Biochemistry, Biophysics, and Structural Biology Commons^™

Effects Of Creatine Supplementation On Muscular Strength And Power Development In Well-Trained Football Players, Josef D. Mcquain

Theses, Dissertations and Capstones

The purpose of this study was to determine the effects of creatine supplementation on strength and power performance in well-trained football players. Twenty-one subjects were randomly assigned to three treatment groups: 1) creatine monohydrate, 2) creatine monohydrate+dextrose, or 3) placebo utilizing a double-blind design. Prior to assignment, subjects were matched according to lean body mass. After a five day loading dose, a maintenance dose was administered for 12 days. Test protocols included three anaerobic work bouts: l) two 30 s Wingate bike tests interspersed with five minutes recovery were used to ascertain relative and absolute power, 2) two 100 yard …

An Immunomodulating Mycotoxin Interferes With The Development Of Autoimmune Diabetes In Diabetes-Prone Bb/Wor Rats, Honggang Liu

Theses, Dissertations and Capstones

Various fungal products have immunomodulating activity and some have been studied regarding prevention of transplantation rejection. Prior to this investigation, the mycotoxin, gliotoxin (GT), has never been investigated as an immunotherapeutic drug for autoimmune disease. GT is a fungal secondary metabolite and a member of the epipolythiodioxopiperazine (ETP) family which has been shown to inhibit phagocytosis, induction of cytolytic T cells and the proliferation of T cells following mitogen stimulation. GT also induces in vitro apoptosis in certain immune cell types. More importantly, GT exhibits selective activity towards cells of hemopoietic origin.

Autoimmune diseases are disorders caused by immune responses …

The Regulation Of Glucose Transport In Cultured Vascular Smooth Muscle Cells By Angiotensin Ii And Glucose, Leslie Ann Quinn

Theses, Dissertations and Capstones

Glucose transport was assessed in vascular smooth muscle (VSM) cells by measuring the uptake of a radiolabeled non-metabolizable glucose analog, [3H]-2-deoxglucose. VSM cells, isolated from rat aortae by enzymatic digestion, were maintained in culture in Dulbecco's Modified Eagle Medium supplemented with 10% newborn calf serum at 37°C with 5% CO2 and air. Angiotensin II (AU) increased glucose transport by 84%. Significant stimulation occurred by two hours of exposure with the maximum effect being observed between six and eight hours. All effects were concentration dependent with a threshold response being detected at 0.1 nM. All-stimulated transport was blocked by an AU …

Regulation Of The Expression Of Hmn-Sod At The Level Of The Mitochondrial Processing Of Precursor Hmn-Sod, Gary Leslie Wright

Theses, Dissertations and Capstones

The mitochondrion imports and processes the vast majority of the proteins which comprise its structural elements and metabolic pathways. Studies in the last decade have been successful at defining the mechanism and protein machinery responsible for recognizing, importing, and processing cytosolic precursor proteins into the mature mitochondrial proteins. Little is known, however, about physiological factors which might influence or compromise mitochondrial protein processing. The objective of this work was to develop a novel approach to study mitochondrial import and assembly of precursor proteins as a process within the context of the cellular environment. To these ends we employed the baculovirus …

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 …

Phosphorylation Within The Amino-Terminal Acidic Domain I Of The Phosphoprotein Of Vesicular Stomatitis Virus Is Required For Transcription But Not For Replication, Asit K. Pattnaik, Leroy Hwang, Tong Le, Nathan Englund, Manjula Mathur, Tapas Das, Amiya Banerjee

School of Veterinary and Biomedical Sciences: Faculty Publications

Phosphorylation by casein kinase II at three specific residues (S-60, T-62, and S-64) within the acidic domain I of the P protein of Indiana serotype vesicular stomatitis virus has been shown to be critical for in vitro transcription activity of the viral RNA polymerase (P-L) complex. To examine the role of phosphorylation of P protein in transcription as well as replication in vivo, we used a panel of mutant P proteins in which the phosphate acceptor sites in domain I were substituted with alanines or other amino acids. Analyses of the alanine-substituted mutant P proteins for the ability to support …

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 …

Modulated Structures In Electrodeposited Superconducting Ba(1-X)K(X)Bio(3), Chad Bryant Huffman

Theses, Dissertations and Capstones

Compositionally modulated crystals of the superconductor Ba_1-x K_xBiO₃ have been grown electrochemically. Modulation was produced through control of the deposition potential.

Modulated crystals were imaged using optical microscopy, backscattered SEM, and STM. The expected linear relationship between the amount of time that current was applied and the modulation thickness was observed, and the growth rates were determined.

Multilayers were differentially etched using an EDTA solution. Etched and unetched samples were imaged using backscattered SEM.

The transition temperature of a modulated sample was determined by SQUID magnetometry to equal the bulk value.

Use Of The (3)H-Tetracycline Rat Model In The Study Of Skeletal Compartmentalization And Metabolism Of Calcium In The Maturing Male And Female Rat And The Role Of The Skeleton In Calcium Homeostasis In The Adolescent Male Rat, Darrin Lee Demoss

Theses, Dissertations and Capstones

The (3)H-tetracycline bone labeling procedure was employed to monitor bone resorption from urinary loss curves in male and female rats of various ages. In addition, whole body dry skeletal mass and the loss of (3)H-tetracycline from individual bones was determined. It was found that the dry skeletal mass/body mass ratio of 24-week-old females was 30-40% greater than that for males. The urinary loss of (3)H-tetracycline was described by a double exponential equation indicating the presence of two distinct and independent exchangeable bone compartments. Both compartments decrease in size with age, but their label loss activities were different. The label loss …

Regulation Of Melanogenesis In B16 Mouse Melanoma Cells By Protein Kinase C (Pkc), Harish Mahalingam

Theses, Dissertations and Capstones

The pigment cell-specific expression of tyrosinase and TRP1 has been shown to be important for the production of melanin in pigmented cells. Using a pigmented cell line, B16 mouse melanoma, we obtained evidence that PKC plays a major role in regulating melanogenesis. Chronic treatment with phorbol dibutyrate (PDBu) leads to downregulation of PKC activity and protein levels. This is accompanied by a loss of pigmentation which is correlated with a 50% reduction and a complete loss of TRP1 and tyrosinase respectively. Similar results were obtained with Northern and Western blotting indicating that PKC may regulate the steady state levels of …

Postnatal Development Of The Neural Retina In A South American Opossum: Monodelphis Domestica, Tracy L. Soltesz

Theses, Dissertations and Capstones

Postnatal retinal development was studied in a marsupial opossum, Monodelphis domestica using light microscopy and 3H-thymidine autoradiography. For the light microscopic study, opossum neonates at 1, 3, 7, 14, 21, 25 and 28 days of age were euthanized, fixed and processed into paraffin sections for hematoxylin and eosin staining. The distinct ganglion cell layer, first observed on postnatal day (P) 7, was separated from the outer neuroblasts by the inner plexiform layer. The neuroblast layer was divided into inner and outer nuclear layers on P25 by the presumptive outer plexiform layer, indicated by discrete intercellular spaces located between the nuclear …

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 …

Bovine Cell Line Resistant To In Vtro Infection By Bovine Viral Darrheavirus And All Other Known Pestiviruses, Ruben O. Donis, Eduardo F. Flores

School of Veterinary and Biomedical Sciences: Faculty Publications

Bovine cell line resistant to infection by the pestiviruses Bovine Viral Diarrhea Virus (BVDV), Hog Cholera Virus (HCV) and Border Disease Virus (BDV) and all progeny and mutants thereof. A bovine cell line (CRIB) that is resistant to infection by cytopathic and non-cytopathic BVDV and by other Pestiviruses due to a stable, recessive genetic defect which blocks infection at the level of viral entry.

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 …

Trophic Transfer Of Fatty Acids, Sterols, And A Triterpenoid Alcohol Between Bacteria, A Ciliate, And The Copepod Acartia Tonsa, Melissa C. Ederington, George B. Mcmanus, H. Rodger Harvey

OES Faculty Publications

The incorporation of lipids into the copepod Acartia tonsa and its eggs was measured when it was fed either a bacterivorous ciliate (Pleuronema sp.) or a diatom (Thalassiosira weissflogii). Egg production was 10-fold higher on the diatom diet, whereas hatch success of eggs was the same for algal and ciliate diets. Adult copepods fed diatoms contained more total fatty acid and sterols than copepods fed the ciliate diet, and individual lipids reflected the dietary source. Eggs from diatom-fed copepods had fewer fatty acids but more sterols than eggs from copepods on a ciliate diet. Ciliate-fed copepods and …

Heterozygosity Mapping Of Partially Congenic Lines: Mapping Of A Semidominant Neurological Mutation, Wheels ( Whl), On Mouse Chromosome 4, Patrick M. Nolan, Patricia J. Sollars, Barbara A. Bohne, Warren J. Ewens, Gary E. Pickard, Maja Bucan

School of Veterinary and Biomedical Sciences: Faculty Publications

We identified a semidominant, chemically induced, mouse use mutation with a complex array of abnormal behaviors including bidirectional circling and hyperactivity, abnormal circadian rhythmicity and abnormal responses to light. In this report, we genetically and phenotypically characterized the circling/ waltzing component of the abnormal behavior. We mapped the locus controlling this trait by heterozygosity mapping of partially congenic lines carrying the mutagenized chromosome outcrossed to different inbred strains for three generations. Analysis of 68 PCR-based markers in 13 affected individuals indicated that the mutant locus, named Wheels (Whl), resides in the subcentromeric portion of mouse chromosome 4. The …

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 …

Restoration Of Circadian Behavior By Anterior Hypothalamic Heterografts, Patricia J. Sollars, Daniel P. Kimble, Gary E. Pickard

School of Veterinary and Biomedical Sciences: Faculty Publications

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus (AH) is a circadian oscillator and an important component of the mammalian circadian system. To determine whether the SCN is the dominant circadian pacemaker responsible for generating a species-typical characteristic of circadian rhythms [i.e., period length (T)], neural transplantation was conducted using fetal AH donors of different species and SCN-lesioned (SCNx) hosts. The circadian behavior of each of the three donor species is clearly distinguishable by its species-typical T. The extent of SCN pacemaker autonomy was assessed by noting whether the period of the restored circadian rhythm following heterograft transplantation was characteristic …

Complementary Gradients In Expression And Binding Of Elf-1 And Mek4 In Development Of The Topographic Retinotectal Projection Map, Masaru Nakamoto

Biology Faculty Publications

Topographic maps with a defined spatial ordering of neuronal connections are a key feature of brain organization. Such maps are believed to develop in response to complementary position-specific labels in presynaptic and postsynaptic fields. However, the complementary labeling molecules are not known. In the well studied visual map of retinal axons projecting to the tectum, the labels are hypothesized to be in gradients, without needing large numbers of cell-specific molecules. We recently cloned ELF-1 as a ligand for Eph family receptors. Here, RNA hybridization shows matching expression gradients for ELF-1 in the tectum and its receptor Mek4 in the retina. …

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 …

Certain Canine Weakly Β-Hemolytic Intestinal Spirochetes Are Phenotypically And Genotypically Related To Spirochetes Associated With Human And Porcine Intestinal Spirochetosis, Gerald E. Duhamel, Nagaraja Muniappa, Michelle R. Mathiesen, J. L. Johnson, J. Toth, R. O. Elder, A. R. Doster

School of Veterinary and Biomedical Sciences: Faculty Publications

Four canine weakly β-hemolytic intestinal spirochetes associated with intestinal spirochetosis (IS-associated WBHIS) were compared with IS-associated human and porcine WBHIS and the type species for Serpulina hyodysenteriae and S. innocens by using phenotypic and genotypic parameters. The IS-associated canine, human, and porcine WBHIS belonged to a phyletic group distinct from but related to previously described Serpulina type species.

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 …

Role Of Heat Shock Protein 70 Kda Cognate In Limiting Thermal Inactivation And Refolding Of Heat-Denatured Nuclear Type I Topoisomerase, Kuo-Kuang Wen

Theses and Dissertations in Biomedical Sciences

Previous studies (Ciavarra et al., 1994) demonstrated that the constitutive 70 kDa heat shock protein (hsc70) protected purified topoisomerase I from thermal injury. In addition, hsc70 was capable of regenerating catalytic activity of heat-denatured topoisomerase I. A whole cell lysate was also active in this reaction assay. The present study demonstrates that heat-denatured topoisomerase I is reactivated by a cytosolic fraction and that this activity is dependent on the presence of cytosolic hsc70. The efficacy of hsc70-mediated refolding of heat-denatured topoisomerase I is greatly enhanced by a cytosolic cofactor(s). In all these refolding reactions, exogenous ATP is not required. Size …

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 …

Morphometric Analysis Of Enteric Lesions In C3h/Hen Mice Inoculated With Serpulina Hyodysenteriae Serotypes 2 And 4 With Or Without Oral Streptomycin Pretreatment, Jagannatha V. Mysore, Gerald Duhamel

School of Veterinary and Biomedical Sciences: Faculty Publications

The segmental distribution and sequential progression and the role of the indigenous bacterial flora in the development of enteric lesions associated with Serpulina hyodysenteriae infection in laboratory mice have not been defined. We examined the distribution and sequential morphometric changes in the large intestine of mice orally inoculated with S. hyodysenteriae serotypes 2 and 4. To determine the role of colonization resistance conferred by the indigenous bacterial flora, 40 female C3H/HeN mice were administered water alone or water containing 5 mg/mL streptomycin sulfate ad libitum for seven days prior to orogastric inoculation either with S. hyodysenteriae or sterile trypticase soy …