Biomedicine & biotechnology senior section.

* Corrigan, G. E. MS MD PhD. The Corrigan Laboratory. The Cytogenetics Of The Benign Lipoma. Previous studies demonstrated the clinical appearance and pathology of the benign lipoma. A complete literature review of lipoma cytogenetics covering the past half-century is fulfilled. Three eras are identified as 1. the Classic Cytogenetic era of Giemsa stained whole cell karyotyping, 2. the era of Molecular Cytogenetics with the use of fluorescent probes as FISH, SKY, and CGH techniques, which overlaps with 3. a concurrent period of Molecular Genetics identified with PCR, RT-PCR, DNA fingerprinting, micro array analysis, flow cytometry, and Northern and Southern Blotting. Works on the lipoma in each era are presented and identify the major cytogenetic qualities of the benign lipoma: cytogenetic heterogeneity with over twenty identified karyotypes; nonconcordance with gross morphology in size, shape, depth, location or sex; broad patterns of congruence with histopathological classifications, and limited utility in behavioral predictability. Frequencies of karyotypic change include an overall incidence of 78% alteration with 25% of all known bands recorded as involved. Gene probes have demonstrated that a major change is the deletion of genetic material in the 12q13-15 region and that breakpoints cluster about the band 13q14. Mitochondrial DNA mutations are recorded in some typical but multiple lipomas.

* Glauser, H.M. and R.C. Garrad. Biomedical Sciences Department, Southwest Missouri State University. The Effect Of C-Terminal Tail Mutations On Desensitixation Of The P2[y.sub.2] Nucleotide Receptor. Cystic Fibrosis (CF) is the most prevalent genetic disorder in Caucasians. CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene encoding the protein that regulates the secretion of chloride ions across the pulmonary epithelium. Present in pulmonary epithelium are P2[y.sub.2] nucleotide receptors of the G protein-coupled receptor (GPCR) family. Activation of the receptor by the agonist (ATP or UTP) leads to an increase in the concentration of intracellular calcium, thereby mediating the opening of calcium-dependent chloride channels and serving as an alternate pathway for chloride secretion. On repeated exposure to the P2[y.sub.2] agonist, the receptor becomes desensitized and is unable to continue to elicit its original response within the cell. The focus of this study is to investigate the effects of mutations of potential phosphorylation sites in the C-terminal tail of the P2[y.sub.2] receptor on desensitization. A better understanding of the process of desensitization could contribute to improved treatments for CF patients. Mutant P2[y.sub.2] receptors have been constructed and expressed in a cell line normally devoid of the receptor, 1321N 1 astrocytoma cells. Effects of these mutations on P2[Y.sub.2] receptor signaling and desensitization are currently being studied using calcium assays, SDS-PAGE, and Western blotting techniques.

* Njau, J., S. Schul, D. Potter, B. Areheart, and L.C. Sudlow. Department of Biological Sciences, Emporia State University. Igf-1 But Not Estrogen Reduces [beta]-Amyloid Toxicity In Cell Cultures. A[B.sub.1-42] is responsible for the toxicity of the [beta]-amyloid neurofibril extracellular plaques of Alzheimer's Disease (Felician and Thomas, 1998, Neuropsych 11:19). 17-[beta]-Estradiol (Manly et al., 2000, Neurology 4:833) and IGF-1 (Dore et al., 1997, PNAS 94:4772) were chosen as ameliorative treatments for A[B.sub.1-42] because levels of both hormones are reduced in the geriatric population and both have been individually demonstrated to improve cell survival during exposure to A[B.sub.1-42]. We sought to determine if treating cells with combinations of more than one hormone would further improve cell survival in the face of A[B.sub.1-42] challenge. Survival of HT-22 hippocampal hybridoma culture cells was measured via the MTT colorimetric assay. Low levels of IGF-1 (<0.1 [micro]M) were sufficient to improve cell survival during A[B.sub.1-42] challenge compared to DMSO or A[B.sub.40-1] reversed-sequence exposure. In contrast, 17-[beta]-estradiol at higher doses (10 [micro]M) contributed to an overall reduction of cell survival during the A[B.sub.1-42] challenge. When both IGF-1 and 17-[beta]-estradiol were applied in combinatorial mixtures, only IGF-1 appeared to contribute to a hormone-dependent elevation of cell survival during A[B.sub.1-42] challenge while 17-[beta]-estradiol provided no protective effect at lower doses. In the HT-22 cultures, only IGF-1 improved HT-22 cell survival in the face of the A[B.sub.1-42] toxicity. It would appear that reduction of IGF-1 levels could serve as a pre-disposing factor towards susceptibility of the cells to the toxic effects of A[B.sub.1-42].

* Stone, D.K.,C.M. Witkowski. Department of Biomedical Sciences, Southwest Missouri State University. Production Of A Functional Collagen Type Iv::Gfp Fusion Protein In Ceanorhabditis Elegans. Collagen type IV is one of the most abundant structural molecules in the basement membrane. We have previously studied collagen type IV in C. elegans using immunofluorescent staining techniques that involve fixation, which kills the animals, or indirectly in live animals using reporter constructs of proteins that are in close proximity to the basement membrane. It is of interest to study the localization, structure, and dynamics of this macromolecule in living worms by directly tagging the collagen type IV molecule with a green fluorescent protein (GFP) reporter that allows the protein to be visualized in vivo. A reporter is being created by using a specialized PCR technique to fuse GFP to collagen type IV. The technique involves sequential PCR reactions, two of which use heterologous primers to create an overhang on the amplified portions of the emb-9 gene, homologous to the gfp gene. During the final two PCR amplifications the overlapping sites produced in the previous reactions are fused producing a product containing the necessary sequence information for translation of a protein containing the functional properties of both collagen type IV and GFP. Sites selected for gfp insertion into emb-9 include an asparagine linked glycosylation site near the 7S domain, a thirteen amino acid interruption in the Gly-X-Y region, and the Gly-X-Y/7S domain junction. The final PCR product will be microinjected along with rol-6, a dominant selectable marker, into the syncytial gonad of C. elegans heterozygous for a collagen type IV null mutation which causes embryonic lethality. Production of a functional fusion protein will be indicated by rescue of the homozygous recessive class with roller and GFP phenotypes.

* Tucker, D.K and R.C Garrad. Department of Biomedical Sciences, Southwest Missouri State University. The Effect Of Protein Kinase C Inhibition On P2[y.sub.2] Receptor Desensitization. Upon agonist (UTP) activation the G protein-coupled P2[Y.sub.2] receptor facilitates an increase in intracellular calcium that stimulates calcium-dependent chloride secretion in airway epithelia. The release of chloride anions via the P2[Y.sub.2] receptor is an alternate pathway that can bypass the defect present in Cystic Fibrosis. This alternative pathway is short lived due to receptor desensitization and internalization. It is known that the P2[Y.sub.2] signaling cascade activates protein kinase C (PKC), however whether PKC plays a specific role in agonist-induced P2[Y.sub.2] desensitization has yet to be determined. Direct activation of PKC by phorbol esters (PMA) or a PMA analog, phorbol 12, 13 dibutyrate, desensitizes the receptor. To investigate the role of PKC we monitored intracellular calcium levels in 1321N1 astrocytoma cells stably transfected with murine P2[Y.sub.2] receptor cDNA using a dual excitation, single emission fluorometric measurement. The PKC inhibitor, GF 109203X caused a concentration-dependent decrease in UTP-mediated desensitization, an effect not produced by the inhibitor RO 31-8220 or by rottlerin. Incubation with GF 109203X may increase the number of receptors on the cell surface, allowing a greater response to the second challenge of UTP. Currently, we are investigating the effects of GF109203X and rottlerin on PMA- and phorbol 12, 13 dibutyrate-mediated desensitization.

* Wijesinghe, R.W., Department of Physics, Pittsburg State University. Comparision Of Electric And Magnetic Techniques For The Determination Of Conduction Velocity Distribution Of Nerve Bundles. There are three main objectives for the research described in this paper. The first is to improve the existing method of predicting Conduction Velocity Distributions of nerve bundles from electrically measured Compound Action Potentials. We accomplished this by using a volume conduction model that includes the effect of an off-center in an isotropic nerve bundle surrounded by an epineurial sheath lying in a saline bath. Furthermore, we allow the transmembrane potential to vary with the temperature and the conduction velocity. The latencies at the stimulating electrodes of the fiber classes are included into our model. Frequency dependent conductivities are used in the volume conduction model to include the dependence of the frequency on the bundle conductivities. Thus far in the literature the calculation the Conduction Velocity Distribution from nerve bundles is based on the electrically measured Compound Action Potentials from the nerve bundles. Therefore, the second objective is to introduce a new method of predicting the Conduction Velocity Distributions of nerve bundles from the Compound Action Currents that are measured magnetically using toroids. The third objective is to compare the variation in the Compound Action Current and Compound Action Potential as the parameters of the model are varied and to compare the Conduction Velocity Distributions predicted from these two signals recorded from nerve bundles with that predicted from histological examination. Based on this detailed analysis of the Compound Action Current, the Compound Action Potential, and the Conduction Velocity Distributions we can conclude that the magnetic technique has certain advantages of the electrical technique.

Colette M. Witkowski

Southwest Missouri State University