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Biomedical/biotechnology section. (Senior Division 2002).

* Chandler, LX, Witkowski, C. Biomedical Sciences Department, Southwest Missouri State University, ANALYSIS OF A PUTATIVE CELL BINDING SITE IN THE [alpha]2(IV) CHAIN OF C. ELEGANS COLLAGEN TYPE IV. The objective of this study is to analyze a possible cell binding site (CBS) composed of two aspartate residues and an arginine residue in the [alpha]1(IV) and [alpha]2(IV) chains of collagen type IV, respectively. In humans, an important CBS on the collagen type IV molecule has been characterized and it is located in the Gly-X-Y triple-helical domain. In C. elegans, the same residues are present only once, located relatively in the same position within the triple-helical domain. This conserved area is hypothesized to play an important role in the recognition of co lagen type IV by cell-surface receptors. Research methods include generating strains containing extra-chromosomal arrays. The let-2 gene encodes the [alpha]2(IV) chain of collagen type IV. The let-2 allele, mn153, causes abnormal collagen type IV assembl y and no secretion, resulting in collapse of muscle cells, and lethality. This mutant strain will be injected with plasmid constructs containing either the wild type let-2 gene or a mutated copy (arginine to lysine substitution at amino acid 476). The marker gene rol-6 and the green fluorescent protein (GFP) will be co-microinjected. Animals containing the extra-chromosomal array will be analyzed using Narmaski optics and immunohistochemical techniques.

Garrad, R.C. (1), F.A. Gonzalez (2) and G.A. Weisman (3). (1.) Biomedical Sciences Department, Southwest Missouri State University. (2.) Department of Chemistry, University of PuertoRico. (3.) Department of Biochemistry, University of Missouri-Columbia. INHIBITION OF P2Y2 RECEPTOR DESENSITIZATIONBY INHIBITION OF PROTEIN KINASE C ACTIVITY. UTP, which activates the G protein-coupled P2Y2 nucleotide receptor has been shown to stimulate calcium-activated anion secretion which can bypass the defect present in CF epithelia. Exposure to UTP desensitizes the P2Y2 receptor to further applications of agonist. The P2Y2 receptor signaling pathway activates PKC and activation of PKC by phorbol esters desensitizes the receptor. We have studied the effect of inhibiting PKC activity on receptor desensitization and sequestration. PKC activity was inhibited either by incubating with 500nM PMA overnight or by treating cells expressing P2Y2 receptors with varying concentrations of GF109203X, a specific PKC inhibitor. Incubation with GF109203X may increase receptor numbers on the cell surface and enable a greater response to a second challenge with UTP. These data suggest a role for PKC in the regulation of receptor cycling in the cell.

Gordon, A.R. Department of Biomedical Sciences, Southwest Missouri State University. IDENTIFYING AGING-RELATED GENES IN DROSOPHILA MELANOGASTER. Aging is a multifactorial process involving environmental and genetic components. Standard Drosophila culture techniques are non-selective for allelic variants of aging-related genes that are phenotypically expressed late in the adult life cycle. Laboratory populations exhibit a wide range of lifespan and associated biochemical parameters suggesting that individuals in aging populations are aging at different rates. In this study, longevity selection based on differences in phototaxic responses among aging adults was used to establish longer-lived and shorter-lived subpopulations. After five generations of selection, longevity differences persisted in subpopulations when maintained without selection. A shorter lifespan in males suggests that a number of aging-related genes are located on the large X-chromosome of Drosophila. Work is in progress to determine whether t hese populations show differences in selected biochemical characters related to aging, such as catalase and superoxide dismutase enzyme activity. Comparisons of specific allelic variants between short-lived and long-lived populations using the known Drosophila genome may help identify genes related to aging when the molecular details of their products are not yet known or identified.

Gordon, J.M. Department of Biomedical Sciences, Southwest Missouri State University. EFFECT OF 5-HYDROXYTRYPTAMINE ON CYTOSKELETAL CHANGES IN ENDOTHELIAL CELLS. It is known that inflammation leads to increased vascular permeability and extravasation of fluid from the capillary bed into the interstitial space. 5-Hydroxytryptamine (5-HT) released by platelets and other cells during inflammation has been found to cause endothelial cell (EC) retraction, resulting in increased intercellular gaps. This study was designed to explore the effect of 5-HT on the morphology of calf pulmonary artery ECs. Using actin-specific rhodamine phalloidin as a fluorescent marker, 5-HT-treated cells were found to have fewer actin filaments at the cell periphery, increased stress fiber formation, and smoother, denser edges than control cells. 5-HT-treated cells had less defined lamellipodia and filopodia than control cells, suggesting changes in the cytoskeleton. Cells pre-treated with SB 206553, a 5-HT receptor inhibitor, prior to 5 -HT incubation showed morphology and actin distribution similar to control cells. This research suggests that 5-HT may have an effect on vascular permeability and extravasation at the EC level related to alteration of the actin cytoskeleton.

House, C. D. and Witkowski, C. M. Department of Biomedical Sciences, Southwest Missouri State University. POTENTIAL INSERTION OF THE GREEN FLUORESCENT PROTEIN CODING SEQUENCE INTO THE C. ELEGANS [alpha]2(IV) COLLAGEN GENE. Type IV collagen is an important structural element in most basement membranes of C. elegans. The formation of type IV collagen networks is partly understood, but not easily visualized in vivo. This study was conducted to explore the potential of expressing type IV collagen tagged with green fluorescent protein in C. elegans. Published literature, database sequences, and computer alignments were used to identify interruptions of the Gly-X-Y triple-helical domain in the [alpha]1 and [alpha]2 subunits of type IV collagen. Computer-simulated restriction enzyme digests were used to identify potential restriction enzyme cut sites located within both genes. Based on the known structure and function of collagen, a restriction enzyme site occurring exactly once on the [alpha]2(IV) gene and located within an interruption of the triple-helical domain was determined to be the best potential site for incorporation of the GFP sequence. Once successfully cloned, this recombinant gene will be used to rescue collagen IV null mutants, and we will be able to select for a functional tagged protein.

Hutcheson, C.J., C.M. Witkowski. Department of Cell and Molecular Biology, Southwest Missouri State University. ANALYSIS OF BODY WALL MUSCLE CELL SHAPE IN WILD-TYPE AND COLLAGEN IV MUTANT STRAINS OF C. ELEGANS. C. elegans body wall muscle cells organize and begin to function at an early stage during embryogenesis. Animals homozygous for a null allele of collagen IV lack this molecule in the basement membrane of muscle cells. These mutants show degeneration of muscle cell ultrastructure as early embryos and die during embryogenesis. The myofilament lattice collapse in these embryos has been analyzed by immunofluorescence with antibodies to myosin A and actin. Research is in progress to produce transgenic C. elegans worms (mutant and wild-type) that contain marker genes rol-6 (su1006) and Green Fluorescent Protein (GFP). GEP expression is driven by a muscle specific promoter from the [beta]1 integrin subunit and is targeted to the plasma membrane allowing GFP to tether to the cells and illuminate specifically m uscle cell membranes in the worm. This study intends to determine whether these muscle cells show degeneration of shape or if they appear normal as compared to wild-type. The results should help determine what effect lack of collagen IV in the basement membrane has on muscle cell shape and organization of the longitudinal body wall muscle quadrants during degeneration of muscle cell ultrastructure.

Islam, [M.R., (1) M. Rodova (2) and J.P. Calvet (2) (1.) Department of Chemistry/Physics, Northwest Missouri State University; (2.) Department of Biochemistry and Molecular Biology, University of Kansas Medical Center. DNA FRAGMENT ISOLATION FROM AGAROSE GELS WITHOUT USING COMMERCIAL KITS. Isolation of DNA fragments is an essential step prior to most cloning works. Several commercial kits available that allow molecular biologists efficient isolation of DNA fragments. Although prices of these kits are not so expensive, the costs are especially important in labs doing a lot of cloning works and in undergraduate teaching colleges/universities where budgets are limited. Here, we describe a fast and efficient method for the isolation of DNA fragments from agarose gels by simply employing centrifugation in microfuge tubes. The yield of linearized 2.95 kb pBluescript ran on 1% agarose in 1x Tris-acetate EDTA was 50%, employing either -20[degrees]C or -80[degrees]C incubations, compare to the 45-60% yields obtained u sing the "Quantum Prep Freeze 'N Squeeze DNA Gel Extraction Spin Columns" from Bio-Rad, and "GenElute Agarose Spin Columns" from Sigma. There was a significant amount of DNA left in the gel slices after the first centrifugation, which could be easily extracted in a small volume by a second centrifugation. The vector DNA isolated in this centrifugation method can be successfully treated with alkaline phosphatase, and ligase to clone a foreign 1.5 kb DNA fragment.

Langiano, L.G., C.M. Witkowski. Department of Cell and Molecular Biology, Southwest Missouri State University. PROPOSED CELL BINDING SITE ANALYSIS OF CAENORHABDITIS ELEGANS COLLAGEN IV. We are using C. elegans as a model organism to analyze the function of a conserved site on the collagen IV molecule. The assembled C. elegans molecule contains a sequence that is homologous to a cell-binding site (CBS) on the human molecule for integrin [alpha]1[beta]1. Our hypothesis is that this may also be an important CBS in C. elegans. We have used transgenic techniques to produce animals bearing a gene for the collagen [alpha]1(IV) chain containing a single point mutation at the proposed CBS in combination with marker genes rol-6 (su1006) and Green fluorescent Protein (GFP). Deletion mutations of the [alpha]1(IV) chain result in a lethal null phenotype. Preliminary data show the CBS mutant transgenes do not rescue the homozygous null animals, while control mutations do. Nomarsky optics and immunohistochemical techniques with antibodies to the [alpha]2(IV) chain, myosin heavy chain, and GFP are being used to analyze the phenotypic defects exhibited by homozygous animals with a CBS mutation. These animals arrest during embryogenesis and show loss of muscle structure, thereby characterizing the CBS as an important conserved feature.

Ralya, A., and C.M. Witkowski, Department of Biomedical Sciences, Southwest Missouri State University. ANALYSIS OF TRANSGENIC C.ELEGANS STRAINS. This project was undertaken to analyze the importance of a putative cell binding site (CBS) on the collagen IV molecule using C. elegans as a model for the human molecules. C. elegans is a non-parasitic nematode that exists either as a maleor a self-fertilizing hermaphrodite, and its entire genome has already been sequenced. Collagen IV exists as two [alpha]1 chains and one [alpha]2 chain, and the human CBS contains conserved aspartate (D) residues on the [alpha]1 chains and an arginine (R) residue on the [alpha]2 chain. These three residues form a 3-dimensional D-R-D conformation that is recognized by the cell surface receptor. Transgenic nematodes were created by injecting three plasmids into the syncytial gonad of the hermaphrodites forming extra-chromosomal arrays. The experimental transgenic genotype was a point mutation of Asp(480) to Glu in the D-R-D site, and the control mutation was Asp(472) to Glu in a Gly-X-Y repeat upstream from the proposed CBS. The other two plasmids received in both injections were marker genes rol-6(su1006) and GFP. Transgenic progeny were then isolated and analyzed using immunofluorescence. Nematode cultures were kept on agar plates with OP-50 E. coli at 15[degrees]C and observed and manipulated under a stereomicroscope. Using these techniques, the function of the CBS can be analyzed.

Witkowski, C.M. Department of Biomedical Sciences, Southwest Missouri State University. USING RNAi TO IDENTIFY A CELL SURFACE RECEPTOR COLLAGEN TYPE IV IN C. ELEGANS. Collagen type IV is one of the most abundant proteins present in basement membranes. Highly conserved regions of this molecule point the importance in providing developmental cues for all organisms. Cells receive infor-mation from the underlying basement membrane through cell surface receptors that interact directly with the basement membrane proteins. In vertebrates, integrins are the cell surface receptors for collagen type IV. The model organism C. elegans, a free living soil worm, expresses collagen type IV. The goal of this research is to identify the C.elegans cell surface receptor for recognition of collagen type IV. Search of the C.elegans genome suggests this organism uses a different mechanism compared to vertebrates. Candidate genes have been identified by BLAST analysis and the reverse genetic technique of RNAi will be used to study these genes. RNAi uses short dRNA oligo-nucleotides to regions of expressed portions of genes to phenocopy a null mutation. Transgenic phenotypes expected will be similar to collagen type IV null animals. Using this model system to understand the functions of collagen type IV will help in understanding the developmental cues provided by collagen type IV. Supported by a SMSU Faculty Research Grant.
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Author:Witkowski, Colette M.
Publication:Transactions of the Missouri Academy of Science
Geographic Code:1U4MO
Date:Jan 1, 2002
Previous Article:Biology section. (Senior Division 2002).
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