Division II

Abstracts Submitted: Division II - Graduate students (1-2 years) - Biomedical Sciences


Mass Spectrometric Identification of Lysines Involved in the Interaction of Human Replication Protein A with ssDNA

Steven M. Shell‡, Sonja Hess¥, Mamuka Kvaratskhelia#, and Yue Zou‡* ‡Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, ¥National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, and #The Ohio State University Health Sciences Center, College of Pharmacy, Center for Retrovirus Research and Comprehensive Cancer Center, Columbus, OH 43210.

Human replication protein A (hRPA), a heterotrimeric single-stranded DNA (ssDNA) binding protein, is required for many cellular pathways including DNA damage repair, recombination, and replication as well as the ATR-mediated DNA damage response. While extensive effort has been devoted to understanding the structural relationships between RPA and ssDNA, information is currently limited to the RPA domains, the trimerization core, and a partial co-crystal structure. In this work, we employed a mass spectrometric protein footprinting method of single amino acid resolution to investigate the interactions of the entire heterotrimeric hRPA with ssDNA. In particular, we monitored surface accessibility of RPA lysines with NHS-Biotin modification in the contexts of the free protein and the nucleoprotein complex. Our results not only indicated excellent agreement with the available crystal structure data for RPA70 DBD-AB-ssDNA complex, but also revealed new protein contacts in the nucleoprotein complex. In addition to two residues: K263 and K343 of p70, previously identified by co-crystallography as direct DNA contacts, we observed protection of five additional lysines (K183, K259, K489, K577, and K588 of p70) upon ssDNA binding to RPA. Three residues: K489, K577, and K588 are located in ssDNA binding domain C and are likely to establish the direct contacts with cognate DNA. In contrast, no ssDNA-contacting lysines were identified in DBD-D. In addition, two lysines, K183 and K259 are positioned outside the putative ssDNA binding cleft. We propose that the protection of these lysines could result from the RPA inter-domain structural reorganization induced by ssDNA binding.


Bacterial Fitness and Conjugative Transfer of Mupirocin Resistance in Staphylococcus

Danielle Barnard and Susan Reynolds, Department of Biological Sciences, East Tennessee State University, Johnson City, TN. 37614

Antibiotic resistance is an increasingly difficult complication in the control of infectious diseases.  In hospitals, methicillin-resistant Staphylococcus aureus (MRSA) causes morbidity and mortality and requires “last-line” antibiotics for treatment.  Control of MRSA has focused on eradication of nasal carriage because most patient MRSA infections are caused by self-inoculation.  The topical antibiotic, mupirocin, has been used for this purpose.  However, in east Tennessee high-level mupirocin resistance (plasmid-encoded) emerged soon after introduction of the antibiotic in the early 1990s and rose dramatically until prescriptions were controlled administratively. 

The population dynamics of antibiotic resistance are dependent upon the rate of antibiotic usage, the fitness cost of resistance to the bacteria and the transmissibility of the resistance determinant.  We have detailed data on local usage patterns for mupirocin in east Tennessee.  The goal of the current study was to estimate the potential fitness cost of mupirocin resistance and to uncover likely transmission pathways within and between two closely related staphylococcal species, the often pathogenic S. aureus and the commensal, S. epidermidisS. epidermidis was included because it also is a frequent nares colonist and it has been shown to represent a local reservoir of mupirocin resistance genes. 

A cost of resistance is most commonly observed as a fitness burden represented by a reduced growth rate.  We estimated growth rates in S. aureus and S. epidermidis using, (i) naturally-occurring sensitive and resistant isolates; (ii) near-isogenic progenitors and their plasmid-cured derivatives, and (iii) near-isogenic susceptible recipient strains and their resistant counterparts that had acquired mupirocin resistance via conjugation.  There was no evidence of a cost of mupirocin resistance in any of the three groups analyzed.  In contrast, significant fitness costs were detected in control assays comparing near-isogenic isolates that differed in sensitivity to the antibiotics, rifampin and fusidic acid. 

Transmission of mupirocin resistance was assessed by filter mating conjugation assays.  Transconjugants were selected on media containing mupirocin and two selectable antibiotic markers, rifampin and fusidic acid.  Putative transconjugants were confirmed by coagulase and disk diffusion tests.  Naturally-occurring S. aureus were capable of transfer to other S. aureus and to S. epidermidis.  In contrast, no S. epidermidis transferred resistance to either species.  However, mupirocin-resistant transconjugant S. epidermidis that had received the mupirocin resistance plasmid from S. aureus were then capable of intra- and inter-specific transfer of the mupirocin resistance. 

The high incidence of mupirocin resistance in S. epidermidis could be a result of no fitness cost or the observation that resistance is not transmissible out of S. epidermidis.  That is, resistance gets “trapped” and accumulates, possibly as a result of chromosomal integration.  While the decline in the population incidence of mupirocin resistance in response to administrative control suggested a fitness cost, our assays have not demonstrated such a cost.  This difference may represent a difference between in vitro and in vivo assessments or an amelioration of a previous fitness cost by compensatory secondary mutations.  Nevertheless, our results show that fitness burdens may not impede the spread of mupirocin resistance but that transmission pathways are limited.


Recognition of ligand-complexed C-reactive protein by the IgG receptor Fc g RIIa

Hima Bindu Radha and Alok Agrawal, Department of Pharmacology, East Tennessee State University, College of Medicine, Johnson City, TN 37614

The primary binding specificity of C-reactive protein (CRP) is for phosphocholine (PCh)-containing ligands such as pneumococcal C-polysaccharide (PnC). Once complexed, CRP exerts one of its two known effector functions: complement activation and phagocytosis. CRP also binds to IgG Fc receptors (FcR) on monocytic cells. Fc g RIIa (CD32), the low-affinity IgG receptor, binds CRP with high affinity. However, due to variations in the purity of CRP and the use of whole IgG antibodies to detect CRP, the data on CRP-FcR interaction were challenged. In this investigation, we tested our hypothesis that CRP might bind FcR but only in its ligand-complexed state. Stable CHO cells expressing Fc g RIIa were generated by transfection with a cDNA encoding Fc g RIIa. Binding of CRP to Fc g RIIa expressed on CHO cells was determined by using Fab fragment of anti-CRP antibody in a luminescense-based assay. Uncomplexed CRP failed to bind Fc g RIIa. PnC-complexed CRP bound Fc g RIIa in a PnC dose-dependent manner suggesting that the ligand was the determinant for CRP-FcR interaction. An anti-CD32 mAb inhibited the binding of CRP showing specificity of the interaction. In contrast to CRP-PnC complexes, CRP complexed with PCh salt did not bind Fc g RIIa. Thus, the ligand-complexed CRP is recognized by FcR g RIIa and that the crowding of CRP on a PCh-rich ligand is necessary for this process. These findings support the absence of CRP on circulating monocytes in vivo and provide a mechanism for CRP-mediated phagocytosis of bacteria.


Humanin is a novel neuroprotective agent against stroke.

Xingshun Xu, Chu Chang Chua, Ronald Hamdy and Balvin Chua. Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614.

Humanin (HN) is a recently identified 24-amino acid peptide that is best known for its ability to protect neurons from damage caused by Alzheimer's disease-related proteins. Given the neuroprotective effects of HN, it is plausible that HN could protect against stroke; however, this possibility has not been fully explored to date. In this study, we examined the neuroprotective effects of HN on focal cerebral ischemia/reperfusion injury in mice. The animals underwent a sham operation or middle cerebral artery occlusion for 75 min followed by 24 h reperfusion. Mice were either pretreated with 0.1 ¦Ìg HN (i.c.v.) 30 min before ischemia; post-treated at 0, 2, 4 h after ischemia; or pretreated with 1 ¦Ìg HN (i.p.) 1 h before ischemia. The cerebral infarct volume was measured by triphenyl tetrazolium chloride staining after 24 h of reperfusion. In our experiments, HN pretreatment with 0.1 ¦Ìg HN (i.c.v.) 30 min before ischemia reduced cerebral infarct volume from 56 ¡À 7% to 25 ¡À 6% (p<0.01). HN post-treatment reduced cerebral infarct volume from 56 ¡À 7% to 34 ¡À 6 % immediately after reperfusion (p<0.01), 42 ¡À 6% at 2 h of reperfusion (p<0.01), and 46 ¡À 7% at 4 h of reperfusion (p<0.05). HN pre-treatment with 1 ¦Ìg HN (i.p.) 1 h before ischemia reduced cerebral infarct volume to 35 ¡À 3% (p<0.01). HN also significantly improved neurological function. Extracellular signal-regulated kinase (ERK), a pathway involved in ischemic neuronal death, was rapidly activated after cerebral ischemia/reperfusion. A significant decrease of phospho-ERK was observed in mice treated with HN. Our results demonstrate that HN offers neuroprotection at least in part by inhibition of ERK activation. These findings have important implications for the therapeutic potential of HN in the treatment of stroke.


Statin and nitric oxide reduce C-reactive protein production while inflammation is ongoing

Bhavya Voleti and Alok Agrawal, Department of Pharmacology, East Tennessee State University, TN 37614

C-reactive protein (CRP) is made in liver and its serum concentration increases in chronic and acute inflammation. Measurement of serum CRP is recommended for use as an indicator of inflammation and predictor of heart complications. Cholesterol-lowering drugs statins also lower CRP. To evaluate statin-mediated CRP reduction and to reassess clinical usefulness of CRP, we investigated regulation of CRP gene expression. Here, we show that pravastatin and nitric oxide (NO) prevent induction of CRP expression in human hepatoma Hep3B cells exposed to inflammatory molecules IL-6 and IL-1β. These findings suggest that the decreased CRP levels in vivo after statin-treatment do not necessarily reflect absence of inflammation, and raise the possibility of utilizing NO-releasing drugs to reduce serum CRP. In addition, we located on the CRP promoter, a regulatory element with overlapping binding sites for transcription factors Oct-1 and NF- қ B (p50-p50 and p50-p65). Basal CRP expression increased dramatically when binding of both NF-қB and Oct-1 was abolished. Results indicate that the binding of Oct-1 to the promoter, facilitated by p50-p50 in a novel way, represses while replacement of Oct-1 by p50-p65 induces CRP transcription. Combined data provide a mechanism for the regulation of CRP expression under acute inflammatory conditions and suggest that the baseline/chronic CRP levels may fluctuate. We propose that the measurement of CRP levels, in normal healthy population and in individuals on statin/NO-therapy, is not as useful as was imagined.  


DIFFERENTIAL INTRACELLULAR TRAFFICKING AND LOCALIZATION OF GLUCANS IN PRIMARY MURINE MACROPHAGES

Matthew P. Goldman*, Tammy R. Ozment-Skelton*, Harry E. Ensley*,^ and David L. Williams*

*Department of Surgery, Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614. ^Department of Chemistry, Tulane University, New Orleans, Louisiana 70115. The innate immune response is an important first line of defense against invading pathogens. Cells in the innate immune system express evolutionarily conserved pattern recognition receptors (PRRs) that recognize and bind pathogen associated molecular patterns (PAMPs) that are only found in microorganisms. Glucans are fungal cell wall PAMPs that are not only associated with fungal infection, but also are known to have immunomodulatory effects. Recent studies have shown that glucans are bound and internalized by the cell surface PRR Dectin-1. It was initially thought that upon internalization glucans were trafficked to the lysosome where they were degraded. However, recent studies have suggested that biologically active glucans, such as glucan phosphate, may be trafficked differently than a biologically inactive glucan, such as laminarin. We have previously shown that glucan phosphate and laminarin are differentially recognized by glucan specific PRRs. In this study, we examined the intracellular trafficking and localization of a biologically active glucan phosphate in primary murine macrophages using confocal microscopy. We compared the trafficking of glucan phosphate with that of an inactive glucan, laminarin. Thioglycollate elicited murine macrophages were incubated with either Alexa Fluor 647 labeled soluble glucan phosphate or laminarin (100 ug/ml) for 30m, 1h, or 3h. The cells were fixed, permeablized, and stained with TRITC conjugated antibodies against endosome, lysosome, and Golgi apparatus associated proteins. The nuclei were counterstained with a SYTOX green nuclear stain. The confocal images were evaluated with Leica multicolor/2D cytofluorogram software. The data were quantified by creation of a binary mask. Co-localization was assessed using mask intensity rate for the ligand fluor. We found that glucan phosphate did not co-localize with endosomes or lysosomes at any time interval. However, by 1 hr the glucan phosphate co-localized to the Golgi apparatus (44.79 + 5.16%). Similarly to glucan....


Exposure to Volatile Organic Compounds and Neurobehavioral Effect.

A.J. Bhanegaonkar, J.W. Flowers, M.B. Hogan, and *T. Wu (East Tennessee State University, Johnson City, TN 37614)

Data from the Priority Toxicant Reference Range Study in the Third National Health and Nutrition Examination Survey were analyzed to examine exposure to volatile organic compounds (VOCs) and the potential for neurobehavioral effects. Self-reported contact to chemical products containing VOCs during the ¡°past three days¡± and blood concentrations of specific chemicals were available. Neurobehavioral function was assessed by simple reaction time test (SRTT), symbol digit substitution test (SDST), and serial digit learning test (SDLT). Among the 1338 respondents, exposures were reported to paint thinner (6.0%); varnish lacquer (7.2%); bug or insect spray (8.7%); weed killer (2.0%); solid toilet bowl deodorant (9.6%); finger nail polish (16.2%); dry-cleaning/spot remover (4.0%); air fresher/room deodorants (33.9%); diesel (10.4%); and gasoline (28.4%). Percentiles of blood VOCs were calculated for 42 chemicals. The 5th, 50th, and 95th percentiles (ug/L) are: benzene 0.021, 0.062, 0.476; 1,1,1- trichloroethane 0.061, 0.132, 0.799; 2-butanone 1.839, 5.422, 16.829; o-xylene 0.045, 0.101, 0.271; and styrene 0.013, 0.041, 0.177. No association was found between the self-reported exposure status and the neurobehavioral assessments. Correlation coefficients with SRTT, SDST, and SDLT were 0.216*, 0.130*, 0.089 for 1,4 dichlorobenzene; 0.058, 0.097*, 0.083 for 1,1,2- trichloroethane; -0.026, 0.010, 0.098* for chloroform; and 0.115*, 0.064, and 0.014 for dibromochloromethane (* p<0.05) suggesting the need for further study. Moreover, this study provided pilot data of exposure status and reference ranges of VOCs for the US population.