Serneels, L. Umans, L. Overbergh, B. DeStrooper, et al. Now that we have documented these tantalizing potential con- “Cloning, characterization, and chromosomal localization to 4pl6 of the human gene (LRPAP1) nections to the Huntington’s disease gene (25) and the tilted- coding for the a2-macroglobulin receptor associated protein and structural comparison with the murine gene coding for the 44-kDa heparin-binding protein.” Genomics 25, 492-500 (1995). mouse gene, our laboratory would like to screen any and all the 25. M.M. Lesperance, J.W. Hall, III, F.H. Bess, K. Fukushima, P.K. Jain, B. Ploplis, et al. “A gene candidate genes that have been mapped to the human 4pl6.3 for autosomal dominant nonsyndromic hereditary hearing impairment maps to 4p 1 6.3.” Hum. region. We are planning to breed the tilted mice to test their hear- Mol. Genet, (in press). ing. If these mice are good candidate models, we would expect to find close syntenic relationships in the mapping of mouse chromosome 5 and the human 4pl6.3 region.

11 — The NIH Catalyst

TTT , — | Hot Methods Clinic

Flow Cytometry: More Than Just Cell Sorting

here are two camps when it comes transferase (TdT) end- labels double- The Method and How It Works to flow cytometry: the believers, who stranded DNA breaks, which are generat- In flow-cytometric analysis, cells in a sin- T appreciate the technique as a fast ed during apoptosis—can be applied to gle cell suspension are stained with multi- lane to tomorrow’s research questions, and large populations of cells by the use of ple fluorescent markers and transported the uninitiated, who haven’t heard all the flow cytometry. Another method of flow- rapidly—routinely 18,000-30,000 cells/min things that are possible through contempo- cytometric apoptosis detection—based on —to intersect a finely focused monochro- rary applications of the technique. After light-scatter characteristics that change matic beam of light of an appropriate fre- getting its start with the crude cell counters when the nucleus condenses during apop- quency. A stream of fluid containing the of the 1930s and ‘40s, contemporaiy flow tosis—requires no manipulations other sample cells is ejected at steady pressure cytometry is no longer just for counting. than preparation of a cell suspension. and rate into a flowing, high-pressure New-age flow cytometry-—which includes “sheath” fluid. The convergence of the but is not limited to fluorescence-activated sample stream with the sheath fluid allows cell sorting (FACS)—couples a highly sensi- for the precise intersection of the sample tive, automated fluorescence-detection stream that contains the single cell suspen- device with sophisticated computerized sion with the laser beam. This is referred analysis of data gleaned at lightning speed to as hydrodynamic focusing. The fluo- for measurements of numerous interesting rochromes attached to the cells absorb properties of large populations of cells. Cell light and emit energy at a longer wave- size, viability—including the presence of length that is specific for the fluo- apoptotic cells, cell-cycle dynamics, kinet- rochrome. For example, fluorescein isoth- ics, and the presence of multiple intracellu- iocyanate (FITC) emits light at a different lar and surface proteins can be determined wave length than phycoerythrin (PE) or for each cell in a sample. peridin-chlorophyll-a-protein (PerCP),

The high sensitivity of this technique, allowing all three indicators to be detected coupled with the ability to rapidly analyze simultaneously in a cell. In addition, light multiple parameters in samples containing is scattered in proportion to the size of the 5,000 to a million cells, allows the detec- cell in the forward direction, much like a tion and definition of unique subpopula- IgM DNA Content shadow. Light is also scattered or reflected tions within a sample. The ability to detect to the side by the intracellular granules; subpopulations with an abnormal pattern Figure 1. Flow cytometric analysis of thus, cells with complex cytoplasm, such of protein expression is useful in diagnos- a murine marginal zone lymphoma. as the granular neutrophils, have a much ing and subclassifying leukemias and lym- Left panels show that the majority of the higher side scatter than do less complex phomas. And because flow cytometry can cells, such as lymphocytes. cells are positiveforfluorescein isothio- routinely pick out one neoplastic cell per Light detectors collecting the forward cyanate (FITC)-staining ofIgM, positive for 1,000 normal cells—and specialized tech- and side scatter and fluorescence emis- phycoerythrin staining of CD5, and posi- niques can improve this sensitivity to find sions thus rapidly gather information for tivefor dim tricolor B220, which, in this one abnormal cell in a million normal each cell on its size, cytoplasmic complexi- case, is indicative of marginal zone lym- cells—the technique is useful in hematol- ty, and fluorescent markers. Three different ogy and hematopathology for detecting phoma. The right upperpanel shows DNA fluorochromes emitting light at three differ- minimal residual disease. content (x-axis) verses cell number (y- ent frequencies are routinely used in clini- By using appropriate standards and axis). The right lowerpanel shows a two- cal laboratories, whereas research facilities controls, flow cytometry can be exquisite- parametric contourplot ofFITC IgM versus may use five or more fluorochromes. Fluo- ly quantitative, allowing researchers to DNA content. Two G1/G0 populations can rochromes may be bound to antibodies for determine the exact number of molecules the detection of specific proteins or of be seen; one is diploid and the other is of fluorescent antibodies and thus the BrdU incorporation in studies of cell-cycle — aneuploid. Both are IgMpositive. molecules of antigen—bound to a cell or kinetics. Alternatively, the fluorochromes within a cell. This makes flow cytometry Reduced DNA content, or hypoploidy, as may be used for direct staining of cellular broadly useful for precise measurement of indicated by propidium iodide (PI) stain- elements. An example of this would be PI the expression of oncogenes, activation ing, is another easy procedure for detect- intercalation into DNA. markers, adhesion receptors, and other ing apoptosis. Even oxidative state and Flow cytometry measures all parameters proteins and, depending on the method of the flux of ions, such as calcium, into cells for all cells passing through the focused staining, allows localization of these pro- can be measured by flow cytometry. The beam, and then the cells can be classified teins to the cell surface or cell interior. power of flow cytometry in cell biology into categories based on any combination Flow cytometry is becoming important lies not only in its sensitivity, but also in of the detected parameters, including pres- for exploring some of the cellular activities its ability to measure multiple characteris- ence of cell-surface lineage-specific mark- that are under intense research scrutiny tics simultaneously on each cell in a pop- ers or activation antigens, oncogene these days. Cell-cycle data can be ulation of 100,000 to a million cells. expression, or DNA content. Complex obtained by DNA-content analysis or by Therefore, for example, the nonapoptotic computer programs then quantify the num- detection of bromodeoxyuridine (BrdU) vs. apoptotic cells in a heterogeneous bers of cells within each defined category incorporation or cell-cycle specific pro- sample can be rapidly compared for levels and record the fluorescent intensity, indi- teins. Flow cytometry is, arguably, the of BCL-2 and p53 protein expression, cating levels of protein expression or quan- most sensitive and easiest method for presence of a lineage-specific surface tities of DNA, for example. The computer detecting apoptosis. The TUNEL method markers, such as T-cell specific antigen, then displays the data in two- or three- in which the terminal deoxynucleotidyl and cell-cycle phase (G1/G0, S, or G2+M). dimensional plots of parameters and calcu-

12 September-October 19 9 5

by Maryalice Stetler-Stevenson, M.D., Ph D.,

NCI, and Gerald E. Marti, M.D., Ph.D ., FDA

lates the percentage of cells falling into any Trouble-Shooting Tips 1200 rpm in swinging-bucket rotor cen- category specified by the investigator. Attention to several technical details helps trifuge at room temperature for 10 min. to prevent problems and to identify the 3. Aspirate the supernatant and repeat Protocols source of problems that do occur. We step 2. above two times. Mention of a specific product in the fol- wash all cells that are derived from living 4. Restore to original volume with PBS. lowing description does not constitute an animals, including humans, to remove Dilute to 2 x 106 cells/mL. endorsement. adherent proteins that may bind labeled Protocols differ depending on the infor- antibodies or dyes in a specific or nonspe- Cell-Surface-Antigen Staining in mation a researcher is seeking—cell-cycle cific manner. Using too high a concentra- Whole Blood or Bone Marrow analysis or cell-surface antigen, for tion of antibody promotes nonspecific 1. Add appropriate amount of antibodies example—and the type of specimen—cell binding. The appropriate concentrations to labeled tubes (usually 5-20 pL). Three lines in media, whole blood, or bone mar- antibodies, each complexed with a differ- row, for example. The two most widely ent color fluorochrome (e.g. FITC, PE, and used protocols are whole-blood lysis PerCP) can be placed in the same tube for (WBL), for surface immunophenotyping of FacScan analysis. For five-color analysis or blood or bone marrow, and PI staining of UV excitation, more complex flow isolated cells, for analyzing the cell-cycle cytometers are required. Add 150 pL PBA (to detect S phase cells) and for detecting (PBS with 0.1% bovine serum albumin aneuploidy. and 0.1% NaN3) or PBS with 10% fetal calf In WBL, a 10- to 100-mL sample of serum (FCS) to each tube. whole blood or bone marrow is stained 2. Add 100 pL of washed whole blood or with two to five conjugated monoclonal bone marrow to each of the prepared reagents. After the staining is complete, reagent tubes and incubate in the dark for the red cells are lysed using either dilute 30 min at room temperature. Because light HC1, hypotonic solution, detergents, bleaches fluorochromes, tubes should be ammonium chloride, or proprietary prepa- kept covered with aluminum foil. rations. The lysed cells are washed and 3. Wash cells by adding 4 mL PBA and fixed for analysis. Compared with centrifuging at 1200 rpm for 8 min in a mononuclear cell isolation followed by swinging-bucket rotor centrifuge at 4-6 °C. antibody staining, WBL is more conve- Figure 2. /. DNA content analysis to Aspirate the supernatant. Approximately nient, quicker, and less likely to cause detect apoptosis in cells after cold 100 pL PBA and cells will remain. selective cell loss, could bias the 4. Lyse the specimens which shock. Cell line A is prone to apoptosis in using a proprietary data. Fixation stabilizes cell membranes, lysing kit (e.g., Immunolyse, Q-prep, and response to cold shock. Sub-diploid apoptot- crosslinks the antibody to its antigen, and Facslyse). We find that manually lysing is ic peak, is shown. Cell line B is resistant to reduces the risk that researchers will be quickest and provides an excellent speci- cold shock-induced apoptosis and shows contaminated with infectious material. men in the hands of an experienced techno sub-diploid apoptotic peak. II. BCL-2 Fixed cells are also stable for longer peri- nician. The manual lysing methods vary expression in cel! lines. Apoptosis-resistant ods than are fresh cells. from product to product but usually entail To stain intracellular antigens, the cells cell line B has higher level ofBCL-2 protein 1) adding a lysing reagent, 2) incubating are permeabilized and stained with conju- expression than apoptosis-prone cell line A. for a precise period of time, 3) adding a gated monoclonal antibodies. Red cells fixative that stops the lysing and 4) wash- are lysed. If the permeabilization method for antibodies are often specified by man- ing the cells to remove the lysing reagent. does not fix the cells, they must be fixed ufacturer or can be empirically determined Timing, in manual lysing, is critical. We after lysis of the red cells. For simple cell- by staining control cells with serial dilu- recommend using an electronic timer and cycle analysis, cells are permeabilized and tions. When working with antibodies, we restricting the number of tubes to be lysed stained with a DNA dye such as PI. Com- use isotypic controls against nonmam- to a number that can be handled prompt- bined antigen-expression and DNA-con- malian proteins to detect nonspecific bind- ly. Experienced technicians in our labora- tent analysis usually consists of staining ing of antibodies. Negative and positive tory do not lyse more than 20 tubes in one with an FITC-labeled antibody, followed controls for all biological processes that batch. Machines that perform the entire by gentle fixation, permeabilization, and are being characterized in a flow-cytome- lysing procedure (e.g., the Coulter Q-prep) PI staining. Both FITC- and PE-labeled try study should be run to ensure sensitivi- are available but can lyse just one tube at reagents can be used together with 7- ty and specificity. Protocols of prepara- a time. The main benefit of the Q-prep is amino-actinomycin D (7AAD) instead of tions for flow-cytometric analysis of cell- that it requires no experience to produce PI, which emits light in the same wave- surface, intracellular, and intranuclear anti- an excellent specimen. Its main drawback length region as PE. Describing the actual gens and DNA content are given below. is that it is not as fast as manual lysing in acquisition and computer analysis of the the hands of an experienced technician. data on the flow cytometer is beyond the Cell Washing 5. After washing the cells with PBA, resus- scope of this article. In our opinion, 1. Add 20 rnL or less of peripheral blood, pend each specimen while vortexing light- acquisition and analysis of flow-cytometry bone marrow, or cell suspension to a 50- ly in 500 pL of 1% paraformaldehyde in data is best done by—or under the super- mL centrifuge tube. With peripheral blood the isotonic solution that the cells will be vision of—someone experienced in the or bone marrow, mark the level. analyzed in. Cover and keep at 4-6 °C in field. Labs with substantial flow-cytometry 2. Add room temperature phosphate- the dark for at least 1 h but, preferably, experience and facilities are listed in the buffered saline (PBS) to bring the volume overnight. “contacts” section of this article. to 45 mL. Invert to mix and centrifuge at continued on page 22.

13 The NIH Catalyst *r

Lab Behind the Leader New NIAID Scientific Director

In its quest for a first-rate scientific infected; mutations in causing potential of director, NIAID didn't have to go far the virus genome, the infected cell. We which spans approxi- We WOULD LIKE TO have cloned provirus- afield— it found just what it needed mately 9 kilobases es from such lethal in its own backyard: noted immunol- UNDERSTAND WHY (kb); variations in host- and nonlethal lines, ogist nomas Kindt. Recognizedfor his virus interactions; the HTLV-1 INFECTION and, through direct contributions to the understanding of genetic background of injection, have used human T-cell leukemia virus-1 (HTLV- the infected subject; or PRODUCES SUCH those DNA clones to 1), Kindt joined NIAID in 1977 and some combination of infect normal human went on to become the chief the Lab- VARIABLE RESPONSES of these parameters. and rabbit cells in oratory of Immunogenetics. Before We have developed IN DIFFERENT vitro and rabbits in arriving at NIH, Kindt, who received in vitro and in vivo vivo. In a further his PhD. from the University of Illinois systems to learn more INDIVIDUALS. effort to pinpoint the at Urhana-Champaign in 1967, held about how HTLV-1 genes that control What events or academic appointments at ne Rocke- exerts its highly vari- HTLV-l’s pathogenici- feller University and Cornell University able effects on the FACTORS DICTATE THE ty, we are now ex- Medical College in New York. He has host. The laboratory changing different received numerous scientific awards, rabbit, which is highly DIFFERENCE BETWEEN component genes susceptible to HTLV-1 the viruses including the Elliot Osserman Award among ASYMPTOMATIC INFECTION infection, is an animal from the lethal and from the Israel Cancer Research Fund model that mimics nonlethal cell lines to and has advised the Howard Hughes AND FATAL OR human infection in sev- create chimeric HTLV-1 Research Scholars Program. Kindt eral ways. As its name CHRONIC DISEASE? strains. offers this description of his research. implies, HTLV-1 infects Our current data T cells, and rabbit T- suggest that a major

ur laboratory studies the human cell lines infected with HTLV-1 survive factor in pathogenicity is the expression retrovirus HTLV-1. Although in culture indefinitely. We have adminis- of certain, as-yet-unidentified cellular O most of the 10 to 20 million tered HTLV-l-infected T-cell lines to genes—expression that is very likely people infected with HTLV-1 world- rabbits and monitored the animals to see induced by infection with the lethal wide suffer no overt whether they develop dis- virus. Our in vitro studies show that disease, about 5% are ease or asymptomatic expression of such genes predisposes afflicted with an acute infections. In an effort the cell to overcoming host resistance. and often fatal leu- to determine what is One route by which this occurs is by kemia, a debilitating responsible for the diver- the apoptosis, or programmed death, of neurologic disease, or gent outcomes, we then host T cells, which are presumably the one of a variety of studied the characteristics cells that keep the infected cells in chronic conditions rang- of the various HTLV- check. ing from arthritis to l-infected cell lines, In the future, our lab, which includes uveitis. In certain areas along with the structure R. Mark Simpson, Tongmao Zhao, Mary of the world, such as of the integrated virus, or Ann Robinson, Michel Leno, and Flo- southern Japan, the provirus, and the func- rence Bowers, will concentrate on prevalence of HTLV-1 tions of the provirus’ exploring the effects of genes from infection may be as component genes. lethal and nonlethal viruses on the high as 20%. This prob- To date, we have expression of host cellular genes. First, lem of endemic HTLV-1 infection, along identified HTLV-1 cell lines that cause both viral and cellular genes that exert with the recent increase in the infection acute disease in rabbits that is similar to either a positive or negative effect on rate in the United States, mainly among human leukemia and, in other cases, pathogenesis will be identified by intravenous drug users, makes HTLV-1 chronic cutaneous lymphoma. We are screening HTLV-l-infected cell lines by a significant health threat. now examining these lines in detail to in vitro molecular and cellular methods. We would like to understand why ascertain how they differ from lines that When candidate genes are found, HTLV-1 infection produces such vari- give rise to asymptomatic infection. We nonlethal HTLV-l-infected cell lines able responses in different individuals. have observed certain small differences transfected with these pathogenesis- What events or factors dictate the differ- in gene expression between the promoting genes and lethal HTLV- ence between asymptomatic infection so-called lethal and nonlethal cell l-infected cell lines transfected with and fatal or chronic disease? Possible lines, but none of those differences pathogenesis-suppressing genes can be candidates include the nature of the cell absolutely correlates with the disease- tested for in vivo effects, m

14 — —

September - October 19 9 5

Tenure Track To make sure the new system is widely many of the best young investigators in continuedfrom page 1 understood, DDIR Michael Gottesman has this country are coming out of our labs

met with groups of postdocs and junior and clinics,” says DDIR Gottesman. “It

tory research decisions, staff, and scientists to explain the new system and would be a travesty if we didn’t snap up resources. But NIH tenure goes further, answer questions. And at each step in the the best of our own for the tenure-track.” actually providing scientists with the new system—starting with intramural Gottesman gives intramural postdocs resources to conduct the research without postdocs just entering NIH for training information on new tenure track positions obliging them to apply for grants, as their there is an emphasis on letting everyone by advertising them on his electronic bul- extramural colleagues must do. know their career status and what NIH letin board a few weeks before the ads Under the old system, intramural staff offers them and expects from them. For appear in journals like Science. NHLBI’s researchers were nominated for tenure by example, all scientists placed on the SD, Edward Korn, says his institute has lab and branch chiefs, then reviewed by tenure track now receive letters congratu- capitalized on the new system to snare their institute, center, or division’s (ICD’s) lating them on their new status and some excellent postdocs for tenure-track promotion and tenure committee. If their describing the resources that will be at positions. “The tenure-track policy pro- scientific director (SD) selected them as their disposal during the six years that vides more opportunities for NIH post- tenure candidates, their packets of cre- they have to establish themselves as inde- docs than the previous policy in that NIH dentials were presented to the Board of pendent scientists on the tenure track. postdocs now can and do compete for Scientific Directors—a group composed One key difference in the system is the many positions outside their own lab- of the SDs for all institutes that meets only beginning to come into play—a new oratory rather than just the few or none biweekly with the deputy director for emphasis on outside recruitment to the that might arise within their own lab.” intramural research (DDIR). The SDs dis- tenure track. When NIH switched from Korn says that of the five nationally cussed each candidate’s merits and voted the old system to the new one, each insti- advertised tenure-track positions NHLBI for or against recommending that the tute was allowed to nominate intramural has filled in the past year, three went to DDIR grant tenure to the candidate. The scientists for “grandfathering” onto the postdocs from other institutes who were DDIR almost invariably approved the can- tenure track based on evidence of high- unknown to NHLBI’s search committee didates that the SDs recommended. About quality, independent research. Around before they applied, and one went to a 95% of the candidates brought to the SDs 180 intramural scientists were placed on postdoc in another NHLBI lab who was were recommended. the tenure track under the grandfather previously unknown to the lab that select-

Data from CTC’s first 14 months sup- clause. Currently, however, the only sci- ed him. port the proposition that the new commit- entists being added to the tenure track David J. Clark, who has been in his tee is no tea party. Of the 28 scientists are those selected as the top candidates tenure-track position at NIDDK for just a whose cases were reviewed, 21—75% in rigorous, nationally couple of months, says were recommended for tenure. Although advertised searches for the his case proves that intra- the percentage of canidates tenured by positions. mural fellows can make the CTC has gone up slightly in the past Some fellows and I it to the tenure track. few months, the new system appears to trainees erroneously be- Clark was selected be tougher. Wyatt says the reasons may lieve that this emphasis on through a national search in part relate to lack of familiarity with the outside recruitment means as the best candidate for new system and a new emphasis on dis- they do not have a crack a tenure-track position tinguishing between independent scien- at the tenure track after within the institute in tists, who may be granted tenure, and their allotted five years as which he was trained, staff or collaborative scientists, who may postdocs in the intramural but in a different lab. “It be granted permanence but not tenure. program. “What is worri- is now more difficult for The most conspicuous change in the some is the fellows who intramural fellows to get system is that the final packets of creden- have this view that there a tenure-track job at the tials no longer go to the SDs, but instead will not be a chance to NIH, but I believe that to a 15-member panel of scientists select- make a life here at NIH if the new system is fairer ed to serve on the CTC by the DDIR. you come in as a postdoc,” says NCI’s and will help to raise the quality of Many of the other changes in the new Elise Kohn, recently tenured by CTC. “We research at NIH,” says Clark, who chose system are not universal or substantive, are starting to see an exodus of outstand- the NIH position over several outside but are refinements intended to make ing postdocs leaving for opportunities offers. tenure policies more uniform among the elsewhere and grave concern among Once scientists make it to the tenure various institutes and understandable to postdocs about coming here.” track, their work is cut out for them. In everyone. “The members of the lab I Although it is likely that in the future, a the course of the next six years, the work in have less anxiety than before smaller percentage of scientists tenured at tenure-track investigator must build up a with respect to the tenuring system,” says NIH will have been trained here, doing a portfolio of research that impresses a

Pat Becerra, who is at the start of NEI’s postdoc stint at NIH is not the kiss of series of judges, starting with the lab or tenure track. “Before, the tenuring system death for one’s intramural tenure branch chief and the institute’s Board of was an unknown. ... Now we all know prospects. “By virtue of the size and Scientific Counselors (BSC)—the panel of where we stand.” excellence of our training programs, outside scientists that convenes at NIH

15 The NIH Catalyst T

every three to four years to review all patents have not been decisive factors in about my record,” says Boguski, who also tenured and tenure-track investigators. If, cases CTC has seen, Dawid says. Wyatt received competing job offers from other based on these evaluations, the scientist says that BSC reviews, SD evaluations, institutions. One bonus of the new system is kept on the tenure track, in six—or and outside letters are all important and for Boguski was that CTC reviewed his perhaps fewer—years, his or her creden- that CTC’s emphasis when it looks at resources and made recommendations to tials will be passed to the institute’s publications is on the quality of a few his SD on appropriate levels of support tenure and review committee, which will key publications rather than sheer num- for his research. “They did a careful job of solicit letters from outside reviewers and bers. Dawid's recommendation to people evaluating me, and once they decided I weigh these along with the BSC review on the tenure track is simple and unsur- was worthy, of seeing that there were and the candidate’s publications and oth- prising: “Do some good work and pub- adequate resources for me to do my job.” er scientific achievements. The ICD lish it.” In the long run, says Dawid, CTC’s tenure committee makes a recommenda- One of the main reasons people have standards for tenure will emerge from tion to the institute's SD, and if the rec- been turned down for tenure by CTC is the cases it reviews and will trickle ommendation is that the candidate weak evidence of scientific independence down to the local tenure-review commit- should be tenured, the SD and the candi- from mentors. The emphasis on this crite- tees, “No one knows exactly what we’re date’s lab or section chief send the candi- rion is new. Under the old system, Wyatt trying to evolve,” says Dawid, but he date's packet to CTC. Two regular CTC notes, there was a fuzzy line between per- anticipates that “a rather high and uni- members plus one ad hoc member are manent, collabo- generally assigned to review the case. At rative scientists the CTC meeting, the SD and the lab and true tenured chief present the candidate’s credentials scientists with to the entire CTC and then leave before independent the committee’s discussion. resources. The

“Some people say [the CTC review] is Board of SDs “did too rigorous,” says Igor Dawid, an not have to NICHD scientist who serves on CTC. “It resolve the issue is definitely quite rigorous. ... Those who of independence are assigned to the cases are preparing fully in every quite carefully and people do take [the case,” Wyatt says. review discussions] very seriously,” Dawid notes that

Dawid says. “The process is supposed to teasing out the take a half hour per case, but it almost issue of indepen- always takes longer.” CTC reviews two dence can be cases per meeting, with meetings called very tough in as often as there are candidates. In instances where addition to the 15 regular members of the work requires CTC, numerous other tenured scientists extensive collabo- have lent their specialized expertise as ad ration or where a hoc members. Ad hoc members are invit- mentor’s achieve- ed to stay for the entire meeting and are ments loom large. welcome to participate in discussion of "In such cases, it both cases under review, but they do not is more important vote. At least eight eligible CTC members to bring to bear must vote on a case, and CTC members other evidence of independence and origi- form set of standards will be applied.” from a candidate’s institute are not nality,” such as outside letters and invita- Korn, who has not yet taken a tenure eligible to vote. tions to speak at international meetings, candidate to CTC, strongly supports the Now the burning question has he says. tenure-track concept but says the feed- become, What is CTC really looking for? Boguski says he was confident as his back of information on tenure standards

“People don’t know what it takes to case came before CTC, even though, as an may not be as good as it used to be. make it at NIH anymore,” says one investigator in the National Center for “This is a loss to the scientific directors,” recently tenured scientist. Kohn says her Biotechnology Information, his achieve- Korn says. He notes that because the recipe for success would include “one ments were made in front of a computer entire board of SDs is only informed part perseverance, one part persistence, screen rather than at a lab bench. “From when a tenure applicant is successful, one part creativity—or more.” Kohn what I hear, there was a good, in-depth there is limited or no feedback to other would also include “independence and discussion of what it means to be an inde- institutes, either on exciting science productivity—but not just publications. pendent scientist at NIH, and how some- being done by the successful tenure can-

... You need to be able to prove that you one like me fits in.” Boguski attributes his didates or, in the case of unsuccessful will be able to sustain the momentum in success to traditional measures of achieve- candidates, on what credentials are your line of investigation with direction ment. “I know what I’ve done, the jour- insufficient to achieve tenure. and focus.” Thus far, teaching and nals where I’ve published. I felt good One scientist who has just started the

16 SEPTEMBE. R-OCTOBER 19 9 5

tenure track worries that, in the final analysis, the judgment of his credentials Tenure-Track, and Tenure-Appointments Process for tenure may be as political as the judg- ments on who would be “grandfathered” STEP 1 The institute, center, or division (ICD) director, scientific director the tenure track.. “The things onto way (SD), and lab or branch chief, in consultation with senior scientists in have gone as I’ve been through this the ICD, determine the need for a new tenure-track position. makes me think it could still be a political process [in the tenure evaluation] at the STEP 2 The SD establishes a search committee with concurrence of the ICD end and may not involve totally objective director and advertises for tenure-track candidates. criteria,” the scientist says. “People have different perspectives on science. What is STEP 3 The search committee evaluates applications, including reference

'hot’ changes dramatically. ... Let’s say you letters, invites promising candidates to campus for interviews and recruit someone in to work on p53 today. seminars, and recommends one to three candidates to the lab or That work might be relegated to special- branch chief, SD, and ICD director. The SD and the ICD director

ized journals in five to 10 years,” when select one name and forward it to the deputy director for intramural the candidate comes up for tenure. research (DDIR) for approval. The DDIR reviews and approves the

Although that scientist is optimistic that selection process and the candidate. the new tenure system will be an improve- ment and that he will, ultimately, achieve STEP 4 The SD and lab or branch chief, in consultation with the potential tenure, he has been saddened by one candidate, prepare and sign a Tenure Track Agreement. A copy is change in his lab that he believes was sent to the DDIR. caused by the new system. “Our lab used signs the Tenure is to fire like an engine; now you have a STEP 5 The candidate Track Agreement and appointed or converted to a tenure-track position, starting the tenure-track bunch of pistons—everyone is tiying to be “clock.” independent,” he says. “I don’t think this

system fosters team work ... but rather cre- STEP 6 Each year, the section or lab or branch chief prepares an oral and ates a possessiveness and territoriality. Peo- written performance evaluation of the candidate. ple are less interactive and more guarded

about what’s theirs and what isn’t.” STEP 7 Approximately every three years, the Board of Scientific Counselors Spiegel, who has had two people (BSC) reviews the candidate’s performance and qualifications for under him come up for tenure before the tenure and decides whether the candidate should be continued in committee, is satisfied that, on the whole, the tenure track, dropped from the track, or advanced for a tenure the new CTC is doing a good job with “to- decision. the-point, incisive, rigorous reviews.” Korn sees the changes in the tenure sys- STEP 8 Before time elapses on the tenure-track clock, the SD and ICD tem as necessary and important. Pointing director review the candidate and decide whether to propose the to the many outstanding and productive candidate for tenure, continue the candidate in tenure track, or drop scientists who were tenured at NIH under him or her from the track. the old system, he says he is optimistic that the new system will be as good or STEP 9 The candidate is informed in writing of the decisions of the BSC, lab better. “It’s like any experiment—we have or branch chief, SD, and ICD director. to wait and see.”

Meanwhile one of NHLBI’s tenure-track STEP 10 If the candidate is advanced for consideration, an ICD Promotion stars, Cynthia Dunbar, echoes her boss’s and Tenure Review Committee is formed to solicit outside letters guarded optimism. Dunbar’s work has and assemble and review credentials. This committee, in been going well and she hopes to be put concurrence with the SD and ICD director, makes a up early for tenure. “I’m not losing sleep recommendation to the NIH Central Tenure Review Committee

over it. I’ve got too many other things to (CTC). worry about,” she says of the tenure system she will be facing. “People STEP 11 CTC reviews the candidate’s credentials and makes a have received more information than previ- recommendation to the DDIR.

ously about how the system is supposed to work, and, in theory, the procedures STEP 12 The DDIR makes a tenure decision. are improved in terms of fairness and reproducibility from institute to institute, STEP 13 The DDIR informs the SD of the decision. In turn, the SD informs the candidate, in writing, of the decision. but only time will tell what the outcome will be.” STEP 14 If the candidate is not approved for tenure or is dropped from tenure track, he or she has one year to wrap up work and find another job.

17 —

NIH and NASA transfer of NASA’s bioreac- AND j tJ monolayer or suspension culture. continued page 1. from tor, a fluid-filled, rotat- The NICHD lab is currently ing-wall cylinder (S’ % origi- using the bioreactor to create a plore the utility of a fiber-optic nally developed to human lymphoid tissue model Co probe for cataract detection allow cells to be cul- for AIDS research. involves no exchange of funds tured in liquid medium The unique properties of the I S. S and resembles traditional collabo- despite the weightless condi- NASA bioreactor, which costs rations between NIH researchers and tions found in the space shuttle—and about $4,500 and is manufactured by scientists in academia. What the two to protect the cell cultures aboard. Synthecon Inc. of Houston, aren’t projects do have in common is that In the first year of the joint project, immediately apparent. Outwardly, the both arose through avenues that which got under way in August 1994, bioreactor looks virtually the same as a NIH researchers often overlook while Zimmerberg’s group, which includes conventional “roller bottle” bulk-cultur- scouting around for collaborative three-dimensional-culturing expert ing device. However, NASA bioengi- opportunities. Leonid Margolis, created a two-room neers have nested a second, slightly core facility in Building 10 that is smaller rotating cylinder within the Space-Age Bioreactors equipped and staffed to assist NIH and roller bottle to form a rotating wall of

Until a friend in academia alerted him NASA researchers who want to use the medium that keeps cultured cells or tis- to one of NASA’s bi-annual “Research bioreactor to address basic biomedical sues in constant, gentle suspension

Announcements,” Joshua Zimmerberg, questions. For its part, NASA has moved rather than allowing them to slosh chief of NICHD’s Laboratory of Theoret- one of its experienced technicians, roughly about as they do in a standard ical and Physical Biology, didn’t know Wendy Fitzgerald, from Houston to roller bottle. The inner wall is semiper- that NIH scientists were even eligible Bethesda to train scientists in the fine meable, to allow the free exchange of for NASA grants, let alone that one of points of using the bioreactor. In addi- oxygen and carbon dioxide within the NASA’s proposal requests dovetailed tion, the collaborative agreement calls bioreactor. neatly with his lab’s interests in three- for Zimmerberg and his colleagues to Although NASA scientists designed the dimensional tissue culture and the use the NASA bioreactor to create tissue bioreactor to protect cells during space imaging of complex tissues [see box]. cultures for other NIH projects that travel, they decided to pursue on-ground The request called for research projects require a higher level of cell organiza- applications after discovering that cells that might facilitate the space-to-ground tion than is available through standard cultured in the bioreactor’s low-turbu-

Histoculture: Entering the Third Dimension by Birgit Art der Lan, NICHD

o adapt John Donne’s familiar phrase, no cell is an island. formed using confocal fluorescence microscopy have much bet- Although many types of cells can be grown through con- ter spatial resolution because they have less out-of-focus light. T ventional culture techniques, such methods provide, at Each plane of focus can also be conveniently stored in a com- best, a pale imitation of the complex microenvironment that puter database, thus facilitating quantitative analysis and allow- influences cell growth and activity within living tissue. Factors ing the reconstruction of three-dimensional images. such as proximity to blood vessels and interactions with other So far, Zimmerberg, Margolis, Boris Baibakov, and Svetlana types of cells don’t come into play when cells are cultured Glushakova have used their three-dimensional histoculture and under standard in vitro conditions. Recognizing a need to devel- imaging techniques to track individual melanoma cells as they op better ways of studying cells in a biomedically relevant con- invade lung tissue. Using cubes cut from human tonsils, the text, Leonid Margolis’ group at NICHD’s Laboratory of Theoreti- researchers have also observed the fusion of healthy T cells cal and Physical Biology has for the past seven years been with various types of human cells expressing the envelope

working with methods that make it possible to culture and glycoprotein of the human immunodeficiency virus (HIV). image three-dimensional blocks of tissue. The lab has also used its culturing and imaging expertise to

It has been known for decades that tiny chunks of tissue can trace the innervation of the epithelial cells in rat tongue tissue be cultured for up to six weeks on collagen sponges floated in by neuroblastoma and trigeminal ganglia cells and to observe growth medium—a system used primarily for studying the tissue differentiation of human breast tissue in vitro. cells’ invasion of the sponges. The NICHD group has gone one step further, however, by using the collagen-substrate system to Additional Reading study cell-cell interactions within tissues. Furthermore, Margolis, L.B. Margolis, S.E. Glushakova, B.A. Biabakov, C. Collin, and.J. Zimmerberg. “Confocal microscopy of cells implanted into tissue blocks: cell migration in long-term histocul- Joshua Zimmerberg, and their colleagues have taken advantage tures.” In Vitro Cell Dev. Biol. 31 221-26 (1995). , of a relatively recent technological advance in light L.B. Margolis, S.E. Glushakova, B.A. Baibakov, C. Collin, and J. Zimmerberg. "Syn- microscopy'—laser confocal fluorescence microscopy-to analyze cytium formation in culture human lymphoid tissue: fusion of implanted HIV glycoprotein 120/41 -expressing cells with native CD4+ cells." AIDS Res. Hum. Retroviruses the native architecture and dynamic processes within such tissue 11 697-704 (1995). specimens, which are too thick for conventional microscopy. , L. Margolis, B. Baibakov, C. Collin, and S.A. Simon. “Dye-coupling in three-dimensional Compared with standard fluorescence microscopy, images epithelium. " Vitro Cell Dev. Biol. 456-61 (1995). histoculture of rat lingual In 31 ,

18 September - October 19 9 5

lence environment on Earth grew into the bioreactor, NIDDK’s three-dimensional masses that bore a Gary and Liliane Striker greater structural and physiologic resem- report promising early blance to natural tissues in vivo than did results in culturing cells cultured by traditional methods. mesangial glomerular Although the fledgling NIH-NASA cells that more closely bioreactor venture has yet to yield any resemble those found publications, Zimmerberg says the most within normal kidneys impressive results from the bioreactor than cells grown by tra- to date fall into two general categories, ditional techniques. “The engineering tissue-like structures from initial bioreactor was too single cells and keeping tissue alive large, says Liliane Strik- with its architecture intact in vitro. er, who says the bioreac- The first set of achievements—mak- tor center helped to ing cells clump together and differenti- modify the device for ate to form tissue-like spheroids of up her group’s experimental to 3 mm in diameter—was accom- needs. plished by using the bioreactor exactly Although he only as NASA envisioned. “At this point in began testing the biore- time ... if someone wants to grow actor a couple of months spheroids for their research, this is the Confocal micrograph of bovine endothelial cells grown ago, NCI’s William best environment in which to grow in the NASA bioreactorfor 1 1 days. Cells attach to Stetler-Stevenson says spheroids,” Zimmerberg says. However, microcarriers and. due to the low shearforces in the biore- that so far, he’s seen the NICHD team’s recent success in actor, bridge them. [Courtesy Leonid Margolis, NICHDJ nothing to make him maintaining 2- to 3-mm fragments of stop exploring the use of lymphoid tissue in the bioreactor for up for a three-year postdoc position. That’s the device in his studies of tumor-cell to three weeks represents a more inno- on top of the training and support ser- invasion. In their experiments, Stetler- vative application of the device. The vices of two technicians and a histolo- Stevenson and his colleagues are ultimate goal of such efforts is to devel- gist funded by NASA. attempting to grow tumor-like spher- op a uniform, controlled environment To date, eight groups of intramural oids using a human melanoma cell in which to study various aspects of scientists have tried or are attempting to line transfected with genes encoding human immunodeficiency virus (HIV) use the NASA bioreactor in their pro- tissue inhibitors of metalloproteinases infection in human lymphoid tissue. jects, with vaiying results. On the disap- (TIMPs), which suppress cell invasion NICHD researchers have also learned pointing side, NHLBI’s Maurice Burg by inhibiting matrix metalloproteinases. a few things about the limitations of says that although the bioreactor did The researchers hope to use those bioreactor technology. For example, keep renal medullary cells alive outside spheroids to examine how varying the Zimmerberg states that a “big draw- of the intact kidney somewhat longer expression of TIMPs affects cell adhe- back” of the bioreactor is its inability to than did other techniques, it did not sion and migration in culture. run multiple controls in the same keep those cells “lively enough long NIA’s Steven Sollott is enlisting the experiment. “In a 96-well plate, you can enough” for Burg’s lab to perform its bioreactor in his efforts to develop bet- have 96 different conditions for cells desired series of experiments. But Burg ter in vitro models for studying cardio- growing, whereas in the bioreactor, it’s says his experience shouldn't dissuade vascular disease. In experiments just just one homogeneous volume,” he others from testing the bioreactor: recently started, Sollott’s group is using says. The lab is currently tiying to get “They [the bioreactor center staff] were bioreactors in attempts to culture vascu- around that problem by wrapping dif- good people to work with—they work lar smooth muscle cells and endothelial ferent types of tissues in separate quickly and well.” Like Burg, NICHD’s cells in a manner that maintains the dif- “envelopes” of agarose before they are David Klein found that the bioreactor ferentiation seen in the body. Under placed in the bioreactor. did little to advance his research. “We standard cell-culture techniques, such The rewards may be substantial for failed to get encouraging results,” says cells de-differentiate and behave differ- those NIH research teams whose Klein, who had hoped the bioreactor ently than they do in vivo. Sollott and research projects happen to mesh well environment would allow pineal gland his colleagues are also co-culturing vas- with the bioreactor’s strengths, accord- cells to proliferate more freely and cular smooth muscle cells and endothe- ing to Zimmerberg. For each of the respond better to challenges from stim- lial cells in the bioreactor in hopes that three or four intramural projects ulants such as norepinephrine than they they may form structures similar to deemed to be the best tests of the do in standard cell-culture conditions. small blood vessels. “We’re very excited bioreactor’s scientific potential, the Others have had a bit more luck. about the potential of the bioreactor,” NASA-NIH center will provide funding Using a scaled-down version of Sollott says.

19 The NIH Catalyst

“It’s hard to test out this technology to the NEI director, says he was for a spur-of-the-moment discussion simply by reading about it,” says Zim- thumbing through TechReach, a with the NASA scientist. merberg, urging more intramural newsletter put out by the Great Lakes “Our realms of expertise are totally researchers to stop by and check out Industrial Technology Center in Cleve- different—I know the biology and he what's going on with the 18 or so NASA land, when an article reprinted from [Ansari] knows the physics. But we may bioreactors the center has on hand. the Federal Lab Consortium Newslink be able to find some common ground Although NASA naturally would like to grabbed his attention. The topic? A in the middle,” says Zigler. see its brain child become a standard small fiber-optic probe—originally NASA’s noninvasive probe, which is fixture in biomedical labs around the designed to measure the growth of about the size of a pencil, consists of globe, Zimmerberg says that space protein crystals in experiments aboard two optical fibers. The first fiber trans- agency officials have not mits a safe, low-power interfered with his research laser beam into the and have made it clear they eye. The second fiber want the assessment of the detects the laser’s light bioreactor to be as objective as it is scattered within as possible. “They were the eye and bounces very excited about the idea back toward the probe. of getting a completely The resulting dynamic unbiased evaluation,” he light-scattering data are says. “I’ve received nothing processed by computer but support from the NASA to generate a three- people. ... There’s no dimensional scan show- ” micromanagement . ing both the size and Other institutions where location of protein NASA bioreactors are being aggregates in the eye’s or have been tested include lens. Some aggregation Harvard University and the of proteins in the lens Massachusetts Institute of is part of the normal Technology in Cambridge, aging process, but Mass., New England Dea- when the clumps of coness Hospital in Boston, proteins grow too

Huntington Medical Re- Schematic drawing of NASA ’s fiber-optic eye diagnostics device. large, they precipitate search Institutes in Pasade- [Courtesy R.R. Ansari, NASA Lewis Research Center, Cleveland.] and create opaque, na, Calif., The Wistar Insti- light-scattering centers. tute in Philadelphia, and the University the space shuttle—now being tested as Such light scattering degrades the of Texas Health Science Centers at San a “compact eye diagnostics device” by optical transparency of the lens, causing Antonio and Houston. But what NASA Rafat Ansari at NASA’s Lewis Research cataracts. Currently, surgical replacement finds particularly appealing about the Center in Cleveland. of the clouded, natural lens with a plastic

NIH center is the easy access it pro- “I decided to call Ansari out of the lens is the only treatment for cataracts, vides to a wide range of top-notch bio- blue and ask him all about it [the and more Medicare dollars—$3-4 billion medical researchers. “What NIH brings device], and he was in 1991 —are used to to the table is a critical mass of scientif- reasonably aggressive f pay for cataract sur- oth- ic intellect that can be pulled together on following up,” says | gery than for any and applied to a given problem,” says Ellwein. During his first 5 er single procedure. Neal Pell is a project director for face-to-face meeting There are no drugs on , biotechnology at NASA’s Johnson Space with Ansari in Bethes- the market in the Unit- Center in Houston. “Within academia, da, Ellwein realized that ed States now to pre- you have to go to multiple universities the NASA probe might vent or slow cataract stretched across the United States to get prove useful in the ani- formation. the same level of expertise, and you mal studies of potential Unlike the clinical often cannot get the same level of anti-cataract drugs collaborations Ansari cooperation.” being conducted in has established with NEI’s Laboratory of the Wills Eye Hospital Seeing Eye to Eye? Mechanisms of Ocular and Drexel University NEI’s interest in another NASA device Disease. He called the in Philadelphia, Zigler using the can be traced to some fortuitous page head of the lab, J. envisions NASA probe primarily Hipping. Leon Ellwein, special adviser Samuel Zigler Jr., over J. Samuel ZiglerJr.

20 September-October 19 9 5

in mouse models to screen for drugs up to seven extramural proposals for The researchers want to use the snail that may halt or slow the progression of “Neurolab” experiments that will use the system to analyze the biophysical prop- cataract formation. Preliminary results space shuttle as a unique environment erties of memory and visual-vestibular from mouse eye lenses that Zigler sent in which to study neurological develop- associative learning under microgravity to Ansari indicate that the fiber probe ment and function. And the list goes on. conditions. Some astronauts have expe- can detect protein aggregations indica- Among the NIH scientists who have rienced short-term memory deficits tive of early-stage cataract formation. It served as consultants on extramural after space travel, but the basic mecha- remains to be seen whether the device projects is NINDS's Daniel Alkon, who nism underlying those deficits is can provide similar data when used on says the contacts he developed through unknown. Before the snails go up in living animals. Currently, researchers such work paved the way for scientific space aboard a U.S. space shuttle—an must rely on slit-lamp exami- event likely to occur within nations, which detect dark the next five years—Alkon areas or shadows in normally says several Japanese scien- homogenous tissue, to make tists will probably come to relatively subjective assess- NIH to study his lab's tech- ments of changes within the niques. In exchange, Alkon animals’ lenses. “If the differ- hopes that he can share his ence between groups [in an Japanese colleagues’ techno- anti-cataract-drug study] is logical advances in high-res- small, it is difficult to estab- olution microscopy to lish anything with current obtain visual images of neu- methods,” Zigler says. “This ronal branches at the same [NASA] device would be time that electrophysiologi- most valuable to us if it cal recordings are being would provide an easy and made. reliable way to monitor In addition to the scien- changes in the lenses of the tific incentives, the growing animals over time.” emphasis on efficiency in In addition to in vivo drug government may serve to studies, Zigler says, he may promote greater interactions try out the probe during in between NASA and NIH’s vitro experiments aimed at better char- exchanges between his lab and the intramural community. “Collaborations acterizing how a molecular chaperone Japanese space agency, NASDA. Alkon will occur whenever an opportunity to called alpha-crystallin acts to prevent has provided the Japanese researchers cooperate and coordinate will move the aggregation of denaturing proteins with his simple-system model of learn- the science forward faster and to a in the lens of healthy eyes. Ansari sug- ing and memory, the snail Hermissen- higher plane and when there will be gests that other possible biomedical da crassicornis. For their part, the cost savings to both agencies in applications of the NASA probe include Japanese scientists are working on accomplishing mutual goals,” says detection of cholesterolosis in the eye’s developing an aquaculture environ- Snow, adding, “Both NASA and NIH aqueous humor and of disorders affect- ment in which the snails can be trained scientists possess enormous expertise ing the vitreous humor. and maintained aboard a spacecraft. and dedication.”

Snails in Space Seeking Postdocs? Consider PRAT To date, the bulk of the interactions Laboratories interested in recruiting a postdoc with pharmacological or related between NIH and NASA on scientific research skills should be aware that the deadline for NIGMS’s Pharmacology projects has been conducted through Research Associate (PRAT) program is Jan. 1. Projects by PRAT fellows may be in extramural programs. NIDCD and NASA the areas of signal transduction, drug metabolism, immunopharmacology, chemistry have established a ground-based center and drug design, structural biology, endocrinology, neuroscience, and clinical phar- for balance and vestibular research at macology, for example. During their two-year appointments, funded by NIGMS, fel- Northwestern University Medical School lows receive competitive salaries, supplies, and travel funds to support research in in Chicago. Through the Space Tissue their preceptors' labs. Postdocs who hope to obtain PRAT funding should apply Loss Program, NIAMS and NASA are together with a preceptor before coming to NIH, even if they plan to come earlier funding extramural projects to conduct through other funding arrangements. Only U.S. citizens or permanent residents are a series of experiments aboard the eligible. Any tenured NIH scientist may identify a PRAT candidate and apply to space shuttle that focus on the changes become a PRAT preceptor. To receive a 1995-96 PRAT Fact Sheet, contact the PRAT in bone and muscle cells during space program assistant (phone: 594-3583; e-mail: [email protected]). flight. NINDS and NASA expect to fund

21 The NIH Catalyst

NIH Collaborators Receive Eastern Bloc Grants

ive researchers who are collaborating with NIH intramural signals and is regulated. Liptak, a professor of biochemistry at the scientists are among the 90 scientists in Eastern Europe and L. Kossuth University in Debrecen, Hungary, is working with F the former Soviet Union chosen to receive a new type of NICHD’s Vince Pozsgay to develop a vaccine against Shigella son- grant from the Howard Hughes Medical Institute (HHMI), based net, a parasite that causes dysentery. Jaskolski, who is an associ- in Bethesda. The grants range from $22,000 to $35,000 annually ate professor at the Institute of Bioorganic Chemistry in Poznan, for five years, and 60 of the grants also provide $2,500 to $3,500 Poland, collaborates with Alexander Wlodawer of NCI's Frederick per year for collaborating scientists. Cancer Research and Development Center (FCRDC) on studying Acknowledging that the grants are small by U.S. standards, the structure of certain enzymes useful for treating leukemia. HHMI President Purnell Choppin says Nedospasov is working with Nancy

he thinks that the money will still go a Rice, also of NCI-FCRDC, on how the long way toward strengthening interna- transcription factor NF-xB/Rel regulates tional scientific ties and helping gene expression by interacting with researchers in former Eastern Bloc DNA. Nedospasov is the head of the nations to modernize their labs and cytokine molecular biology unit at the undertake new experiments. The funds V.A. Engelhardt Institute of Molecular can be used to pay for salaries, travel, Biology in Moscow. Together with supplies, equipment, computer and NIEHS’s Michael Resnick, Gordenin is communication services, and journals. exploring the genetic consequences of Selected from more than 2,000 inverted DNA repeats, which are com- applicants, the grant recipients with mon in many organisms. Gordenin is a collaborative links to NIH intramural leading research fellow in the physio- scientists are Laszlo Hunyady and logical genetics laboratory at St. Peters- Andras Liptak of Hungaiy, Mariusz Jaskolski of Poland, and Sergei burg State University in Russia. Nedospasov and Dmitry Anatoly Gordenin of Russia. “We were veiy impressed with the quality of their [the grant

Hunyady is an associate professor in the physiology depart- recipients’] research, especially since so many of them are work- ment of Semmelweis University of Medicine in Budapest. In con- ing under extremely difficult conditions,” HHMI’s Choppin says.

junction with NICHD’s Kevin Catt, Hunyady is examining how the receptor for angiotensin, a polypeptide in the blood, generates -Anne Blank, N1CHD, and Loma Heartley 4- IK

Hot Methods (MPO)] to tubes and vortex lightly. Incu- centrifuging at 1200 rpm in swinging- continued from page 13- bate in an ice bath for 1 h in the dark. bucket rotor centrifuge at 4-6 °C for 10 6. Wash cells by adding 2 mL PBA, cen- min, decanting supernatant, and resus- Staining Cell-Surface, Intracellu- trifuging at 1400-1600 rpm in a swinging- pending pellet by agitating tube or light lar, and Intranuclear Antigens bucket rotor centrifuge at 4-6 °C, and vortexing. Repeat wash. 1. Place 5-20 mL of PE-conjugated mono- decanting supernatant. 4. Add to resuspended cells 1 mL 70% clonal antibody for the detection of the 7. Resuspend pellet in 0.5 mL 1% cold (4-6 °C) ethanol per 1 x 106 cells antigen of interest (e.g., CD3 for T cells) paraformaldehyde in isotonic saline, pH while vortexing gently. Incubate in the into labeled, flow-cytometer-compatible 7.4. Analyze samples on flow cytometer dark overnight at 4-6 °C to fix cells. tubes. Add 150 mL PBA and 100 mL within 24 hours. Other permeabilization 5. Wash by adding 4 mL cold PBS (4-6 °C), washed cells. Incubate for 30 min at room reagents can be used, but method must centrifuging at 1600 rpm in swinging- temperature in the dark. be optimized for each reagent. bucket rotor centrifuge at 4-6 °C for 5 2. Add 2 mL lx Ortho PermaFix* to each min, decanting supernatant, and resus- tube, vortex for a second or two, and Surface-Antigen and DNA- pending pellet by agitating tube or by incubate for 40 min at room temperature Content Detection light vortexing. in the dark. 1. For whole blood and bone marrow, 6. Add 500 mL PI/RNase solution (50 3- Centrifuge for 8 min at 1400-1600 rpm begin by separating mononuclear cells by mg/mL PI, 200 units RNase/mL in PBS). in a swinging-bucket rotor centrifuge at density gradient (e.g., by using Ficoll Incubate 45 min to 1 h at room tempera- 6 °C. Aspirate off the supernatant and Hypaque). Wash this cell suspension or ture. Analyze samples on flow cytometer vortex the pellet thoroughly. others, such as cells from a cultured cell within 4 h. 4. If specimen is whole blood or bone line or cells teased from lymph node, marrow, lyse the red blood cells by with PBS prior to staining. Contacts Maryalice Stetler-Stevenson, NCI adding 2 mL PBS, vortexing thoroughly, 2. Place 100 mL PBS in labeled, flow- Phone: 402-1424, fax: 402-7762 and incubating at room temperature for 10 cytometer-compatible tubes and add an E-mail: [email protected] min in the dark. Vortex again and cen- appropriate amount of FITC-conjugated Gerald Marti, FDA trifuge for 8 min at 1400-1600 rpm in a antibody for detection of the surface anti- Phone : 827-0453, fax: 827-0449 swinging-bucket rotor centrifuge at 4-6 °C. gen of choice. Add one million to two E-mail: [email protected] Decant supernatant and vortex gently. million cells in 100 mL. Incubate in the Robert Wersto, NCI 5. Add 20 mL appropriately diluted anti- dark at 4-6 °C for 30 min. Phone 496-3776, fax: 402-0043 body [e.g. TdT or myeloperoxidase 3. Wash by adding 4 mL cold PBS (4-6 °C),

22 ! — .

September-October 19 9 5

A FARE Deal Tapping the Talents of “Re-entry” Scientists Fellows Research Award

ostdoctoral and clinical fellows he research community is encouraged to attend a special workshop

should start fine-tuning their this fall showcasing the scientific achievements of participants in the abstracts now for the second P T Offiice of Research on Women’s Health’s (ORWH’s) Re-entiy Pro- annual Fellows Award for Research gram. The 1 1/2-day workshop, entitled "Re-Entry Into Biomedical Research Excellence (FARE) competition. Last Careers,” will take place Nov. from 8:30 a.m to p.m. and Nov. 14 year, about 450 fellows applied and 30 13 5:00 were chosen to receive the merit-based from 8:30 a.m to 12:00 p.m. at the Natcher Building Conference Center.

awards, which provide up to $1,000 The workshop’s first day will feature keynote speakers, scientific presen- toward travel expenses to a domestic tations, and poster sessions by scientists who were awarded grants through scientific meeting. The deadline for sub- ORWH’s Re-entiy Program. The second day will focus on career options, mitting applications for this year’s FARE NIH resources, and mentoring and networking to facilitate productive sci- awards is Dec. 15, and the winners will be announced in February. All postdoc- ence careers. Issues, concerns, and experiences of principal investigators toral and clinical fellows—including for- and mentors will also be discussed. The workshop, sponsored by ORWH, eign and visiting fellows are invited to — is free and open to the public. For more information, contact Joyce Rudick apply for the awards, which are based (phone: 402-1770). on a peer review of submitted abstracts. ORWH has established two pilot programs—intramural and extramural Application forms and further information are available from each ICD’s repre- to encourage fully trained women and men to resume active research sentative on the N1H Fellows Committee careers after taking a break to meet family demands. Since 1992, the re-

and from the Office of Science Educa- entry program for intramural scientists, which was developed in conjunc- tion (phone: 496-3887). n tion with the Office of Education, has supported the placement of three sci-

entists, two at NCI and one in NINDS. The re-entry program for extramural

scientists has supported the placement of 26 researchers over those same Thomas Fleisher, CC Phone: 497-4120, fax: 402-1884 three years. E-mail: [email protected]

The Flow Cytometry Consortium Web Page: http://www.cber.nih.gov/welcome.html

References Ns-Tionzl TrC-t\icdeS 'The. FIVE YEARS of "wELUjItS NOT DEMT 1. H.M. Shapiro. “How a Flow Cytometer Works." In: Practical J>ungeorv lyrionmCS ^ COLLEGE ySIX LIKE I'M ANM. t>- Flow Cytometry 2nd ed. H.M. Shapiro, ed. New York: Alan YEARS OR STEVIE — R. Liss. (1994)." of rtfAbufiTT School, rt>WLy I'M JUST A You HEAR THAT? 2. R.W. Vogt, Jr., G.E. Marti, and A. Schwartz. “Quantitation Four ANb yeARs Of poST-boc. 1'n\ calibration of fluorescence intensity for clinical and PoST-t>oc7oR/)(_ YOU'RE GoiNG-To research applications of immunophenotyping by flow JUST A&flUWT— wor k— And TH ATi> M6BICAL SCHOOL/ cytometry." In: Reviews of Biotechnology and Bioengineer- A FooT-SoLbiER ing. vol. H.W. Tyler, ed. Norwood, Ablex Publish- ALL THE Space 1, N.J.: IN 1H£ WARo/V ing Corp. (1995). you 66T? ITS CANCER. IM 3. M.A. Moltz, Gong, F. Taraganos, and Z. Darzynkiewicz. J. UKEA MONK'S r>ow/v IN THE “Flow cytometric detection of apoptosis: comparison of the CELL/ TRENCHES /Z'm, assays of in situ DNA degradation and chromatin changes.” wH/ havew't Cytometry 15, 237-44 (1994). THEY S-IVENyou DOING-THE biRTy 4. J. Gong, F. Traganos, and Z. Darzynkiewicz. “A selective W/oRK THAT N0- procedure for DNA extraction from apoptotic cells applica- Bobf CARESABoiCT. ble for gel electrophoresis and flow cytometry.” Analy. t Don't aim Biochem 218, 314-19 (1994). Ays

5. L.L. Wheeless, J.S. Coon, C. Cox, A.D. Deitch, R.W. deVere UKEtTj Som6T)/hes White, Y, Fradet, et al. “Precision of DNA flow cytometry i HA-mrr. but in inter-institutional analysis.” Cytometry 12, 405—12 (1991) XT's MY Jog — 6. R.P. Wersto, R.L. Liblit, and L.G. Koss. “Flow cytometric DNA analysis of human solid tumors: a review of the inter- My lurry to My youboNTiwbegsimC

pretation of DNA histograms.” Pathol. 22, 1085-98 ' Human CouNTRy! iM-the last owE (1991). of b&fgwsE/ yviA 7. M. Stetler-Stevenson, L.J. Medeiros, and E.S. Jaffe. KNOWj-WBAEARE “Immunophenotypic methods and findings in the diagnosis strange Forces of lymphoproliferative diseases.” In: Surgical Pathology of OUT WERE. CMiyop the Lymph Nodes and Related Organs. 2nd ed, Philadelphia: MOUTH E CHAOS? W.B. Saunders Co., pp, 22-57 (1995). IN here like rats! [MOTHERjboN let' 8. R.P Wersto V and M. Stetler-Stevenson. “Debris compensation Your S0NSC1Z0WUP I Kg SApt>iNES/ of DNA histograms and its effect on S-phase analysis." JL ^ f™B£?OST-J)ocs! Cytometry 20, 43-52 (1995). j

23 . The NIH Catalyst

Catalytic Reactions

n this issue, we are asking 1) What do you think poses the greatest health or safety risk to NIH staff? What specific sugges- for your reactions in four I tions do you have for improving safety and security at NIH? areas: safety and security, NCI’s changes, tips for our Hot Methods Clinic, and postdoc concerns. Send your responses on these 2) What is your reaction to the changes under way in NCI’s intramural program? What advice topics or comments on would you give the institute’s new director? other intramural research concerns to us via e-mail: [email protected]. gov; fax: 402-4303; or mail: Building 1, Room 334. 3) Do you have any suggestions or comments about the flow cytometry techniques featured in this issue’s Hot Methods Clinic? What methods would you like to see covered in future issues?

In Future Issues. .

Postdoc Life: When Dreams And Reality Collide

Linking Scientific 4) We plan to devote our next issue to postdoc concerns, so now is the time for postdocs and Devices to Computers their mentors to fire away. What do you think is the biggest challenge facing postdocs at NIH Cultural Crossroads, today? What can be done to improve the postdoc experience? And, postdocs, what are your pet The NIH Experience peeves about life at NIH? a Chromosome Mapping: Stretching the DNA

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