Biochemistry, Biophysics, and Structural Biology Commons^™

In Vivo Incorporation Of Tritium To Measure Lipogenesis In Red Skeletal Muscle: Significance Of A Nonlipogenic Diet In La/N-Fa(K)("Corpulent") And Zucker Fa Rat Strains, Karin Traci Mann

Theses, Dissertations and Capstones

Skeletal muscle lipogenesis in rodents may equal that of liver but its importance in the obese animals is not known. We evaluated the rate of fatty acid and triglyceride synthesis and storage content in red skeletal muscles, liver, inguinal fat, and retroperitoneal fat of the LA/N-fa^k (“corpulent”) and Zucker fa obese rat strains. Obese-lean pairs of both rat strains, weaned at 32 days, fed a nonlipogenic diet (54% starch for 21 days) were either dosed with 2 µCi 3H-H² O per gram of body weight and tested after 60 minutes for whole body 3H-incorporation into fatty acids and …

Abnormalities In Post-Translational Processing Of Platelet Rap 1b In Niddm: A Possible Cause Of Platelet Hyperactivity And Cardiovascular Disease In Diabetes, Elizabeth Ann Hall

Theses and Dissertations in Biomedical Sciences

Post-translational processing is critical for the appropriate subcellular localization and function of platelet G-proteins. The majority of the platelet responses to agonists are mediated through specific receptor/G-protein complexes. Therefore, G-protein activity is central to "normal" platelet activity (i.e. aggregation). We have shown that Simvastatin, the in vivo inhibitor of HMG CoA Reductase and therefore isoprenoid synthesis, inhibits the post-translational processing of specific platelet G-proteins and alters platelet responses to agonists. These results show the importance of post-translational processing of G-proteins to platelet activity. Altered post-translational processing of specific G-proteins may explain platelet hyperactivity and the increased incidence of cardiovascular disease …

The Drosophila Melanogaster Rad54 Homolog, Dmrad54, Is Involved In The Repair Of Radiation Damage And Recombination, Rolf Kooistra, José B. M. Zonneveld, Anja De Jong, Jan C. J. Eeken, Chris J. Osgood, Jean-Marie Buerstedde, Paul H. M. Lohman, Albert Pastink

Biological Sciences Faculty Publications

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRAD54-deficient flies develop normally, but the females …

Numa Assembles Into An Extensive Filamentous Structure When Expressed In The Cell Cytoplasm, Alejandro Saredi, Louisa Howard, Duane A. Compton

Dartmouth Scholarship

NuMA is a 236 kDa protein that participates in the organization of the mitotic spindle despite its strict localization in the nucleus during interphase. To test how cells progress through mitosis when NuMA is localized in the cytoplasm instead of the nucleus, we have deleted the nuclear localization sequence of NuMA using site-directed mutagenesis and transiently expressed this mutant protein (NuMA-DeltaNLS) in BHK-21 cells. During interphase, NuMA-DeltaNLS accumulates in the cytoplasm as a large mass approximately the same size as the cell nucleus. When cells enter mitosis, NuMA-DeltaNLS associates normally with the mitotic spindle without causing any apparent deleterious effects …

Modulation Of Queuine Uptake And Incorporation Into Trna By Protein Kinase C And Protein Phosphatase, Rana C. Morris, Bonnie J. Brooks, K. Lenore Hart, Mark S. Elliot

Chemistry & Biochemistry Faculty Publications

It has been suggested that the rate of queuine uptake into cultured human fibroblasts is controlled by phosphorylation levels within the cell. We show that the uptake of queuine is stimulated by activators of protein kinase C (PKC) and inhibitors of protein phosphatase; while inhibitors of PKC, and down-regulation of PKC by chronic exposure to phorbol esters inhibit the uptake of queuine into cultured human fibroblasts. Activators of cAMP- and cGMP-dependent kinases exert no effect on the uptake of queuine into fibroblast cell cultures. These studies suggest that PKC directly supports the activity of the queuine uptake mechanism, and that …

Investigation Of The Substrate Recognition Characteristics And Kinetics Of Mammalian Mitochondrial Dna Topoisomerase I, Zeki Topcu

Theses and Dissertations in Biomedical Sciences

Topoisomerases are DNA-modifying enzymes found in prokaryotes, eukaryotes, viruses and organelles such as chloroplast and mitochondria. Information about these enzymes in eukaryotic systems is mostly limited to nuclear enzymes, although our laboratory has been characterizing the biochemical and biophysical properties of the mammalian mitochondrial topoisomerases. We have determined the polarity of the attachment of mitochondrial topoisomerase I to its substrate DNA. To study the substrate preference and kinetic parameters of mitochondrial topoisomerase I, selected regions of mammalian mitochondrial DNA (mtDNA) were inserted into pGEM plasmid vectors following a series of modification and optimization experiments of currently available methods for PCR-cloning. …

In Vitro Expression Of Human Zona Pellucida Protein 3, Chih-Wei Chen

Theses and Dissertations in Biomedical Sciences

Human zona pellucida protein 3 (hZP3) is the putative receptor on the zona pellucida of the mature oocyte that recognizes and binds sperm, and therefore plays a critical role in fertilization.

A full length cDNA of hZP3 (1278 bp) was amplified from the human ovary mRNA by reverse transcription-polymerase chain reaction (RT-PCR). The hZP3 cDNA was subcloned into PSK and pREP4 expression vectors. The cDNA of hZP3 was further characterized by restriction mapping, PCR, auto-sequencing and Southern blot analysis by using an internal oligonucleotide probe, and found to be identical to the one reported by J. Dean. Using autosequencing, 289 …

A Kinetic Investigation Of The Mechanism Of Muscle Contraction With A Series Of Nucleotides, Wei Jiang

Theses and Dissertations in Biomedical Sciences

Muscle contraction is thought to be accomplished by sliding of myosin filaments along actin filaments. Although actomyosin naturally uses ATP as an energy source, it can also use other nucleoside triphosphates (NTP) as substrates. In this work, the dependence of rate and equilibrium constants of the various steps in the muscle contraction mechanism upon nucleotide structure was investigated to unravel the dependence of the mechanical properties of the muscle upon changes in different biochemical steps of the mechanism.

The experiments measuring the dissociation of actomyosin by NTP (or mant-NTP) shows that both the rate constant of actomyosin-S1 dissociation and second …

Fish Analysis On Spontaneously Arising Micronuclei In The Icf Syndrome, Michael W. Stacey, M. S. Bennett, M. Hulten

Bioelectrics Publications

The ICF syndrome is a rare disorder where patients show undercondensation of the heterochromatic blocks of chromosomes 1, 9, and 16 along with variable immunodeficiency. The undercondensation of the heterochromatic block appears to be restricted to a portion of PHA stimulated T cells. Patients with this syndrome also show an increase in micronuclei formation. We have used dual colour FISH to investigate the chromosomal content of these micronuclei in PHA stimulated peripheral blood cultures, an EBV transformed B cell line, and also micronuclei observed in vivo from peripheral blood smears. Chromosome 1 appears to be present in a higher proportion …

Activation Of Transfer Rna-Guanine Ribosyltransferase By Protein Kinase C, Rana C. Morris, Bonnie J. Brooks, Panayota Eriotou, Deborah F. Kelly, Sandeep Sagar, K. Lenore Hart, Mark S. Elliot

Chemistry & Biochemistry Faculty Publications

Transfer RNA-guanine ribosyltransferase (TGRase) irreversibly incorporates queuine into the first position in the anticodon of four tRNA isoacceptors. Rat brain protein kinase C (PKC) was shown to stimulate rat liver TG Rase activity, TGRase preparations derived from rat liver have been observed to decrease in activity over time in storage at -20 or -70°C, Contamination of the samples by phosphatases was indicated by a p-nitrophenylphosphate conversion test, The addition of micromolar concentrations of the phosphatase inhibitors sodium pyrophosphate and sodium fluoride into TGRase isolation buffers resulted in a greater return of TGRase activity than without these inhibitors, Inactive TGRase preparations …

In Situ Regulation Of Cytosolic Phospolipase A₂, Beverly A. Rzigalinski

Theses and Dissertations in Biomedical Sciences

The 85 kDa cytosolic phospholipase A₂ (cPLA₂) is an agonist-responsive effector for intracellular signal transduction through the arachidonate cascade. In vitro studies have demonstrated that this enzyme is regulated by sub-micromolar calcium and is specific for arachidonate as the sn-2 fatty acyl group of phospholipid substrates. However, very little data is available regarding in situ mechanisms which govern the activity of cPLA₂. The primarily objective of these studies was to develop an in situ system for the study of cPLA₂, and investigate mobilization of arachidonate during signal transduction events.

Dimethylsulfoxide differentiation of the …

Translational Regulation Of The C-Jun Proto-Oncogene, Anil Sehgal

Theses and Dissertations in Biomedical Sciences

The v-jun oncogene was originally isolated from the ASV17 virus in 1987. Ever since its isolation, extensive work has been done to understand the role of the v-jun oncogene in cell transformation. The c-Jun protein is a transcription factor which binds to the DNA target TGACTCA. The c-Jun protein binds to DNA in the form of dimers. It can form homodimers with itself and heterodimers with Jun family (JunB and JunD), Fos family (FosB, Fra1 and Fra2), or with CREB family members through the leucine zipper motif. Because the c-jun proto-oncogene plays an important role in cell transformation, extensive work …

Binding Of Matrix Attachment Regions To Lamin Polymers Involves Single-Stranded Regions And The Minor Groove., M. E. Eva Ludérus, Jan L. Den Blaauwen, Oncko J. De Smit, Duane A. Compton, Roel Van Driel

Dartmouth Scholarship

Chromatin in eukaryotic nuclei is thought to be partitioned into functional loop domains that are generated by the binding of defined DNA sequences, named MARs (matrix attachment regions), to the nuclear matrix. We have previously identified B-type lamins as MAR-binding matrix components (M. E. E. Ludérus, A. de Graaf, E. Mattia, J. L. den Blaauwen, M. A. Grande, L. de Jong, and R. van Driel, Cell 70:949-959, 1992). Here we show that A-type lamins and the structurally related proteins desmin and NuMA also specifically bind MARs in vitro. We studied the interaction between MARs and lamin polymers in molecular detail …

Unusual Structure Of A Human Middle Repetitive Dna, Duminda D. Ratnasinghe

Electronic Theses and Dissertations

The L2Hs sequences are a polymorphic, interspersed, middle repetitive DNA family unique to human genomes. Genomic fingerprinting indicates that these DNAs vary from one individual to another and between tissues of the same individual. Sequence analysis reveals that they are AT-rich (76%) and contain many unusual sequence arrangements (palindromes, inverted and direct repeats). These sequence properties confer on the L2Hs elements the potential to fold into non-B-form structures, a characteristic of recombination hot spots. To test this hypothesis carbodiimide, osmium tetroxide and S$\sb1$ nuclease were used as single-strand specific probes to study a recombinant plasmid, pN6.4.39, containing a single L2Hs …

The Isolation And Identification Of Polyphenolics From Grape Seeds And Their Activity Towards Specific Cell Lines, Joseph A. Reddy

Chemistry & Biochemistry Theses & Dissertations

Grape seed extract is being marketed in France for cardiovascular use and skin care. The active constituents of both these drugs are certain polyphenols called procyanidins. A similar seed extract has now been obtained from Virginia Chardonnay grape seeds. Ten of the major components in this extract have been isolated in their pure form by the semi-preparative HPLC separation on a YMC ODS-AQ (Octadecylsilane-AQ) column. The ODS-Aq column has been shown to have a better resolving capacity compared to the Spherisorb ODS (Octadecylsilane) column previously described in the literature. This has been attributed to the ODSAQ support which has both …

The Cytotoxic Effect Of Tropical Plants On Human And Rat Hepatoma Cells, Mark Tafazoli

Honors Capstone Projects and Theses

No abstract provided.

Analysis Of The Proteoglycan Content In Fresh And Cryopreserved Porcine Cardiovascular Tissues, Yun Hee Shon

Theses and Dissertations in Biomedical Sciences

The purpose of this research was to study the effects of cryopreservation on the proteoglycan (PG) and mineral content of aorta conduit tissue. Proteoglycans from fresh and cryopreserved porcine aorta conduit tissues were isolated by extraction with 4 M guanidine (Gdn) - hydrochloride (HCl) for 48 hours. Quantitative analysis of extracted proteoglycans revealed that the content of proteoglycan material from cryopreserved tissue, measured as uronate and protein per unit weight of wet tissue, was similar to that from fresh tissue. Gel permeation column chromatography studies suggested that proteoglycans present in three CsCl fractions from cryopreserved tissues had molecular weights similar …

The Cγ Subunit Is A Unique Isozyme Of The Camp-Dependent Protein Kinase, Stephen J. Beebe, Paul Salomonsky, Tore Jahnsen, Yixin Li

Bioelectrics Publications

There are at least three isozymes (Cα, Cβ, and Cγ) of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase (PKA) (Beebe, S., Oyen, O., Sandberg, M., Froysa, A., Hansson, V., and Jahnsen, T. (1990) Mol. Endocrinol. 4, 465-475). To compare the Cγ and Cα isozymes, the respective cDNAs were expressed in permanently transformed Kin-8 PKA-deficient Y1 adrenal cells using the mouse metallothionein promoter. The recombinant C subunits were characterized as immunoreactive, zinc-inducible, cAMP-dependent kinase activities. In contrast to Cα, histone was a better substrate than Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) for Cγ. Furthermore, Cγ histone kinase activity was not inhibited by the …

Characterization Of Two Temperature-Sensitive Mutants Of Escherichia Coli Exhibiting An Altered L22 Ribosomal Protein, Bonnie A. Burnette-Vick

Electronic Theses and Dissertations

Analysis of E. coli strains SK1047 and SK1048 have shown them to be temperature-sensitive, protein-synthesis deficient. An alteration in ribosomal protein L22 was detected in both strains using two dimensional gel electrophoresis. Protein L22 was purified from both strains by reversed phase high performance liquid chromatography and from two dimensional electrophoretic gels. Purified ribosomal protein L22 was labeled by reductive methylation and used in 23S RNA binding assays with and without ribosomal protein L4. At the permissive temperature, protein L22 from SK1047 bound less efficiently than the control while protein L22 from SK1048 bound as efficiently as the control. At …

Mouse Mast Cell Proteases: Induction, Molecular Cloning, And Characterization, Wei Chu

Electronic Theses and Dissertations

Tryptase, a mast cell-specific serine protease with trypsin-like specificity, has been identified in a mouse mast cell line (ABFTL-6) based on it's enzymatic activity, inhibition properties, and cross-reactivity to a human mast cell tryptase antibody. The effects of fibroblast-conditioned medium and sodium butyrate on ABFTL-6 mast cell differentiation and tryptase expression have been examined. ABFTL-6 mouse mast cells undergo phenotypic changes upon culturing in media supplemented with fibroblast-conditioned media at 50% or 1 mM sodium butyrate. The induced cells increased in size, had larger and more metachromatic cytoplasmic granules, and increased their total cellular protein about four-fold. Tryptase activity increased …

Cell Surface-Binding Sites For Progesterone Mediate Calcium Uptake In Human Sperm, Peter F. Blackmore, Joseph Neulan, Frank Lattanzio, Stephen J. Beebe

Bioelectrics Publications

Recent studies (e.g. Blackmore, P. F., Beebe, S. J., Danforth, D. R., and Alexander, N.) (1990) J. Biol. Chem. 265, 1376-1380) have shown that in human sperm, progesterone produces a rapid increase in intracellular free calcium ([Ca2+]i) and an induction of the acrosome reaction (e.g. Osman, R. A., Andria, M. L., Jones, A. D., and Meizel, S. (1989) Biochem. Biophys. Res. Commun. 160, 828-833). In this study, the location of progesterone receptors on the cell surface of human sperm was identified using progesterone immobilized on bovine serum albumin (BSA) (progesterone 3-(O-carboxymethyl)oxime:BSA) as well as progesterone and its 3-O-carboxymethyloxime derivative. Using …

Laminin Potentiates Differentiation Of Pcc4uva Embryonal Carcinoma Into Neurons, T. M. Sweeney, Roy C. Ogle, C. D. Little

School of Medical Diagnostics & Translational Sciences Publications

The embryonal carcinoma PCC4uva differentiates into neurons in response to treatment with retinoic acid and dbcAMP. We used this in vitro model system to study the effects of laminin on early neural differentiation. Laminin substrata markedly potentiate neural differentiation of retinoic acid and dbcAMP-treated cultures. Only laminin induced more rapid neural cell body clustering, neurite growth and neurite fasciculation as compared to type IV collagen, type I collagen, and fibronectin substrata. Exogenous laminin substrata promoted greater cell attachment, cellular spreading and growth to confluence than type IV collagen, type I collagen, fibronectin and glass substrata. Laminin-induced effects were inhibited by …

Effects Of Chemical Aneuploidogens On Taxol Purified Drosophila And Mouse Brain Microtubules Polymerization And Depolymerization In Vitro, Anil Sehgal

Biological Sciences Theses & Dissertations

The effects of aneuploidogens (aneuploidy causing agents) on taxol-purified microtubules from Drosophila and mouse brain in vitro were studied by using a spectrophotometric assay and electron microscopy. Colchicine, acetonitrile, propionitrile, acrylonitrile, dimethyl sulfoxide (DMSO), griseofulvin and cadmium chloride inhibited microtubule polymerization whereas methoxyethyl acetate (MEA) and methyl mercuric chloride (MMC) did not. All aneuploidogens tested (at 50mM) resulted in reduced rate of elongation of mouse brain microtubules. MMC, cadmium chloride and DMSO resulted in increased rates of Drosophila microtubule elongation whereas the rest of the drugs resulted in decreases. The in vitro results from Drosophila correlate well with the previously …

The Role Of Small Peptides In Cancer Physiology And Chemotherapy, Bao-Ling Tsay

Theses and Dissertations in Biomedical Sciences

The targeting of proven anticancer drugs specifically to cancer cells would provide a unique opportunity to restrict neoplasms without damaging the cancer patient. The present research utilizes the phenomenon of illicit transport, i.e. the coupling of normally impermeant metabolites to permeant metabolites, in targeting the drug melphalan to mouse Ehrlich ascites tumor cells. The dipeptide beta-alanyl-melphalan was synthesized and tested in vitro for toxicity towards mouse Ehrlich ascites tumor cells, mouse liver cells, and mouse 3T3 embryonic cells. The parent compound, melphalan, was used as a control treatment. In addition, both melphalan and beta-alanyl-melphalan were utilized in in vivo chemotherapeutic …

Collagen Binding Proteins Derived From The Embryonic Fibroblast Cell Surface Recognize Arginine-Glycine-Aspartic Acid, Roy C. Ogle, Charles D. Little

School of Medical Diagnostics & Translational Sciences Publications

Several cell surface proteins (Mr = 120,000, 90,000, 63,000 and 47,000) apparently integral to embryonic fibroblast plasma membranes were extracted with detergent and isolated by collagen affinity chromatography. Certain of these proteins (Mr = 120,000, 90,000, and 47,000) were specifically eluted from collagen affinity columns by synthetic peptides containing the amino acid sequence arginyl-glycyl-aspartic acid (RGD). These data show that a number of collagen binding proteins exist on the embryonic fibroblast cell surface. Some of the proteins may be collagen receptors binding to RGD sequences in the collagen molecule while at least one of the proteins (Mr = 63,000) recognizes …

A Flow Cytometric Approach To Assessing The Environmental And Physiological Status Of Phytoplankton, Serge Demers, Kimberly Davis, Terry L. Cucci

CCPO Publications

No abstract provided.

A Light And Electron Microscopic Study Of The Rat Olfactory Tubercle: Normal Morphology And Acetylcholinesterase Localization, James Curtis Woodley

Biological Sciences Theses & Dissertations

A cytoarchitectural analysis of the rat olfactory tubercle using Nissl-stained coronal sections revealed that the dense cell layer (DCL) consisted of medium sized striatal cells in the cortical regions and small sized "granule" cells in the cap regions. Also delineated from this experiment was a rim of neuropil, nearly devoid of neurons as well as neuronal processes, outlining the islands of Calleja. Acetylcholinesterase (AChE) localization utilizing light microscopy revealed that the olfactory tubercles (OT) contained AChE-positive fibers that were orientated dorsoventrally in the molecular and multiform layers. The DCL consisted of only fibers en passant and putative terminals. Diisopropylfluorophosphate (DFP) …

Modulation Of Queuine Uptake In Cultured Human Fibroblasts By Phorbol Esters And Interferons, Debra L. Crane

Chemistry & Biochemistry Theses & Dissertations

Queuosine, a nucleoside found exclusively in the first position of the anticodon of transfer RNA (tRNA), is generated post-transcriptionally in an exchange of guanine for queuine by the modifying enzyme tRNA-guanine ribosyltransferase. Phorbol ester tumor promoters were shown to induce queuine hypomodified tRNA, and that phorbol ester action was due, in part, to inhibition of queuine transport across the cell membrane. An unidentified protein designated conditioned medium factor (CMF) that potentiated a phorbol-induced inhibition of queuine uptake was also documented. We suggest here that phorbol ester-induced inhibition of queuine uptake is not a significant factor in inducing queuine-deficient tRNA although …

Use Of 52Cr For The Quantitative Determination Of Red Cell Volume, Claude Michael Hanbury

Chemistry & Biochemistry Theses & Dissertations

Chromium-52 has recently been suggested for use as a new agent to label red cells for the in vivo quantitation of red cell volume (1). In this paper, the development and validation of a routine ⁵²cr labeling procedure is described.

The accuracy, precision, and detection limits of chromium analysis by Zeeman effect atomic absorption spectroscopy was evaluated in the concentration range of 1 - 10 ug Cr/L.

Red cell chromium uptake was evaluated as a function of time, temperature, and concentration. Red cells labeled with a 2.5 mg/L chromium solution for 30 minutes at room temperature exhibited optimal label …

Laminin Receptors For Neurite Formation, H. K. Kleinman, Roy C. Ogle, F. B. Cannon, C. D. Little, T. M. Sweeney, L. Luckenbill-Edds

School of Medical Diagnostics & Translational Sciences Publications

Laminin, a basement membrane glycoprotein promotes both cell attachment and neurite outgrowth. Separate domains on laminin elicit these responses, suggesting that distinct receptors occur on the surface of cells. NG108-15 neuroblastoma-glioma cells rapidly extend long processes in the presence of laminin. We report here that 125I-labeled laminin specifically binds to these cells and to three membrane proteins of 67, 110, and 180 kDa. These proteins were isolated by affinity chromatography on laminin-Sepharose. The 67-kDa protein reacted with antibody to the previously characterized receptor for cell attachment to laminin. Antibodies to the 110-kDa and 180-kDa bands demonstrated that the 110-kDa protein …