DOCSLIB.ORG

Structural Characterization and Subunit Communication Of

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

STRUCTURAL CHARACTERIZATION AND SUBUNIT COMMUNICATION OF ESCHERICHIA COLI PYRUVATE DEHYDROGENASE MULTIENZYME COMPLEX By Jaeyoung Song A dissertation submitted to the Graduate School-Newark Rutgers, The State University of New Jersey In partial fulfillment of requirements for the degree of Doctor of Philosophy Graduate Program in Chemistry Written under the direction of Professor Frank Jordan and approved by Newark, New Jersey May, 2011 ABSTRACT OF THE THESIS Structural Characterization and Subunit Communication of Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex By Jaeyoung Song Thesis Director: Professor Frank Jordan The pyruvate dehydrogenase multienzyme complex (PDHc) from Escherichia coli (E. coli) is the best characterized of the 2-oxoacid dehydrogenase complexes. The complex plays a role as catalyst for the conversion of pyruvate to acetyl Coenzyme A (acetylCoA) by three enzyme components in the complex. The complex is comprised of 24 copies of the dimeric pyruvate dehydrogenase (E1ec; 99,474 Da), a cubic core of 24 copies of dihydrolipoamide acetyltransferase (E2ec; 65,959 Da), and 12 copies of dihydrolipoamide dehydrogenase (E3ec; 50,554 Da) (1-3). The crystal structure of the E. coli pyruvate dehydrogenase complex E1 subunit (E1ec) has been deterimined, and there were three missing regions (residues 1-55, 401-413, and 541-557) remaining absent in the model due to high flexibilities of these regions (4). Most bacterial pyruvate dehydrogenase complexes from either Gram-positive or Gram-negative bacteria have E1 components with an 2 homodimeric quaternary structure. In a sequel to our previous publications (5-8), the first NMR study on the flexible regions of the E1 component from Escherichia coli and its biological relevance ii was presented. In the study, sequence-specific NMR assignments for six residues in the N-terminal 1-55 region, and for a glycine in each of the two mobile active center loops of the E1 component, a 200 kDa homodimer was made. This was accomplished by using site-specific substitutions and appropriate labeling patterns, along with a peptide with the sequence corresponding to the N-terminal 1-35 amino acids of the E1 component. To study the functions of these mobile regions, the spectra were also examined in the presence of: (a) a reaction intermediate analog known to affect the mobility of the active center loops, (b) an E2 component construct consisting of a lipoyl domain (LD) and peripheral subunit binding domain (PSBD) and (c) a peptide corresponding to the amino acid sequence of the E2 peripheral subunit binding domain. Deductions from the NMR studies are in excellent agreement with our functional finding, providing clear indication that the N-terminal region of the E1 interacts with the E2 peripheral subunit binding domain, and that this interaction precedes reductive acetylation. The results provide the first structural support to the notion that the N-terminal region of the E1 component of this entire class of bacterial pyruvate dehydrogenase complexes is responsible for binding the E2 component. Among three components of PDHc, E2ec consists of 24 chains, and in the overall reaction, the lipoyl domain is reductively acetylated by E1ec and pyruvate, and S- acetyldihydrolipoyl domain transfers acetyl group to Coenzyme A leading acetylcoenzyme A production. Even though the precise number of E2 subunits is still ambiguous, in many cases including human PHDc (9), the sum is a multiple of three chains indicating that multiples of chains can affect the acetyl transfer to CoA by interchain acetyl transfer. To answer this question, the E2 component from E. coli iii specifically designed with only a single lipoyl domain (LD, 1-lip E2ec), rather than the three lipoyl domains found in the wild type enzyme (3-lip E2ec) was used. Earlier, it was shown that the activities of these two enzymes are virtually the same, while the 1-lip E2ec provides obvious advantages for mechanistic studies (10). At the same time, it is also important to point out that there indeed are other sources of the E2 component with only a single domain, such as from Mycobacterium tuberculosis. To study the question, two constructs of the 1-lip E2ec were prepared, one in which the lysine on the LD ordinarily carrying the lipoic acid is changed to alanine (henceforth K41A), and a second one in which the histidine believed to catalyze the transacetylation in the catalytic domain (CD) is substituted to A or C, H399C and H399A. The first is incompetent towards posttranslational ligation of the lipoic acid, hence towards reductive acetylation. The second one is incompetent towards acetylCoA formation, by virtue of the absence of the catalytic histidine residue. This is a biochemical version of a classical crossover experiment, as should the reaction proceed within one chain the two constructs should each be inactive either together or individually. On the other hand, should the reaction proceed by an interchain mechanism, addition of the two constructs should produce measurable activity. Both kinetic and mass spectrometric evidence supported the second scenario. Hence, plausible model/explanation for the multiples of three chains present in each E2 component as well as for their assembly was suggested. iv ACKNOWLEDGEMENTS First and foremost, I would like to express my sincere gratitude to my advisor Prof. Frank Jordan for the continuous support, motivation, enthusiasm, and immense knowledge of my Ph.D study. I would like to thank to Dr. Natalia Nemeria for the help and the contribution of didomain E2ec, thank to Dr. Yun-Hee Park for the contribution of human didomain and tridomain, and thank to Dr. Sachin Kale for the contribution of His6-tag E1ec. Last but not least, I would like to thank to my thesis committees, Prof. Edgardo T. Farinas, Prof. Richard Mendelsohn, and Prof. Darren Hansen for kind consideration of my thesis, to my family for the immense support, to Dr. Lazaros Kakalis for the help with NMR, to Dr. Roman Brukh for the help with FTMS, and to Anand Balakrishnan for sharing of research interests and discussion. v TABLE OF CONTENTS ABSTRACT OF THE THESIS .......................................................................................... ii ACKNOWLEDGEMENTS ................................................................................................ v LIST OF TABLES ............................................................................................................ xii LIST OF FIGURES ......................................................................................................... xiii LIST OF SCHEMES........................................................................................................ xvi ABBREVIATIONS ........................................................................................................ xvii CHAPTER 1. Structural and Functional Characterization of N-Terminal Region in the E1ec Component of the Pyruvate Dehydrogenase Complex .............................................. 1 1.1. INTRODUCTION ................................................................................................. 1 1.2. MATERIALS and METHODS ............................................................................. 6 1.2.1. Materials ......................................................................................................... 6 1.2.2. Plasmid purification ........................................................................................ 7 1.2.3. Site directed mutagenesis ................................................................................ 9 1.2.4. Protein expression ......................................................................................... 14 1.2.5. Protein purification ....................................................................................... 16 1.2.6. 1-lip E2ec purification .................................................................................. 18 1.2.7. His6-tag didomain of 1-lip E2ec (E21-190) purification ................................. 20 1.2.8. Purification of His6-tag lipoyl domain of 1-lip E2ec .................................... 22 1.2.9. E3ec purification ........................................................................................... 23 vi 1.2.10. SDS-PAGE ................................................................................................ 24 1.2.11. Concentrating protein using Millipore Centriprep Centrifugal Filter Units 26 1.2.12. Determination of protein concentration by Bradford assay ....................... 27 1.2.13. Overall activity measurement .................................................................... 29 1.2.14. Structural analysis of mobile regions of E1ec using Nuclear Magnetic Resonance .................................................................................................................. 29 1.3. RESULTS ............................................................................................................ 31 1.3.1. Kinetic properties of the C-terminal His6-tag E1ec indicates an acceptable surrogate for the non His6-tag version ....................................................................... 31 1.3.2. 1H-15N TROSY-HSQC spectrum shows well dispersed resonances, but only a limited number from E1ec ...................................................................................... 34 1.3.3. Glycine auxotroph assisted in the assignment of resonances to glycines ..... 36 1.3.4. 3D HNCO revealed Glycine 47 in N-terminal region
Recommended publications
  • Ribonuclease and Deoxyribonuclease Activities in Experimental and Human Tumors by the Histochemical Substrate Film Method*

    Ribonuclease and Deoxyribonuclease Activities in Experimental and Human Tumors by the Histochemical Substrate Film Method*

    Ribonuclease and Deoxyribonuclease Activities in Experimental and Human Tumors by the Histochemical Substrate Film Method* R. DAOUSTJANDHARUKOAMANOÕ (Laboratoires de Recherche, Institut du Cancer de Montréal,Hôpital Notre-Dame et Universitéde Montréal,Montréal,Canada) SUMMARY The ribonuclease and deoxyribonuclease activities of 65 experimental and human tu mors (32 different types) have been examined by histochemical substrate film methods. A same general pattern was obtained for the distribution of both nucleases in the various types of experimental and human tumors. The connective tissue stroma and the necrotic regions of the tumor masses showed various levels of nuclease activity, whereas the neoplastic cells showed no demonstrable activity. It appears that deficien cies in ribonuclease and deoxyribonuclease activities represent general properties of cancer cells. The possible significance of the losses of nuclease activities in carcinogenesis is dis cussed. Studies on nucleases by histochemical methods MATERIALS AND METHODS have shown that losses of ribonuclease (RNase) The experimental tumors used in the present and deoxyribonuclease (DNase) activities take study were mostly rat, mouse, and hamster trans- place in rat liver during azo-dye carcinogenesis (1, plantable tumors (see Table 1). The tumor-bearing 6). The loss of RNase activity is progressive and animals were obtained from commercial or private occurs before parenchymal cells become cancerous, sources, and the tumors were used as supplied or whereas the loss of DNase activity is abrupt and closely associated with the neoplastic transforma TABLE1 tion of parenchymal cells. EXPERIMENTALTUMORS If a loss of RNase or DNase activity plays an important role in tumor formation, the lack of SpeciesRat"""MouseHamsterTumorPrimary demonstrable nuclease activity observed in rat hepatomaNovikoff primary hepatomas should also be observed in a hepatomaWalker variety of tumors.
  • HOXB6 Homeo Box B6 HOXB5 Homeo Box B5 WNT5A Wingless-Type

    HOXB6 Homeo Box B6 HOXB5 Homeo Box B5 WNT5A Wingless-Type

    5 6 6 5 . 4 2 1 1 1 2 4 6 4 3 2 9 9 7 0 5 7 5 8 6 4 0 8 2 3 1 8 3 7 1 0 0 4 0 2 5 0 8 7 5 4 1 1 0 3 6 0 4 8 3 7 4 7 6 9 6 7 1 5 0 8 1 4 1 1 7 1 0 0 4 2 0 8 1 1 1 2 5 3 5 0 7 2 6 9 1 2 1 8 3 5 2 9 8 0 6 0 9 5 1 9 9 2 1 1 6 0 2 3 0 3 6 9 1 6 5 5 7 1 1 2 1 1 7 5 4 6 6 4 1 1 2 8 4 7 1 6 2 7 7 5 4 3 2 4 3 6 9 4 1 7 1 3 4 1 2 1 3 1 1 4 7 3 1 1 1 1 5 3 2 6 1 5 1 3 5 4 5 2 3 1 1 6 1 7 3 2 5 4 3 1 6 1 5 3 1 7 6 5 1 1 1 4 6 1 6 2 7 2 1 2 e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S HOXB6 homeo box B6 HOXB5 homeo box B5 WNT5A wingless-type MMTV integration site family, member 5A WNT5A wingless-type MMTV integration site family, member 5A FKBP11 FK506 binding protein 11, 19 kDa EPOR erythropoietin receptor SLC5A6 solute carrier family 5 sodium-dependent vitamin transporter, member 6 SLC5A6 solute carrier family 5 sodium-dependent vitamin transporter, member 6 RAD52 RAD52 homolog S.
  • Datasheet for T7 Exonuclease (M0263; Lot 0031212)

    Datasheet for T7 Exonuclease (M0263; Lot 0031212)

    Source: Purified from an E. coli strain containing a Unit Assay Conditions: 50 mM potassium Physical Purity: Purified to > 95% homogeneity TYB12 intein fusion acetate, 20 mM Tris-acetate, 10 mM magnesium as determined by SDS-PAGE analysis using T7 Exonuclease acetate, 1mM dithiothreitol (pH 7.9) and 0.15 mM Coomassie Blue detection. Supplied in: 10 mM Tris-HCl (pH 8.0), sonicated duplex [3H] DNA. 0.1 mM EDTA, 5 mM DTT and 50% glycerol. RNase Activity (Extended Digestion): A 10 µl Quality Control Assays 1-800-632-7799 reaction in NEBuffer 4 containing 40 ng of [email protected] Reagents Supplied with Enzyme: Single Stranded Deoxyribonuclease Activity flourescein labeled RNA transcript and 10 units www.neb.com 10X NEBuffer 4. (FAM Labeled Oligo): A 50 µl reaction in of T7 Exonuclease incubated at 37°C. After M0263S 003121214121 NEBuffer 4 containing a 20 nM solution of a incubation for 4 hours, > 90% of the substrate Reaction Conditions: fluorescent internal labeled oligonucleotide and a RNA remains intact as determined by gel minimum of 50 units of T7 Exonuclease incubated 1X NEBuffer 4. electrophoresis using fluorescence detection. M0263S Incubate at 25°C. for 16 hours at 37°C yields < 5% degradation as determined by capillary electrophoresis. 1,000 units 10,000 U/ml Lot: 0031212 Heat Inactivation: No 1X NEBuffer 4: RECOMBINANT Store at –20°C Exp: 12/14 50 mM potassium acetate Endonuclease Activity: Incubation of a 50 µl References: reaction containing 100 units of T7 Exonuclease Description: T7 Exonuclease acts in the 5´ to 20 mM Tris-acetate 1.
  • Q 297 Suppl USE

    Q 297 Suppl USE

    The following supplement accompanies the article Atlantic salmon raised with diets low in long-chain polyunsaturated n-3 fatty acids in freshwater have a Mycoplasma dominated gut microbiota at sea Yang Jin, Inga Leena Angell, Simen Rød Sandve, Lars Gustav Snipen, Yngvar Olsen, Knut Rudi* *Corresponding author: [email protected] Aquaculture Environment Interactions 11: 31–39 (2019) Table S1. Composition of high- and low LC-PUFA diets. Stage Fresh water Sea water Feed type High LC-PUFA Low LC-PUFA Fish oil Initial fish weight (g) 0.2 0.4 1 5 15 30 50 0.2 0.4 1 5 15 30 50 80 200 Feed size (mm) 0.6 0.9 1.3 1.7 2.2 2.8 3.5 0.6 0.9 1.3 1.7 2.2 2.8 3.5 3.5 4.9 North Atlantic fishmeal (%) 41 40 40 40 40 30 30 41 40 40 40 40 30 30 35 25 Plant meals (%) 46 45 45 42 40 49 48 46 45 45 42 40 49 48 39 46 Additives (%) 3.3 3.2 3.2 3.5 3.3 3.4 3.9 3.3 3.2 3.2 3.5 3.3 3.4 3.9 2.6 3.3 North Atlantic fish oil (%) 9.9 12 12 15 16 17 18 0 0 0 0 0 1.2 1.2 23 26 Linseed oil (%) 0 0 0 0 0 0 0 6.8 8.1 8.1 9.7 11 10 11 0 0 Palm oil (%) 0 0 0 0 0 0 0 3.2 3.8 3.8 5.4 5.9 5.8 5.9 0 0 Protein (%) 56 55 55 51 49 47 47 56 55 55 51 49 47 47 44 41 Fat (%) 16 18 18 21 22 22 22 16 18 18 21 22 22 22 28 31 EPA+DHA (% diet) 2.2 2.4 2.4 2.9 3.1 3.1 3.1 0.7 0.7 0.7 0.7 0.7 0.7 0.7 4 4.2 Table S2.
  • However, There Was Substantial Falloff in Terms Of

    However, There Was Substantial Falloff in Terms Of

    446 Technical Briefs accuracy; however, there was substantial falloff in terms disease, myocardial infarction, and ischemic cerebrovascular disease. Six case-control studies from the Copenhagen City Heart Study. Ann Intern Med of performance with new vs used chips. This lowers the 2001;134:941–54. cost per SNP to almost one-third of the cost of using a new 5. Twyman RM, Primrose SB. Techniques patents for SNP genotyping. Pharma- microelectronic chip and more lowers the cost compared cogenomics 2003;4:67–79. 6. Thistlethwaite WA. Rapid genotyping of common MeCP2 mutations with an with RFLP analysis by more than one-half. This is approx- electronic DNA microchip using serial differential hybridization. J Mol Diagn imately the same cost reported by others for detecting 2003;5:121–6. eight SNPs on one test site simultaneously (€1.62 per SNP) 7. Gilles PN, Wu DJ, Foster CB, Dillon PJ, Chanock SJ. Single nucleotide polymorphic discrimination by an electronic dot blot assay on semiconductor (6). It should be noted, however, that purchase of the microchips. Nat Biotechnol 1999;17:365–70. Nanogen NMW 1000 Nanochip Molecular Biology Work- 8. Nagan N, O’Kane DJ. Validation of a single nucleotide polymorphism station is not included in these calculations. genotyping assay for the human serum paraoxonase gene using electroni- cally active customized microarrays. Clin Biochem 2001;34:589–92. The microelectronic chip examined in this study was 9. Sohni YR, Dukek B, Taylor W, Ricart E, Sandborn WJ, O’Kane DJ. Active limited by the number of test sites per chip. Increasing the electronic arrays for genotyping of NAT2 polymorphisms.
  • Oc I Heov4 Strand

    Oc I Heov4 Strand

    MAPPING THE 5'-TERMINAL NUCLEOTIDES OF THE DNA OF BACTERIOPHAGE X AND RELATED PHAGES* BY RAY WUt AND A. D. KAISER DEPARTMENT OF BIOCHEMISTRY, STANFORD UNIVERSITY SCHOOL OF MEDICINE, PALO ALTO, CALIFORNIA Communicated by Arthur Kornberg, October 28, 1966 The two ends of a molecule of X DNA have a structure which enables them to join together to form circular monomers1-' or linear dimers, trimers, etc.' Many, if not all, temperate bacteriophages have cohesive ends including coliphages 480,4 21, 186, 424, 434,5 and P2.6 The thermal reversibility of cohesion1'7 and the response of the ends of X DNA to Escherichia coli DNA polymerase and exonuclease III8 suggest that each end has a short single strand, about 15 nucleotides long, which protrudes beyond a long double-stranded interior of about 50,000 nucleotide pairs. The single strand at one end is judged to be complementary to the single strand at the other end because cohesion occurs only between a "left" end and a "right" end.31 Figure 1 shows how a molecule with complementary single strands protruding from its ends can form a circular molecule by base-pairing between the two single strands. left half right holf' sus sus 31 HO I W) V oCO = heov4 Strand I i qht strand FIG. L.-Structure of X DNA and formation of circular molecules. The structure of the 5'- termini is represented diagrammatically;88MMSA, SU8SB, and ix are genetic markers;7 other sym- bols are described in the text. Soon after infection of a sensitive or a lysogenic bacterium, the two ends of the DNA molecule of an infecting phage cohere to each other and the single-strand inter- ruptions are closed by covalent bonds.9' 10 Thus cohesive ends provide a mechanism by which DNA molecules can be joined transiently or permanently.
  • An Enriched Environment Ameliorates Oxidative Stress and Nitrated A-Syn in PD

    An Enriched Environment Ameliorates Oxidative Stress and Nitrated A-Syn in PD

    Original Article Cell Transplantation 2018, Vol. 27(5) 831–839 ª The Author(s) 2018 An Enriched Environment Ameliorates Reprints and permission: sagepub.com/journalsPermissions.nav Oxidative Stress and Olfactory Dysfunction DOI: 10.1177/0963689717742662 in Parkinson’sDiseasewitha-Synucleinopathy journals.sagepub.com/home/cll Soohyun Wi1,2, Jang Woo Lee3,4, MinGi Kim1,2, Chang-Hwan Park5, and Sung-Rae Cho1,2,6,7 Abstract Parkinson’s disease (PD) features nonmotor symptoms such as olfactory dysfunction referred to as hyposmia, an initial sign of disease progression. Metabolic dysfunction can contribute to neurodegenerative diseases, and various xenobiotics and endogenous compounds are also involved in the pathogenesis of PD. Although aerobic exercise was found to induce preservation or improvement in olfactory function in PD patients in a recent study, the exact underlying mechanism for this effect is not clear. We aimed to investigate the influence of an enriched environment (EE) on olfactory dysfunction especially via metabolic pathways related to detoxification enzymes. Eight-month-old transgenic (Tg) PD mice that overexpress human A53T a-synuclein (a-syn) were randomly allocated to an EE or standard conditions for 2 mo. The buried food test showed that EE group had significantly improved olfactory function compared to the control group. Reverse transcription polymerase chain reaction (PCR) and real-time quantitative PCR showed that expression of the detoxification enzymes––cytochrome P450 family 1 subfamily A member 2, para- oxonase 1, alcohol dehydrogenase 1, UDP glucuronosyltransferase family 2 member A1 complex locus, aldehyde oxidase homolog 2,and aldehyde glutathione peroxidase 6––was significantly increased in the olfactory bulb (OB) of the PD control group, but these enzymes were normalized in the EE group.
  • 12) United States Patent (10

    12) United States Patent (10

    US007635572B2 (12) UnitedO States Patent (10) Patent No.: US 7,635,572 B2 Zhou et al. (45) Date of Patent: Dec. 22, 2009 (54) METHODS FOR CONDUCTING ASSAYS FOR 5,506,121 A 4/1996 Skerra et al. ENZYME ACTIVITY ON PROTEIN 5,510,270 A 4/1996 Fodor et al. MICROARRAYS 5,512,492 A 4/1996 Herron et al. 5,516,635 A 5/1996 Ekins et al. (75) Inventors: Fang X. Zhou, New Haven, CT (US); 5,532,128 A 7/1996 Eggers Barry Schweitzer, Cheshire, CT (US) 5,538,897 A 7/1996 Yates, III et al. s s 5,541,070 A 7/1996 Kauvar (73) Assignee: Life Technologies Corporation, .. S.E. al Carlsbad, CA (US) 5,585,069 A 12/1996 Zanzucchi et al. 5,585,639 A 12/1996 Dorsel et al. (*) Notice: Subject to any disclaimer, the term of this 5,593,838 A 1/1997 Zanzucchi et al. patent is extended or adjusted under 35 5,605,662 A 2f1997 Heller et al. U.S.C. 154(b) by 0 days. 5,620,850 A 4/1997 Bamdad et al. 5,624,711 A 4/1997 Sundberg et al. (21) Appl. No.: 10/865,431 5,627,369 A 5/1997 Vestal et al. 5,629,213 A 5/1997 Kornguth et al. (22) Filed: Jun. 9, 2004 (Continued) (65) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2005/O118665 A1 Jun. 2, 2005 EP 596421 10, 1993 EP 0619321 12/1994 (51) Int. Cl. EP O664452 7, 1995 CI2O 1/50 (2006.01) EP O818467 1, 1998 (52) U.S.
  • Supplementary Materials For

    Supplementary Materials For

    Supplementary Materials For: Altered expression and localization of tumor suppressive E3 ubiquitin ligase SMURF2 in human prostate and breast cancer Andrea Emanuelli, Dhanoop Manikoth Ayyathan, Praveen Koganti, Pooja Anil Shah, Liat Apel-Sarid, Biagio Paolini, Rajesh Detroja, Milana Frenkel-Morgenstern and Michael Blank Figure S1. SMURF2 gene and protein expressions in human organs and tissues. (a) The mRNA expression levels of SMURF2 in a panel of human normal organs and tissues: HPA, GTEx and FANTOM datasets. (b) Comparative analysis of IHC-based SMURF2 protein expression versus its gene expression. 1 Figure S2. The expression and molecular localization of SMURF2 in human breast cell models. (a) Western blot analysis of SMURF2 expression in protein fractions prepared from non-tumorigenic mammary epithelial MCF10A cells and metastatic breast carcinoma cell models. Right panel shows the cytoplasm/nucleoplasm ratio of SMURF2 in the cytoplasmic (CYT) and nucleoplasmic (NUCL) compartments of the cells, normalized to coomassie staining. (b) SMURF2 expression analysis conducted as in (a), but incorporating three cellular fractions: cytoplasm, nucleoplasm and insoluble, chromatin- containing, fraction solubilized with sonication. All samples were run on the same SDS-PAGE and probed with the indicated antibodies. Coomassie gel staining was also conducted, and used for sample normalization. The quality of fractionation is demonstrated by sample probing with anti-TOP1 (nuclear marker), anti-α-TUBULIN (cytoplasmic marker), and anti-histone-H2B (chromatin marker) antibodies. Right panel shows the cytoplasm/nucleoplasm ratio of SMURF2 expression in untransformed and breast cancer cells normalized to coomassie. 2 Figure S3. Examination of SMURF2 turnover rate in the cytoplasmic and nuclear fraction of MCF10A cells.
  • POLSKIE TOWARZYSTWO BIOCHEMICZNE Postępy Biochemii

    POLSKIE TOWARZYSTWO BIOCHEMICZNE Postępy Biochemii

    POLSKIE TOWARZYSTWO BIOCHEMICZNE Postępy Biochemii http://rcin.org.pl WSKAZÓWKI DLA AUTORÓW Kwartalnik „Postępy Biochemii” publikuje artykuły monograficzne omawiające wąskie tematy, oraz artykuły przeglądowe referujące szersze zagadnienia z biochemii i nauk pokrewnych. Artykuły pierwszego typu winny w sposób syntetyczny omawiać wybrany temat na podstawie możliwie pełnego piśmiennictwa z kilku ostatnich lat, a artykuły drugiego typu na podstawie piśmiennictwa z ostatnich dwu lat. Objętość takich artykułów nie powinna przekraczać 25 stron maszynopisu (nie licząc ilustracji i piśmiennictwa). Kwartalnik publikuje także artykuły typu minireviews, do 10 stron maszynopisu, z dziedziny zainteresowań autora, opracowane na podstawie najnow­ szego piśmiennictwa, wystarczającego dla zilustrowania problemu. Ponadto kwartalnik publikuje krótkie noty, do 5 stron maszynopisu, informujące o nowych, interesujących osiągnięciach biochemii i nauk pokrewnych, oraz noty przybliżające historię badań w zakresie różnych dziedzin biochemii. Przekazanie artykułu do Redakcji jest równoznaczne z oświadczeniem, że nadesłana praca nie była i nie będzie publikowana w innym czasopiśmie, jeżeli zostanie ogłoszona w „Postępach Biochemii”. Autorzy artykułu odpowiadają za prawidłowość i ścisłość podanych informacji. Autorów obowiązuje korekta autorska. Koszty zmian tekstu w korekcie (poza poprawieniem błędów drukarskich) ponoszą autorzy. Artykuły honoruje się według obowiązujących stawek. Autorzy otrzymują bezpłatnie 25 odbitek swego artykułu; zamówienia na dodatkowe odbitki (płatne) należy zgłosić pisemnie odsyłając pracę po korekcie autorskiej. Redakcja prosi autorów o przestrzeganie następujących wskazówek: Forma maszynopisu: maszynopis pracy i wszelkie załączniki należy nadsyłać w dwu egzem­ plarzach. Maszynopis powinien być napisany jednostronnie, z podwójną interlinią, z marginesem ok. 4 cm po lewej i ok. 1 cm po prawej stronie; nie może zawierać więcej niż 60 znaków w jednym wierszu nie więcej niż 30 wierszy na stronie zgodnie z Normą Polską.
  • Polyamines Mitigate Antibiotic Inhibition of A.Actinomycetemcomitans Growth

    Polyamines Mitigate Antibiotic Inhibition of A.Actinomycetemcomitans Growth

    Polyamines Mitigate Antibiotic Inhibition of A.actinomycetemcomitans Growth THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Allan Wattimena Graduate Program in Dentistry The Ohio State University 2017 Master's Examination Committee: Dr John Walters, Advisor Dr Purnima Kumar Dr Sara Palmer Dr Shareef Dabdoub Copyright by Allan Wattimena 2017 Abstract Polyamines are ubiquitous polycationic molecules that are present in all prokaryotic and eukaryotic cells. They are the breakdown products of amino acids and are important modulators of cell growth, stress and cell proliferation. Polyamines are present in higher concentrations in the periodontal pocket and may affect antibiotic resistance of bacterial biofilms. The effect of polyamines was investigated with amoxicillin (AMX), azithromycin (AZM) and doxycycline (DOX) on the growth of Aggregatibacter actinomycetemcomitans (A.a.) Y4 strain. Bacteria were grown in brain heart infusion broth under the following conditions: 1) A.a. only, 2) A.a. + antibiotic, 3) A.a. + antibiotic + polyamine mix (1.4mM putrescine, 0.4mM spermidine, 0.4mM spermine). Growth curve analysis, MIC determination and metatranscriptomic analysis were carried out. The presence of exogenous polyamines produced a small, but significant increase in growth of A.a. Polyamines mitigated the inhibitory effect of AMX, AZM and DOX on A.a. growth. Metatranscriptomic analysis revealed differing transcriptomic profiles when comparing AMX and AZM in the presence of polyamines. Polyamines produced a transient mitigation of AMX inhibition, but did not have a significant effect on gene transcription. Many gene transcription changes were seen when polyamines were in the presence of AZM.
  • Potential of Human PON2 As an Anti- Pseudomonal Therapy

    Potential of Human PON2 As an Anti- Pseudomonal Therapy

    Potential of human PON2 as an anti- Pseudomonal therapy by Naseem Mohammad Ali School of Medicine Submitted in fulfilment of the requirements for the Doctor of Philosophy (Medical Studies) University of Tasmania June, 2015 DECLARATION OF ORIGINALITY This thesis contains no material which has been accepted for a degree or diploma by the University or any other institution, except by way of background information and duly acknowledged in the thesis, and to the best of my knowledge and belief no material previously published or written by another person except where due acknowledgement is made in the text of the thesis, nor does the thesis contain any material that infringes copyright. Signature Date - 02/11/15 [i] AUTHORITY OF ACCESS This thesis may be made available for loan. Copying and communication of any part of this thesis is prohibited for two years from the date this statement was signed; after that time limited copying and communication is permitted in accordance with the Copyright Act 1968. Signature Date - 02-11-15 [i] STATEMENT OF ETHICAL CONDUCT The research associated with this thesis abides by the international and Australian codes on human and animal experimentation, the guidelines by the Australian Government's Office of the Gene Technology Regulator and the rulings of the Safety, Ethics and Institutional Biosafety Committees of the University [i] Abstract ABSTRACT Cystic Fibrosis (CF) is the most common life-limiting single gene disorder in Caucasian populations. CF results from mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, which leads to accumulation of thick and sticky mucus in the airways of people with CF, ultimately dampening immune clearance of potential respiratory pathogens.