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Abstracts Submitted:Division II - Graduate students (1-2 years) - Biomedical Sciences
Modification of the 1-Phosphate of Helicobacter pylori Lipid A
A. X. Tran 1, M. Karbarz 2, C. R. Raetz 2, S. McGrath 3 , R. Cotter 3 and M. S. Trent 1;
1 J. H. Quillen Coll. of Med., ETSU, Johnson City, TN, 2 Duke Univ. Med. Ctr., Durham, NC, 3Johns Hopkins Univ. School of Med., Baltimore, MD.
Pathogenic bacteria modify the lipid A portion of their LPS to help evade the host innate immune response. Modification of the negatively charged phosphate groups of lipid A aids in resistance to cationic antimicrobial peptides that target the bacterial cell surface. The lipid A of Helicobacter pylori contains a phosphoethanolamine (pEtN) unit directly linked to the 1-position of the dissacharide backbone. This is in contrast to the pEtN units found in Escherichia coli, Salmonella typhimurium, and Neisseria meningitidis lipid A, which are attached to the lipid A phosphate group to form a pyrophosphate linkage. This study describes two enzymes involved in the modification of the 1-phosphate of H. pylori lipid A. Using an in vitro assay system, we demonstrate the presence of lipid A 1-phosphatase in membranes of H. pylori. In an attempt to identify possible lipid A phosphatases, we cloned four distant orthologs of E. coli PgpB, the phosphatidylglycerol-phosphate phosphatase, from H. pylori 26695. One of these orthologs, HP0021, is the structural gene for the lipid A 1-phosphatase and is required for removal of the 1-phosphate from mature lipid A in an in vitro assay system. Heterologous expression of HP0021 in E. coli K-12 resulted in the highly selective removal of the 1-phosphate from E. coli lipid A as demonstrated by mass spectrometry. We have also identified the structural gene for the H. pylori lipid A pEtN transferase (HP0022). Mass spectrometric analysis of the lipid A isolated from E. coli expressing both HP0021 and HP0022 showed the addition of a single pEtN group at the 1-position, suggesting that HP0022 is responsible for the addition of a pEtN unit at the 1-position in H. pylori lipid A. In conclusion, we have demonstrated that modification of the 1-phosphate of H. pylori lipid A requires two enzymatic steps: (1) removal of the 1-phosphate by a specific phosphatase and (2) addition of a pEtN unit directly to the glucosamine dissacharide backbone. The addition of the pEtN unit to the 1-position of H. pylori lipid A represents a novel mechanism in the modification of Gram-negative bacterial lipid A.
STRUCTURE-ACTIVITY RELATIONSHIPS FOR THREE MACROLIDE ANTIBIOTICS IN HAEMOPHILUS INFLUENZAE
Susan Mabe, Jessica Eller, and W. Scott Champney, Department of Biochemistry and Molecular Biology East Tennessee State University, College of Medicine, Johnson City, TN 37614
A prior study examining differences in the activity of erythromycin and azithromycin on cellular functions in the gram-negative pathogen, Heamophilus influenzae, revealed a marked difference in their inhibitory activities. The study revealed that protein synthesis and 50S subunit assembly were equal targets for inhibition by azithromycin while erythromycin was a preferential inhibitor of translation. This contrast in inhibitory activities stimulated a comparative analysis of three additional antibiotics clarithromycin, flurithromycin and roxithromycin. A goal was to correlate structural differences to differences in inhibitory activities of each drug on H. influenzae. Each compound was tested over a concentration range for inhibitory effects on cellular processes. Translation rates were measured by 35S-methionine incorporation into proteins and cell viability by colony counting after dilution. Ribosomal subunit formation was determined after 3H uridine labeling of rRNA and sucrose gradient centrifugation to separate ribosomal subunits. Of the three compounds examined clarithromycin was the most effective inhibitor of protein synthesis with an IC50 of 5.6 g/ml followed by flurithromycin at 6.0 at g/ml, and roxithromycin at 9.0 g/ml. IC50 values for antibiotic effects on viable cell counts and growth rate were similar to those obtained for translational inhibition. Flurithromycin had the strongest effect on 50S subunit formation with an IC50 of 8.0 g/ml followed by clarithromycin and roxithromycin at 9.0 g/ml and 12.5 g/ml respectively. 30S ribosomal subunit formation in cells treated with flurithromycin and roxithromycin was also reduced to some extent. Pulse and chase labeling kinetics examining subunit assembly rates verified the slower synthesis rate of the subunits in the presence of each macrolide. Clarithromycin behaved like azithromycin targeting protein synthesis and 50S subunit assembly with near equality. Flurithromycin and roxithromycin preferentially targeted subunit assembly whileerythromycin affected translation with greater efficacy. The results are discussed in terms of structural disparities of these macrolides and their differential inhibitory effects on both cellular targets.
EFFECTS OF DNA ADDUCT STRUCTURE AND SEQUENCE CONTEXT ON STRAND OPENING OF REPAIR INTERMEDIATES AND INCISION BY UVRABC NUCLEASE
Steven M. Shell1, C. D. Utzat 2, C. Luo 1, Z. Yang 1, N. E. Geacintov 3, A. K. Basu 2, and Yue Zou 11 J.H. Quillen Coll. of Medicine, ETSU, Johnson City, TN, 2 University of Connecticut, Storrs, CT, 3 New York University, New York, NY.
DNA damage recognition of nucleotide excision repair (NER) in Escherichia coli is achieved by at least two steps. In the first step, a helical distortion is recognized, which leads to a strand opening at the lesion site. The second step involves the recognition of the type of chemical modification in the single-stranded region of DNA during the processing of the lesions by UvrABC. In the current work, by comparing the efficiencies of UvrABC incision of several types of different DNA adducts, we show that the size and position of the strand opening are dependent on the type of DNA adducts. Optimal incision efficiency for the C8-guanine adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) was observed in a bubble of three mismatched nucleotides, whereas the same for C8-guanine adduct of 1-nitropyrene and N2-guanine adducts of benzo[a]pyrene diol epoxide (BPDE) was noted in a bubble of six mismatched nucleotides. This suggests that the size of the aromatic ring system of the adduct might influence the extent and number of bases associated with the opened strand region catalyzed by UvrABC. We also showed that the incision efficiency of the AF or AAF adduct was affected by the neighboring DNA sequence context, which, in turn, was the result of differential binding of UvrA to the substrates. The sequence context effect on both incision and binding disappeared when a bubble structure of three bases was introduced at the adduct site. We therefore propose that these effects relate to the initial step of damage recognition of DNA structural distortion. The structure-function relationships in the recognition of the DNA lesions, based on our results, have been discussed.
DEGRADATION OF 23S rRNA IN AZITHROMYCIN-TREATED RIBONUCLEASE MUTANTS OF ESCHERICHIA COLI
Jessica Eller and W. Scott Champney. J.H. Quillen Coll. of Medicine, ETSU, Department of Biochemistry and Molecular Biology, Johnson City, TN 37614
Azithromycin is an azalide, a subclass member of the macrolide class of antibiotics derived from erythromycin. Like other macrolides, azithromycin binds to the 50S ribosomal subunit of bacterial ribosomes and inhibits translation. Azithromycin also prevents 50S ribosomal subunit assembly by binding to a 50S ribosomal subunit precursor particle. When exposed to azithromycin, several ribonucleases in wild-type Escherichia coli cells degrade the antibiotic-bound 50S precursor. Presumably, cells expressing one or more mutated ribonucleases will degrade the antibiotic-bound precursor less efficiently. It is thought that this reduced turnover of 50S precursor particles in mutant cells will result in increased sensitivity to azithromycin. To test this, eight ribonuclease deficient strains of Escherichia coli were grown in the presence or absence of azithromycin. All strains, including the wild-type, demonstrated a decline in protein synthesis rate, growth rate, and cell viability when exposed to azithromycin, with the extent of inhibition depending on the strain. Hybridization experiments were performed to analyze the patterns of ribosomal RNA degradation in both wild-type and mutant cells grown in the presence or absence of the macrolide. Cell lysates were centrifuged on 5-20% sucrose density gradients and each gradient was collected in three fractions, labeled top, 30S, and 50S. The ribosomal RNA from each gradient fraction was resolved on agarose gels and northern blots of the gels were hybridized with a biotinylated 23S DNA probe. Partially degraded 23S ribosomal RNA was found to accumulate in all of the mutant strains when exposed to azithromycin. The effects of the antibiotic were comparable in the wild-type strain and the ribonuclease I and III mutants. However, ribonuclease II, ribonuclease E, and polynucleotide phosphorylase mutants demonstrated hypersensitivity to the antibiotic and showed a greater extent of 23S rRNA accumulation, suggesting that these three ribonucleases play a significant role in the degradation of rRNA in azithromycin-inhibited Escherichia coli.
INTERLEUKIN-1BETA AFFECTS EXPRESSION OF ANGIOGENIC GENES INCLUDING MATRIX METALLOPROTEINASE-2 IN CARDIAC MICROVASCULAR ENDOTHELIAL CELLS: ROLE OF MAPK AND SRC
Deidra J.H. Mountain, Mahipal Singh, Ph.D., and Krishna Singh, Ph.D., Department of Physiology, James H. Quillen College of Medicine, East Tennessee State University, James H. Quillen Veterans Affairs Medical Center, Johnson City, TN 37614
Angiogenesis is essential in the repair of the heart following myocardial infarction (MI). Matrix metalloproteinases (MMPs), a family of extracellular endopeptidases, are implicated in angiogenesis due to their role in extracellular matrix remodeling. Interleukin-1beta (IL-1B), increased in the heart post MI, plays a protective role in post MI remodeling by promoting wound healing. Here we studied expression of various angiogenic genes affected by IL-1B in Cardiac Microvascular Endothelial Cells (CMECs), and investigated the signaling pathways involved in IL-1B-stimulated increases in MMP expression and activity. Methods: Primary cultures of CMECs, isolated from adult rat hearts, were exposed to IL-1B (4 ng/ml) for 24h. Total RNA was reverse transcribed and resulting cDNAs were hybridized with a SuperArray membrane containing 96 angiogenesis-related genes. MMP activity in the conditioned media was measured using gelatin in-gel zymography. Western blot analyses were used to examine the expression of MMP-2 and activation of MAPK (mitogen activated protein kinases; ERK1/2 and JNKs) and Src. Results: Out of 96 angiogenic genes, IL-1B increased the expression of 11 genes (>2-fold), including MMP2, integrin alpha5, integrin alphaV, TGF beta1, TIMP1, and VCAM-1, while decreasing the expression of 4 genes, including FGF16, VEGF-D, and TGF beta3. IL-1B increased MMP-2 activity by 1.6-fold (p<0.05 vs control). IL-1B had no significant effect on MMP-9 activity. IL-1B increased MMP-2 protein levels by 5.9-fold (p<0.05 vs control) in the cell lysates. UO126 (10 uM; inhibitor of ERK1/2 pathway), and SP600125 (10 uM; inhibitor of JNKs) inhibited IL-1B-stimulated increases in MMP-2 expression by 42% and 47% (p<0.05 vs IL-1B), respectively. PP2 (20 uM; inhibitor of Src) increased IL-1B-stimulated increases in MMP-2 protein levels by 35% (p<0.05 vs IL-1B). IL-1B activated ERK1/2 and JNKs, not Src, within 15 min of treatment. Angiotensin II (AII), also increased in the heart post MI, increased phosphorylation ofSrc but failed to increase MMP-2 expression. Interestingly, AII inhibited IL-1B-stimulated increases in MMP-2 expression. Conclusion: IL-1B affects expression of angiogenic genes, including MMP2, in CMECs. The increase in MMP-2 expression is mediated via the activation of ERK1/2 and JNKs, while activation of Src negatively regulates MMP-2 expression. Thus, IL-1B may play a protective role post MI by affecting expression of angiogenic cytokines and cell adhesive molecules.
INTERACTIONS OF HUMAN REPLICATION PROTEIN A WITH DAMAGED AND UNDAMAGED SINGLE-STRANDED DNA
Y. Y. Liu1, Z. G. Yang 1, C. D. Utzat 2, Y. Liu 1, N. E. Geacintov 3, A. K. Basu 2, and Y. Zou 1
1Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, 2Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, and 3Department of Chemistry, New York University, New York 1003, USA.
Human replication protein A (hRPA), a protein of three subunits of 70, 32 and 14 kDa, plays indispensable roles in DNA repair and replication, and has been suggested to be one of the DNA damage sensors in nucleotide excision repair (NER). However, controversial models have been proposed regarding the spatial arrangement and function of RPA in the early steps of NER due to the different observations of RPA-damaged/undamaged ssDNA interactions. Although the conformational change of RPA in the formation of RPA-ssDNA complex has been rigorously studied recently, the structural alternation or structures of the damaged ssDNA upon binding remain unknown. To gain a comprehensive understanding of the mechanism of RPA-damaged ssDNA interactions, we have examined the binding of RPA to well-defined DNA substrates by employing the non-invasive fluorescence spectroscopic methods under the true equilibrium conditions. Upon binding of RPA, the fluorescence of BPDE-adducted ssDNA was enhanced by 7-8 folds, indicating that the original stacking between the BPDE molecule and the neighboring bases in the ssDNA was largely disrupted most likely due to a local bending of the ssDNA. This bending may serve as a structural determinant for the RPA recognition of DNA damage in NER. Our study also supported that the binding of RPA to ssDNA occurs progressively in the direction of 5 to 3. Comparison of RPA bindings to a series of damaged and undamaged ssDNA indicated that the preferential binding of RPA to damaged or undamaged ssDNA over one another depends on the types of adduct.
CHARACTERIZATION OF A MICROSPORIDIA PROTEIN THAT POTENTIALLY FUNCTIONS IN SPORE ATTACHMENT AND INFECTION OF HOST CELLS GROWN IN VITRO
C. E. Jolly, T. R. Southern, and J. R. Hayman,Department of Microbiology,JamesH.QuillenCollegeof Medicine,Johnson City,TN,37614
Microsporidia are obligate intracellular opportunistic protists capable of infecting invertebrate and vertebrate hosts via an environmentally stable spore. The microsporidian Encephalitozoon cuniculi causes disease in a variety of animals including immunocompromised humans. Previous studies from our laboratory indicate that microsporidia spores adhere to in vitro grown host cell surfaces and spore adherence is directly associated with infection. Spore adherence and subsequent infection can be inhibited by exogenous proteoglycans such as heparin. The purpose of this study is to identify E. cuniculi proteins that are involved in the attachment of spores to host cells. A candidate 36kDa protein was identified via a western blot of protein from in vitro grown host cells incubated with biotin labeled spore protein lysate. Using this same method, a similar sized protein (40kDa) was identified in another microsporidian species, E. intestinalis. In addition, the 40kDa protein of E. intestinalis was shown by affinity chromatography to bind heparin. Lectin binding assays using agarose bound Concanavalin A (ConA) and Wheat Germ Agglutnin (WGA) demonstrated that the 36kDa protein of E. cuniculi is likely a glycoprotein containing residues of N-acetylglucosamine which are known to react with WGA. Studies are underway to identify the 36kDa E. cuniculi protein. The identification and characterization of proteins involved in spore attachment will aid in understanding the interaction between the spore and host during the infection process.
INDUCTION OF C-REACTIVE PROTEIN GENE EXPRESSION BY NF-KB
Bhavya Voleti, Mahua Chakraborty and Alok Agrawal, Department of Pharmacology, East Tennessee State University, TN 37614
C-reactive protein (CRP) is an acute phase protein produced by hepatocytes and whose rate of synthesis increases dramatically during inflammatory conditions. In human hepatoma Hep3B cells, interleukin (IL)-6 induces CRP gene expression modestly while IL-1, which alone has no effect, synergistically enhances the effects of IL-6. The first 125 bases of the proximal region of the CRP promoter are sufficient for IL-1 synergy. IL-6-induced CRP expression is mediated by the transcription factors STAT3 and C/EBP-beta and the IL-1 effects may be mediated by the activation of classical NF-kappa B (NF-kB; heterodimer of p50 and p65). Our previous studies showed that NF-kB enhanced CRP transcription and that it did so by acting through the -125/+1 region of the promoter. The purpose of this study was to identify the kB site responsible for NF-kB-mediated induction of CRP expression. Careful inspection of -125/+1 region of the promoter revealed a potential kB site located at position -74/-63. To determine the capability of this site to bind NF-kB, we performed electrophoretic mobility shift assays: an oligonucleotide (-81/-57) derived from the CRP promoter and containing the potential kB site was used as the radiolabelled probe and the nuclear extracts from IL-1-beta-treated Hep3B cells were used as the source of NF-kB. Three specific DNA-protein complexes were formed on this probe. Formation of one of these complexes could be competed by an unlabelled consensus NF-kB-binding oligonucleotide indicating the presence of NF-kB proteins in the complex. Supershift analysis using antibodies confirmed the presence of p50 and p65 in this complex. Thus, the sequence -74/-63 is the kB site, and its location within the first 125 bases strongly indicates that this site could be the IL-1 response element on the CRP promoter.
ANGIOGENESIS AND MYOGENESIS FOR CHRONIC ISCHEMIC HEART
Esha D. Ibrahim, Janet Davis, and Race L. Kao, Dept. of Surgery, East Tennessee State Univ., Johnson City, TN 37614.
Heart disease is Americas leading killer. Following a myocardial infarction, scarring consequently reduces contractility and functionality. The proposed study combines two revolutionary procedures: Transmyocardial laser revascularization (TMR) and autologous stem cell implantation to re-establish the heart tissues health and function. The experiment to evaluate this involves three surgical procedures on thirty miniswine: (1st) placement of ameroid occluder on the left anterior descending coronary artery to induce a myocardial infarction and biopsy of skeletal muscle (2nd) Use of a carbon dioxide heart laser on the ventricular wall of the heart and implantation of labeled cells (3rd) Termination of the animal and removal of the heart followed by data analysis. The animals were divided into three groups of ten and designated: (Group 1) Ischemia (Group 2) Ischemia + TMR (Group 3) Ischemia + TMR + Cells. Stimulated angiogenesis in tissues which are affected by chronic ischemia can promote tissue healing. Preliminary data analysis of the cardiac output (stroke volume x heart rate) illustrated a significant difference between the controls (receiving induced ischemia and mock injection after baseline recording) and the treated animals (receiving induced ischemia + laser + cells). Digital sonomicrometry and Millar microtip pressure transducers are used to determine contractility, left ventricular pressure, pressure-volume loops, and pressure-length loops.
AUGMENTATION OF MICROSPORIDIA SPORE ADHERENCE BY DIVALENT CATIONS
Timothy Southern and J. Russell Hayman, Department of Microbiology, East Tennessee State University, James H. Quillen College of Medicine, Johnson City, TN 37604Microsporidia are obligate intracellular opportunistic protists that infect vertebrate and invertebrate hosts via environmentally resistant spores. Human microsporidiosis is often attributed to Encephalitozoon intestinalis and is characterized by severe diarrhea and wasting in the immunocompromised. Current hypotheses on the microsporidial infection process in humans make no concession for spore attachment to host cells, a process observed during in vitro culture of spores. Our previous studies show that E. intestinalis spores bind sulfated glycosaminoglycans on the host cell surface and that the addition of certain divalent cations to the incubation medium augments this binding. These results suggest that a receptor or ligand involved in adherence may be activated by divalent cations. The goal of the current study is to determine the effects of the divalent cations Mn 2+, Mg 2+, and Ca 2+ on E. intestinalis spore adherence to Vero host cells. The efficiency of spore adherence was measured using an assay that quantifies spore adherence to host cell surfaces by immunofluorescence. All assays were conducted in an incubation medium that does not contain divalent cations (HEPES buffered saline with 1% bovine serum albumin). The data indicate that the addition of 0.1-1mM Mn 2+ or Mg 2+ increases spore adherence at least 2 fold compared to spore adherence without the addition of divalent cations. Ca 2+, however, does not augment spore adherence to Vero cells. Spore adherence assays were also used to determine the effects of combined divalent cations on spore adherence. Interestingly, spore adherence to Vero cells increases at least 3 fold when 0.001-1mM Ca 2+ is combined with 1mM Mn 2+ or 1mM Mg 2+, as compared to 0.001-1mM Ca 2+ alone. Spore adherence increases at least 2 fold when 0.001-0.1mM Mn 2+ is combined with 1mM Ca 2+ or 1mM Mg 2+, as compared to 0.001-0.1mM Mn 2+ alone. Spore adherence also increases at least 2 fold when 0.001-0.1mM Mg 2+ is combined with 1mM Mn 2+ or 1mM Ca 2+, as compared to 0.001-0.1mM Mg 2+ alone. Finally, data from spore adherence assays using fixed Vero host cells suggest that Mn 2+ may play a significant role in activation of a ligand on the surface of the spore and not the host cells. Collectively, these data show that Mn 2+ or Mg 2+, but not Ca 2+, can independently augment spore adherence to host cells. Assays with divalent cations in combination reveal that virtually any combination of two divalent cations, independent of concentration, will facilitate a higher level of spore binding compared to any single divalent cation. These data provide a better understanding of spore ligand activation by divalent cations during attachment to host cells and may eventually contribute to the development of novel therapeutic interventions.