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Appalachian Student Research Forum

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Division III

Abstracts Submitted:Division III - Graduate students (2 + years) - Biomedical Sciences


INHIBITION OF ACYL-COA: CHOLESTEROL ACYLTRANSFERASE PREVENTS OXYSTEROL INDUCED APOPTOSIS IN MACROPHAGES

Natalie E. Freeman, Antonio E. Rusinol, Michael S. Sinensky, and Douglas Thewke, Department of Biochemistry and Molecular Biology, East Tennessee State University, Quillen College of Medicine, Johnson City, TN 37614

Components of oxidized low-density lipoproteins, such as oxysterols, are thought to mediate many of the pathological events associated with the development of atherosclerosis. Oxysterols induce apoptosis in several cell types by a poorly understood mechanism. Previous work in our laboratory has demonstrated that oxysterols, 25-hydroxycholesterol and 7-ketocholesterol, exert their initial effects through the activation of a Ca2+ influx, which activates cytosolic phospholipase A2 (cPLA2), producing a release of free arachidonic acid (AA) from membrane phospholipids. The aim of this study was to further elucidate the oxysterol apoptosis signal transduction pathway in murine monocyte-macrophage cell lines (P388D1 and Raw 264.7). Confirming previous results, calcium antagonists (nifedipine and MRS-1845), and a cPLA2 inhibitor (AACOCF3) prevented oxysterol-induced apoptosis in these cell lines as determined by measuring Caspase 3 activity. Eicosatetraynoic acid (ETYA), a global inhibitor of AA metabolism, reduced oxysterol-induced apoptosis suggesting that an AA metabolite may be required for induction. However, pharmacological inhibition of eicosanoid synthesis did not impair the ability of oxysterols to induce apoptosis in the macrophage cell lines and peritoneal macrophages isolated from cycloxygenase and lipoxygenase knockout mice were not resistant to the induction of apoptosis by oxysterols. Another potential fate of the released AA is conversion to a fatty acyl-CoA by archidonoyl-CoA synthetase and the subsequent esterification to free cholesterol by acyl-CoA: acyltransferase (ACAT). Inhibitors of both arachidonoyl-CoA synthetase (Triacsin C) and ACAT (58035) effectively blocked oxysterol-induced apoptosis. In addition, 58035 inhibited the accumulation of cholesteryl and oxysteryl esters from radiolabeled precursors confirming the inhibition of ACAT activity under these conditions. MRS-1845 and AACOCF3 also inhibited the accumulation of cholesteryl and oxysteryl esters.These results strongly suggest that an ACAT derived product is necessary for induction of apoptosis by oxysterols in P388D1 and Raw 264.7 macrophages.


THE DISTRIBUTION OF SEVOFLURANE IN A SEVOFLURANE INDUCED DEATH

David L. Burrows1, M.S.; Andrea Nicolaides 1, B.S.; Gretel C. Stephens 2 , M.D.; Kenneth E. Ferslew 1, Ph.D.

Section of Toxicology, Department of Pharmacology 1, Department of Forensic Pathology 2, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614

The distribution of sevoflurane (fluoromethyl 2,2,2,-trifluoro-1-(trifluoromethyl) ethyl ether) in blood, urine, liver, kidney, vitreous fluid, and tracheal aspirate is presented from a subject with a sevoflurane induced death. Sevoflurane is a nonflammable general anesthetic administered by inhalation of vaporized liquid. Although general inhalation anesthetics have the potential to be fatal if not properly administered, the incidence of abuse is minute in comparison to other illicit drugs. Currently, there are no citations in the literature defining the body distribution of sevoflurane in a sevoflurane induced death. The decedent was found lying in a bed with an oxygen mask containing a gauze pad secured to his face. Three empty bottles and one partially full bottle of Ultane (sevoflurane) were found with the body in addition to two pill boxes containing a variety of prescription and non-prescription drugs. Serum, urine and gastric contents from the deceased were screened for numerous drugs and metabolites using a combination of thin layer chromatographic, colorimetric and immunoassay techniques. Analysis of biological specimens from the deceased revealed the presence of: amphetamine, caffeine, pseudoephedrine, nicotine, nicotine metabolite, and valproic acid. Sevoflurane concentrations were determined by head space gas chromatography with flame ionization detection and revealed concentrations of 26.2 mcg/mL in the blood, 105.2 mcg/mL in the urine, 31.9 mcg/mL in the tracheal aspirate, 86.7 mcg/mL in the vitreous, 30.8 mg/kg in the liver, and 12.8 mg/kg in the kidney. The decedent had pathologies consistent with respiratory suppression including pulmonary atelectasis, pulmonary edema, and neck vein distention. The official cause of death was respiratory suppression by sevoflurane and the manner of death was unclear.

Key Words: Ultane, Partitions, Anesthetic, Lethal, Forensic

ZMPSTE24: AN ENZYME WITH TWO DISTINCT PROTEOLYTIC ACTIVE SITES THAT CAN FULLY PROCESS PRELAMIN A.

Douglas Corrigan, Danuta Kuszczak, Michael Sinensky, Doug Thewke, and Antonio Rusinol, Department of Biochemistry and Molecular Biology, East Tennessee State University, College of Medicine, Johnson City, TN 37614

Lamin A, the proteolyzed form of prelamin A, is a nuclear envelope scaffolding protein which provides structural rigidity and form to the nuclear envelope. Lamin A is first synthesized as the 74 kDa precursor prelamin A which then becomes prenylated with a farnesyl isoprenoid, and then proteolyzed in two steps. The first proteolytic event removes the last three c-terminal residues residing within the prenylated CAAX box and exposes the prenylated cysteine as the new c-terminus. In a second endoproteolytic event that occurs 15 amino acids upstream from the c-terminal farnesylated cysteine residue, the precursor substrate prelamin A is fully converted to its mature form lamin A. To date, the protease(s) responsible for these two substrate cleavages have not been identified, however, recent mice knockout experiments have implicated Zmpste24 (FACE-1) as either the first AAXing enzyme, the endoprotease, or both. Until now, however, human Zmpste24 has not been assayed in-vitro to test these possibilities. Data obtained from this most current set of in-vitro experiments employing reactions between recombinant Zmpste24, the purified substrate prelamin A, and a farnesylated CAAX tetrapeptide, clearly demonstrate that Zmpste24 is a novel enzyme with the ability to perform both endoproteolytic events. Even more surprising is the observation from inhibitory profiles and mass spectral analysis that these endoproteolytic activities are arising from two distinct active sites within Zmpste24: a zinc dependent metalloprotease site responsible for the first AAXing reaction; and a newly identified chymotrypsin-like active site which catalyzes the second endoproteolytic event. Inhibition profiles indicate that these two proteolytic functionalities are mechanistically uncoupled and, therefore, separate. The uniqueness of this novel protease is demonstrated by its ability to process prelamin A at two distinct points along its prenylation dependent maturation pathway by means of two functionally distinct active sites.


STUDIES ON THE BINDING OF ERYTHROMYCIN TO THE 23rRNA, METHYL TRANSFERASE ENZYME AND TO A 50S RIBOSOMAL SUBUNIT PRECURSOR PARTICLE

Indira Pokkunuri and Scott Champney, Department of Biochemistry and Molecular Biology, East Tennessee State University, College of Medicine, Johnson City, TN 37614.

The macrolide antibiotic erythromycin inhibits not only mRNA translation but also 50S ribosomal subunit assembly in cells. It is also known that an important mechanism of erythromycin resistance is 23S rRNA methylation by erm methyl transferase. We are interested in investigating what is the true substrate for methylation since it is known from our work and others that 50S subunits are not the substrates for methylation. We have recently published a model for 50S ribosomal subunit formation where, the precursor particle which accumulates in erythromycin treated cells, is the target for methyl transferase activity. We are now investigating the role of the precursor particle as substrate for erm E methyl transferase activity. In our studies, we expressed a cloned erm E methyl transferase with a His- tag in E.coli and purified the enzyme on a Ni-affinity chromatography column. Purity of the enzyme was assessed by SDS-polyacrylamide gel electrophoresis. A 43 KDa protein, that was about 90% pure, was found to be the major purification product. As substrates for the enzyme activity, we used 23S rRNA, partially purified 50S precursor particle and 50S ribosomal subunits isolated from S. aureus cells. In our assays using 3 [H] S-Adenosyl methionine as a methyl group donor, we found that erythromycin inhibited methylation of both protein free 23S rRNA and the precursor particle. The concentration of erythromycin that gave 50% inhibition of methylation was found to be 17nM for the precursor particle and 31nM for the 23S rRNA.

A series of assays were done to look at 14C-erythromycin binding to four substrates, the methylase enzyme, 23S rRNA, the precursor particle and the 50S ribosomal subunits. For the three RNA s, the stoichiometry of binding was found to be 0.5 mole of 14C-erythromycin / mole of the substrate. Binding to the enzyme and the precursor particle are being studied further. The precursor particle is a new target for the methylase activity and is likely to be the true substrate for the enzyme in the cells treated with erythromycin. Studies of binding of erythromycin to both these substrates are important and this should give interesting insights into the methylase mediated erythromycin resistance.


NEOMYCIN AND PAROMOMYCIN INHIBIT 30S RIBOSOMAL SUBUNIT ASSEMBLY IN VITRO

Cerrone Foster and Scott Champney, Department of Biochemisty and Molecular Biology, East Tennessee State University, College of Medicine, Johnson City, TN 37614

The bacterial ribosome is a large macromolecular complex responsible for cellular protein synthesis. This complex is composed of a large and small ribosomal subunit responsible for the cells translational activivity. The small subunit sediments at 30S and is composed of 16S rRNA and 21 ribosomal proteins. It binds mRNA, initiation factors, as well as the large subnit. The small subunit also participates in tRNA selection, thus playing a critical role in translational fidelity. Neomycin and paromomycin are aminoglycoside antibiotics that specificaly stimulate the misreading of mRNA by binding to the decoding site of 16SrRNA. Mehta and Champney have shown that both neomycin and paromomycin inhibit 30S subunit assembly in Escherichia coli and Staphylococcus aureus cells. Antibiotic treatment stimulated the acumulation of a 30S assembly intermediate precursor with a sedimentiation coefficient of 21S. Collectivelly the work from our lab has shown that ribosomal subunit assembly is an antibiotic target, but how this process is imparied remains unclear. Ribosomal reconstitution was used to explore 30S assembly inhibition by neomycin and paromomycin in vitro. Reconstitution allows the generation of ribosomal subunits from their constituent rRNA and ribosomal proteins. Increasing the magnesium concentration and temperature of the reaction, allows the rRNA to undergo a conformational change forming active subunits. Control reconstitution gave 30S subunits and 21S intermediates at 40? and 0? C respectively. Reconstitution was performed in the presence of neomycin and paromomycin at a 104excess of antibiotic to rRNA. Results showed that in the presence of the antibiotic total reconstitution was prevented and a particle was produced with a sedimentation coefficient of 25S. This particle was similar to the 21S precursor particle isolated from E.coli cells grown with 7.5 ug/ml of neomycin or 6.5 ug/ml of paromomycin. Two-dimensional gel electrophoresis is being performed to charcterize the protein content of both the protein content of both the in vivo and in vitro precursors. The results of this work show that amino glycosides inhibit 30S subunit assembly in vitro and the precursor particle formed is similar to the precursor isolated from growing cells. This work is intended to further characterize the steps of30S subunit assembly inhibition as an antibiotic target.


ACUTE ETICLOPRIDE TREATMENT ELIMINATES COGNITIVE DEFICITS PRODUCED BY NEONATAL QUINPIROLE TREATMENT.

Thompson, K. N., Department of Anatomy and Cell Biology; Click, I. R.; Best, R. A. C.; Thacker, S. K.; Brown, R. W. Department of Psychology, East Tennessee State University, Johnson City, TN 37614 USA.

This study was designed to investigate the effects of acute eticlopride (0.02 mg/kg, D2 antagonist) treatment, given immediately before training, in rats neonatally treated with quinpirole, which has been shown to produce long-term D2 receptor supersensitization. Rats were given quinpirole (1mg/kg) or saline treatment from P1-21. Beginning on P22, rats were administered eticlopride or saline (i.p.) fifteen mins before each of seven days of training. Rats were tested on the Morris water task (MWT). For the first three consecutive days, rats were tested on the place version of the MWT with a stationary platform. Animals were given 24 training trials followed by a probe trial, and swim patterns were analyzed with platform removed. The next day, animals began testing on the match-to-place version for four consecutive days and two daily trials were given with the platform moved to a new location each day. On both the search time and target visit measures of the probe trial, animals neonatally treated with quinpirole demonstrated a deficit, and eticlopride eliminated this deficit. Interestingly, animals neonatally treated with saline but given eticlopride before training also demonstrated a deficit on both measures. On the match-to-place version, the difference in latency to locate the platform between the two daily trials served as the dependent measure. Similar to the MWT place version, eticlopride treatment eliminated deficits produced by neonatal quinpirole treatment on this task, and eticlopride produced a deficit in saline controls. This study demonstrates that in a model of dopamine D2 supersensitivity, it appears that the increased sensitivity of the D2 receptor is important for cognitive function.


PEPTIDE ANTISERA GENERATION AGAINST THREE CHLAMYDIA TRACHOMATIS HSP60 HOMOLOGUES

R. W. LaRue and J. E. Raulston, Department of Microbiology, East Tennessee State University, Quillen College of Medicine, Johnson City, TN37614

Chlamydia trachomatis is the leading cause of preventable blindness worldwide and the leading bacterial agent involved in sexually acquired infections in the United States. Infections with Chlamydia can lead to the development of scarring trachoma in men and women, pelvic inflammatory disease, salpingitis, tubal factor infertility, and ectopic pregnancy in women, and urethritis, epididymitis, and sterility in men. Chlamydia likely encounter an iron-limited environment inhuman hosts. Recently our laboratory showed that Chlamydia exhibits increased expression of heatshock protein 60kDa (chsp60) in response to iron limitation. Subsequent genome sequencing revealed three genes encoding chsp60s. The objectives of this study were to (i) amplify the open reading frames (ORFs) coding for Chsp60 using PCR and ligate the PCR-products into a plasmid expression vector, (ii) sequence recombinant chlamydial DNA from E. coliLMG194 cells transformed with the recombinant plasmids, (iii) examine the DNA sequence for each C. trachomatis serovar E chsp60 and compare with existing genome sequences, (iv) examine amino acid sequences for the location of peptides unique to each chsp60. Synthetic peptides were used to generate antisera; the resultant sera were purified by affinity chromatography and adsorbed to reduce cross-reactivity and increase monospecificity. Antisera were evaluated against each recombinant chsp60 protein by Western blotting. Reactivity against native chsp60s were visualized by transmission electron microscopy. Initial experiments indicate that expression of the second chsp60 (encoded by groEL_2) is increased during iron limitation. The production of chsp60 antibodies in human patients is associated with damaging sequelae in chlamydial genital and ocular infections.


DECTIN-1 EXPRESSION IS DECREASED IN GLUCAN TREATED SEPTIC MICE.

Tammy Ozment-Skelton, DVM and David Williams, East Tennessee State University, Department of Surgery

Introduction: We have reported that the carbohydrate ligand, glucan, decreases morbidity and mortality in a murine model of CLP induced polymicrobial sepsis. The mechanisms by which glucan confers protection are not fully understood. Recent data indicate that glucans are bound by membrane associated pattern recognition receptors including Dectin-1. Ligation of glucan by this receptor leads to stimulation of NF-kB activity by Toll-Like Receptor 2 (TLR2) dependent and independent mechanisms. Dectin is expressed on multiple inflammatory cell types including dendritic cells, macrophages, and neutrophils and is considered a sentinel receptor for fungal infections. How this receptor may be involved in the protective effects of glucan in sepsis is unknown. We examined Dectin-1 and TLR2 expression in CLP sepsis in the presence and absence glucan treatment.

Method: Ninety male ICR/HSD adult mice were divided into 6 groups: i) control no surgery, ii) sham (laparotomy only), iii) cecal ligation and puncture (CLP), iv) glucan no surgery, v) sham glucan, and vi) CLP glucan. Glucan phosphate (40 mg/kg) was administered IP one hour prior to surgery. At 0, 3, 6, 12, and 24 hours post-operatively three mice from each group were sacrificed. Spleens and peritoneal cells were harvested. Dectin-1 and TLR2 expression were assessed in peritoneal cells and splenocytes by flow cytometric analysis with antibodies to Dectin-1 or TLR2.

Results: CLP increased Dectin-1 expression on peritoneal cells (113%) when compared to sham surgery (12 h; p<0.01). CLP decreased splenic Dectin-1 expression (19%) when compared to sham surgery (12h; p<0.01). Glucan in conjunction with CLP or sham surgery decreased Dectin-1 expression in peritoneal cells at 3 and 12 h in sham (59%, and 62%; p<0.05) and 12 h in CLP (66%; p<0.01) mice. Glucan treatment coincident with sham or CLP also decreased splenic Dectin-1 expression in sham surgery (47% and 42%; p<0.01) at 6 and 12 h and in CLP (34%; p<0.01) at 6 and 12 h. Glucan treatment alone decreased peritoneal cell Dectin-1 expression at 3 (32%) and 6 (52%) h, respectively (p<0.05). Neither surgery, glucan treatment, nor surgery with glucan treatment altered splenocyte or peritoneal cell TLR2 surface expression.

Conclusions: Glucan treatment, which is known to confer protection in polymicrobial sepsis, decreases Dectin-1 expression on the surface of peritoneal cells in the presence and absence of sepsis. Glucan also decreased splenocyte Dectin-1 expression in the presence of sepsis. Whether this decrease in Dectin-1 surface expression relates to increased survival in sepsis is unknown; however, modulation of this receptor may play a role in the response to polymicrobial sepsis.


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