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Molecular Biology 9:00 am Saturday, April 6, 2002 Battelle Hall 144 Daniel J. Kaser-Presiding.

9:00 NITRIC OXIDE AND ENDOTHELIAL NITRIC OXIDE SYNTHASE MAY PRODUCE A HYPOTENSIVE EFFECT IN A LOW BLOOD PRESSURE LINE OF WISTAR-KYOTO (WKY) RAT. Colin M. Everhart, (Daniel L. Ely, Dept. of Biology, University of Akron, Akron OH 44325-3908. The hypothesis tested was that a greater amount of endothelial nitric oxide synthase (eNOS) protein and nitric oxide (NO) will be produced in the WKY/Low blood pressure (LBP) strain than in both the WKY/Normal blood pressure (NBP) and Spontaneously Hypertensive Rat (SHR) strains (n=8/group). Blood pressure (BP) was measured by a tail-cuff method and recorded at 12 and 20 weeks of age and correlated to NO and eNOS levels. Aortas, hearts, and livers were removed and frozen at -70 [degrees] C. Tissues were homogenized and examined by Western Blot for levels of eNOS activity. Urine (24 hour) was collected from the animals and examined by a gas-phase chemiluminescence's detection technique to determine total NO output. The mean BP for WKY/LBP was 115 [+ or -] 2 mmHg, WKY/NBP was 127 [+ or -] 4 mmHg, SHR was 162 [+ or -] 5 mmHg. The mean NO output for the WKY/LBP was 62 [+ or -] 30 [micro]mol/day N[O.sub.3]-. WKY/NBP was 29 [+ or -] 20 [micro]mol/day N[O.sub.3]-, SHR was 7 [+ or -] 7 [micro]mol/day N[O.sub.3]-. The relationship between BP and NO output between SHR and WKY/LBP was statistically significant (p<0.001, [r.sup.2]=0.43). This suggests that NO production may produce a hypotensive effect in a LBP line of WKY rats.

9:15 EFFECTS OF A MIXTURE OF TWO SPECIFIC PCB MOLECULES ON GENERAL DEVELOPMENT, THYROID STATUS, AND A NEUROCHEMICAL MEASURE IN 15-DAY OLD SPRAGUE-DAWLEY RATS. Chelsea S. Combs, combs Michelle J. Meadows, Katherine Kusnyer, Terri Provost, Christa Bowen, and Lee A. Meserve. Bowling Green State University, 217 Life Science Building, Bowling Green OH 43403-0212.

Polychlorinated biphenyls (PCB) were used in industry for over 30 years. Although their manufacture and use has been prohibited for the last 20 years, lipophilic and stable natures of PCB continues to result in its bioconcentration in the fatty tissues of animals and humans. Depending on the amount of chlorine, there are 209 possible forms (congeners) of PCB molecules. Thyroid hormone (TH) status is a target for endocrine-disrupting effects of PCB, so developmental and neurochemical alterations result from ingestion. The present study examined effects of a mixture of PCB 47 (minimally thyroid disruptive) and PCB 77 (thyroid disruptive) on developmental, hormonal, and neurochemical measures in 15-day old Sprague-Dawley rats (Rattus norvegicus). Dams were continuously fed a control diet, 1.25 ppm, 12.5 ppm, or 25.0 ppm of PCB 47/77 from gestational day 1, and sacrificed on postnatal day 15. Ingestion of PCB 47/77 significantly increased liver weights and decreased spleen weights, indicating the widespread effects of this toxicant. PCB 47/77 significantly depressed levels of both [T.sub.4] and [T.sub.3] with pronounced effects on [T.sub.4] levels. Choline acetyltransferase activity was elevated in basal forebrain and hippocampus. Thus, exposure of 15-day old rats to PCB 47/77 reduced body weights, altered organ weights, depressed thyroid hormone levels, and elevated ChAT activity, suggesting that PCB 47/77 disrupts thyroid status and other developmental measures, and inappropriately enhances ChAT activity. This enhancement may relate to increased incidence of behavioral alterations in human populations ingesting PCB.

9:30 THE MECHANISM OF CASODEX-INDUCED CELL DEATH? Rachel A. Schallhorn, rschallh@capital Dept. of Biology, Capital University, 2199 E. Main St., Columbus OH 43209-2394.

Prostate cancer is the second leading cause of cancer related death for men in the United States. Anti-androgens such as Casodex[R] are a widely used form of treatment. Patients treated with anti-androgens ultimately become resistant to the drug and develop hormone refractory tumors. Studies have shown a positive correlation between increased levels of Bcl-2, a protein involved in the regulation of the mitochondrial apoptotic pathway, and prostate cancer progression. Two sublines of LNCaP non-invasive human prostate cancer cells over-expressing Bcl-2 were treated with varying concentrations of tumor necrosis factor [alpha] (TNF-[alpha]) and Casodex[R]. Crystal violet assays were used to determine relative viability and MTT assays to determine the relative mitochondrial activity. Western blot analyses were run to determine the androgen receptor status of the Bcl-2 over-expressing cells. The Bcl-2 over-expressing cell lines treated with TNF-[alpha] had a significantly lower amount of death than the wild-type cell line. Treatment with Casodex[R] resulted in similar amounts of death in each of the three cell lines. Western blots showed androgen receptors were absent in the nuclei of the cells treated with Casodex[R]. These results indicate over-expression of Bcl-2 protects LNCaP cells against death from TNF-[alpha] but not from Casodex[R]. Therefore, the mechanism by which Casodex[R] induces death in LNCaP cells prevents androgen receptors from entering the nucleus and is unaffected by Bcl-2 levels.

9:45 VALIDATION OF DIFFERENTIAL GENE EXPRESSION IN NF1-MUTANT SCHWANN CELLS USING REAL-TIME, QUANTITATIVE RT-PCR. John F. Vitullo, Dept. of Biology, Capital University, 2199 E. Main St., Columbus OH 43209-2594.

NF1 is an acronym for the gene responsible for Neurofibromatosis type 1, an inherited disorder that often results in permanent physical and mental impairment to those afflicted. Patients with NF1 have a predisposition to developing a variety of benign and malignant tumors, many of which affect the peripheral and central nervous systems (PNS and CNS). NF1 patients can also exhibit cognitive deficits and many other manifestations unrelated to cancer, usually affecting neural-crest derived tissues outside of the PNS. This seems to indicate that the NF1 gene is important to growth control and developmental functions in a wide range of cell and tissue types. Schwann cells are the major cell population in benign neurofibromas, tumors that disfigure human patients with NF1. It is not yet known how molecular abnormalities in neurofibroma Schwann cells contribute to tumor formation. By increasing our understanding of this tumor development through molecular genetic and other cell biological approaches, we should begin to see an integrated picture of the normal role of NF1. The objective of my study was to confirm differential expression of several select genes identified in a cDNA microarray analysis. Quantitative, real-time RT-PCR was utilized to assess the relative gene expression of Schwann cell cDNA in normal wild-type mice as compared to transformed NF1-mutant mice. Quantitative analysis confirmed differential expression in eight genes. However, the magnitude of these changes showed marked variation. Seven of the eight genes showed a positive correlation to our microarray expression data, thus confirming our data for those genes.

10:00 DISSECTION OF MEMBRANE TARGETING MECHANISMS FOR CYTOPLASMIC DYNEIN Albert E. Chaffin, Capital University, and Kevin T. Vaughan, University of Notre Dame, Dept. of Biology, Notre Dame IN 46556.

Cytoplasmic dynein (CD) is a microtubule motor protein responsible for multiple aspects of membrane transport and chromosomal segregation during mitosis. The Vaughan Laboratory at the University of Notre Dame is interested in mechanisms that cytoplasmic dynein uses to bind cargo. Several subunits have been implicated in mediating organelle binding, including the intermediate chains (ICs), light intermediate chains (LICs) and light chains (LCs). We have focused on the ICs because previous studies revealed that they interact with the p[150.sup.Glued] subunit of dynactin and that this interaction is regulated via IC phosphorylation at serine-84. To determine the impact of IC phosphorylation, site-directed mutants that mimic either phosphorylated (S84D) or dephosphorylated (S84A) IC-2C were transfected into COS-7 cells and tested for membrane transport defects. S84A mutants perturbed dynein-mediated transport by competing with intact dynein for organelle binding. In contrast, S84D mutants failed to compete with dynein for organelle binding or disrupt dynein-mediated transport. Because previous studies had been performed using full-length ICs, which can associate with the dynein LC subunits, we prepared truncated ICs that lack LC-binding sites and tested for organelle transport defects by transfection. The results using truncated ICs were different from results gathered using full-length ICs, with the S84A mutants displaying less disruption of dynein-mediated transport, and S84D mutants displaying more disruption. Although further work will be needed to clarify these observations, these findings suggest that the N-terminus of the ICs and interacting proteins could contribute to organelle binding.

10:15 SUBCLONING OF THE LEUKOCYTE ANTIGEN RELATED GENE INTO A BACTERIAL EXPRESSION PLASMI D. Albert E. Chaffin, Jens Hemmingsen, Capital University, Dept. of Chemistry, Columbus OH 43209-2394.

Enzyme-catalyzed phosphorylation and dephosphorylation of tyrosine residues act as regulatory mechanisms in many intracellular signal transduction pathways. In Humans, domain 1 of the Leukocyte Antigen Related Protein (LAR-D1)is a tyrosine phosphatase involved in cell growth regulation. The LAR-D1 gene has been subcloned into the plasmid pSTBlue-1 for blue white colony screening. When cultured, subcloned Eschericia coli colonies were white. This suggests that LAR-D1 was inserted into pSTBlue-1. Having isolated the LAR-D1 gene into the screening plasmid, pSTBlue-1, we then subcloned the gene into an expression plasmid. This will allow us to collect ample amounts of the LAR-D1 protein to use for enzyme kinetic studies. To achieve this next step, we attempted to ligate LAR-D1 into the plasmid pGEX-4T-1 as a fusion to Glutathione-S-transferase. This allows expression of the LAR-D1 protein in Eschericia coli bacteria and makes collecting the protein easier. LAR-D1 has been successfully subcloned into pGEX-4T-1 according to restriction digest mapping. However, successful cloning will be confirmed by DNA sequence analysis and protein electrophoresis. Once we have isolated the enzyme, future studies will focus on the enzymology and inhibition of LAR-D1. By studying the chemical interactions of this protein, possibilities arise to develop inhibitors to treat disorders such as cancer, diabetes, immune and other diseases.

10:30 DEVELOPMENT OF OPIOID RECEPTORS IN THE CHICK AUDITORY BRAINSTEM. Erika N. Oleson, (Kerry Cheesman, Ph.D. Capital University, 2199 E Main St, Columbus OH43209.

Opioid receptors are located in the nucleus magnocellularis, an element of the auditory brainstem, of the chick species Gallus domesticus. Traditionally found in the somatosensory system, the receptors are now believed to be located in additional sensory systems. The aim of this study is to determine the location of the kappa, delta, and mu-opioid receptors within the nucleus magnocellularis of the auditory brainstem. The study will follow the development of each opioid receptor from embryonic age 11 days to post-hatch age 23 days. Standard immunohistochemical procedures are performed on each auditory brainstem tissue and then stained. Initial results indicate that the kappa-opioid receptor is found in the cytoplasm of the nucleus magnocellularis. Mu-opioid receptors have been found in the nucleus of embryonic age 11 days chicks. At post-hatch age 7 days the staining transitions into the nucleus and cytoplasm, continuing through post-hatch age 23 days. Delta-opioid receptors give a general amorphous stain, indicating that they may not be present in the nucleus magnocellularis.

10:45 CHARACTERIZATION OF ADRENAL NICOTINICRECEPTOR SUB-POPULATIONS USING [[sup.3]H]EBIPATIDINE BINDING AND PROTECTION ASSAYS. Daniel J. Kaser, (Dennis B. McKay, Ph.D., The Ohio State University, Division of Pharmacology, 500 W 12th Ave, Columbus OH 43210.

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that pervade the central and peripheral nervous systems and are implicated in several neurological disorders such as Alzheimer's Disease, Parkinson's Disease, and schizophrenia. Multiple nAChR subtypes exist, each with a distinct pharmacological and functional profile. The hypothesis is that bovine adrenal chromaffin cells express multiple subtypes that can be identified using receptor protection assays. Exposure to subtype-specific compounds prevents irreversible inactivation of the reduced binding site via alkylation with bromoacetylcholine. By protecting certain subtypes from alkylation, it is then possible to perform radiolabeled binding assays to pharmacologically characterize this population. Our laboratory has demonstrated the utility of antagonist protectants on cultured chromaffin cells; the studies documented here, however, involve the use of a membrane preparation. Unlike an intact cell system, agonistic desensitization does not occur in homogenate tissue, allowing a broader scope of drugs to be examined. Specific binding of [[sup.3]H]epibatidine to the adrenal membrane preparation reaches equilibrium within 30 min at room temperature, and saturation binding fits a model for a single site with a Hill coefficient close to 1. Initial results demonstrate that alkylation eliminates binding within 5%, and a reduction followed by reoxidation control group can yield a recovery of binding above 90%. Two agonists, nicotine and carbachol, offer near complete protection from alkylation, consistent with the expected results since the compounds are not hypothesized to be subtype-specific like mecamylamine and d-tubocurarine.
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Publication:The Ohio Journal of Science
Geographic Code:1USA
Date:Mar 1, 2002
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