Molecular Biology Commons^™

The Stromal Cell-Derived Factor-1alpha/Cxcr4 Ligand-Receptor Axis Is Critical For Progenitor Survival And Migration In The Pancreas., Ayse G. Kayali, Kurt Van Gunst, Iain L. Campbell, Aleksandr Stotland, Marcie Kritzik, Guoxun Liu, Malin Flodström-Tullberg, You-Qing Zhang, Nora Sarvetnick

Journal Articles: Regenerative Medicine

The SDF-1alpha/CXCR4 ligand/chemokine receptor pair is required for appropriate patterning during ontogeny and stimulates the growth and differentiation of critical cell types. Here, we demonstrate SDF-1alpha and CXCR4 expression in fetal pancreas. We have found that SDF-1alpha and its receptor CXCR4 are expressed in islets, also CXCR4 is expressed in and around the proliferating duct epithelium of the regenerating pancreas of the interferon (IFN) gamma-nonobese diabetic mouse. We show that SDF-1alpha stimulates the phosphorylation of Akt, mitogen-activated protein kinase, and Src in pancreatic duct cells. Furthermore, migration assays indicate a stimulatory effect of SDF-1alpha on ductal cell migration. Importantly, blocking …

Perilipin A Is Essential For The Translocation Of Hormone-Sensitive Lipase During Lipolytic Activation. J Cell Biol, John Tansey

Chemistry Faculty Scholarship

A key step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase (HSL) from the cytosol to the surface of the lipid storage droplet. Adipocytes from perilipin -null animals have an elevated basal rate of lipolysis compared with adipocytes from wild-type mice, but fail to respond maximally to lipolytic stimuli. This defect is downstream of the -adrenergic receptor–adenylyl cyclase complex. Now, we show that HSL is basally associated with lipid droplet surfaces at a low level in perilipin nulls, but that stimulated translocation from the cytosol to lipid droplets is absent in adipocytes derived from embryonic fibroblasts of …

Hybridization Between The Watersnakes Nerodia Sipedon And Nerodia Fasciata, In The Carolinas: A Morphological And Molecular Approach, Konrad Mebert

Biological Sciences Theses & Dissertations

A few traditionally applied diagnostic characters of color pattern were compared with an additional set of morphological and genetic characters to evaluate differences between Nerodia sipedon and N. fasciata and to study the dynamics across their hybrid zone in the Carolinas. Many of the morphological characters exhibited significant interspecific differences, although only the number of dorsally complete crossbands (CBa) was diagnostic by itself. A discriminant function analysis of morphological characters was successful in separating both taxa. Species-specific nuclear markers, identified by the AFLP (Amplified Fragment Length Polymorphism) technique were nearly fixed and served as diagnostic markers. They revealed extensive introgression …

Hyaluronate Lyase Gene Of Streptococcus Pyogenes: Molecular Characterization And Regulation, Sheryl Lynne Walton

Theses and Dissertations in Biomedical Sciences

Streptococcus pyogenes causes an assortment of diseases ranging from pharyngitis to necrotizing fasciitis. This bacterium has the ability to elaborate several extracellular products capable of causing tissue damage; one of which is a hyaluronate lyase. Little information is available regarding the regulation of streptococcal hyaluronidase. As such, the expression of hylA in the hyaluronate lyase-producing strain ATCC 10403 and in strains that do not produce detectable enzyme was analyzed by RT-PCR and quantitative PCR. Hyaluronate lyase transcript was detected throughout growth for all strains; however, expression of hylA in the enzymatically inactive strains, 71698 and SF370, was determined …

The Quantitative Assessment Of Osteoinductivity Of Human Demineralized Bone Matrix And Cdna Array Analysis Of Osteogenic Differentiation In Human Periosteal Cells, Sittisak Honsawek

Theses and Dissertations in Biomedical Sciences

Demineralized bone matrix (DBM) has been extensively utilized in orthopeadic, periodontal, and maxillofacial applications. DBM comprises bone morphogenetic proteins (BMPs) that are essential regulators for endochondral bone formation. The current study was undertaken to establish the quantitative in vitro assays for determining the osteoinductive potential of human DBM and the use of cDNA gene expression array technology for examining gene expression profiles during osteoblast differentiation in human periosteal cells.

BMP-4 is one of the most osteoinductive factors and has been investigated for clinical applications. For this reason, BMP-4 was designated as the osteoinductive protein for use in the quantitative in …

Purified Particulate Methane Monooxygenase From Methylococcus Capsulatus (Bath) Is A Dimer With Both Mononuclear Copper And A Copper-Containing Cluster, Raquel L. Lieberman, Deepak B. Shrestha, Peter E. Doan, Brian M. Hoffman, Timothy L. Stemmler, Amy C. Rosenzweig

Biochemistry and Molecular Biology Faculty Publications

Particulate methane monooxygenase (pMMO) is a membrane-bound enzyme that catalyzes the oxidation of methane to methanol in methanotropic bacteria. Understanding how this enzyme hydroxylates methane at ambient temperature and pressure is of fundamental chemical and potential commercial importance. Difficulties in solubilizing and purifying active pMMO have led to conflicting reports regarding its biochemical and biophysical properties, however. We have purified pMMO from Methylococcus capsulatus (Bath) and detected activity. The purified enzyme has a molecular mass of ~200 kDa, probably corresponding to an a2b2g2 polypeptide arrangement. Each 200 kDa pMMO complex contains 4.8 ± 0.8 copper ions and 1.5 ± 0.7 …

Androgen-Induced Recruitment Of Rna Polymerase Ii To A Nuclear Receptor–P160 Coactivator Complex, Maggie C. Louie, Hong Qiong Yang, Ai-Hong Ma, Wei Xu, June X. Zou, Hsing-Jien Kung, Hong-Wu Chen

Collected Faculty Scholarship

The androgen receptor, like other nuclear receptors, activates target genes by binding to hormone-responsive enhancers. Here we demonstrate that androgen induces robust recruitment of androgen receptor, members of the p160 coactivator family, and CREB-binding protein/p300 specifically at the distant enhancer of prostate-specific antigen (PSA) gene. Unexpectedly, we found that RNA polymerase II (Pol II) is directly recruited to the enhancer in a hormone-dependent manner, independent of the proximal promoter, and that the isolated PSA enhancer can mediate efficient androgen induction of transcription. Inhibition of the Pol II carboxyl-terminal domain kinase activity with low concentrations of flavopiridol blocks Pol II transfer …

Rec12 (Spo11) Recominase Of Fission Yeast Promotes A Backup, Distributive Pathway For Chromosome Segregation In Meiosis I, Wallace D. Sharif, Mari K. Davidson, Wayne P. Wahls

Journal of the Arkansas Academy of Science

We studied the relationship between recombination and segregation in the fission yeast Schizosaccharomyces pombe. In meiosis, chromosomes undergo two rounds of segregation to produce haploid meiotic products. Crossover meiotic ecombination (chiasmata) promotes chromosome segregation during meiosis I; achiasmatic chromosomes often suffer on disjunction in meiosis I. Recl2 protein is a topoisomerase IIortholog that introduces double-strand DNA breaks that nitiate recombination. The red2 null (deletion) and active site (Y98F) mutants lack recombination and crossovers, and onsequently suffer meiosis I nondisjunction. However, null mutants chromosomes segregate to opposite poles more frequently than predicted. Thus, fission yeast has a backup, "distributive segregation" pathway …

Protocols For Disease Classification From Mass Spectrometry Data, Michael Wagner, Dayanand Naik, Alex Pothen

Mathematics & Statistics Faculty Publications

We report our results in classifying protein matrix-assisted laser desorption/ionizationtime of flight mass spectra obtained from serum samples into diseased and healthy groups. We discuss in detail five of the steps in preprocessing the mass spectral data for biomarker discovery, as well as our criterion for choosing a small set of peaks for classifying the samples. Cross-validation studies with four selected proteins yielded misclassification rates in the 10-15% range for all the classification methods. Three of these proteins or protein fragments are down-regulated and one up-regulated in lung cancer, the disease under consideration in this data set. When cross-validation studies …

Multiple Transporters Associated With Malaria Parasite Responses To Chloroquine And Quinine, Jianbing Mu, Michael T. Ferdig, Xiaorong Feng, Deirdre A. Joy, Junhui Duan, Tetsuya Furuya, G. Subramanian, L. Aravind, Roland A. Cooper, John C. Wootton, Momia Xiong, Xin-Zhuan Su

Biological Sciences Faculty Publications

Mutations and/or overexpression of various transporters are known to confer drug resistance in a variety of organisms. In the malaria parasite Plasmodium falciparum, a homologue of P-glycoprotein, PfMDR1, has been implicated in responses to chloroquine (CO), quinine (ON) and other drugs, and a putative transporter, PfCRT, was recently demonstrated to be the key molecule in CO resistance. However, other unknown molecules are probably involved, as different parasite clones carrying the same pfcrt and pfmdr1 alleles show a wide range of quantitative responses to CO and ON. Such molecules may contribute to increasing incidences of ON treatment failure, the molecular basis …

Ccr4-Bearing T Cells Participate In Autoimmune Diabetes., Soon H. Kim, Mary M. Cleary, Howard S. Fox, Icos Coporation, Nora Sarvetnick

Journal Articles: Regenerative Medicine

Chemokine receptor expression is exquisitely regulated on T cell subsets during the course of their migration to inflammatory sites. In the present study we demonstrate that CCR4 expression marks a pathogenic population of autoimmune T cells. CCR4 was found exclusively on memory CD4(+) T cells during the progression of disease in NOD mice. Cells expressing the CCR4 ligand TARC (thymus- and activation-regulated chemokine) were detected within infiltrated islets from prediabetic mice. Interestingly, neutralization of macrophage-derived chemokine (MDC) with Ab caused a significant reduction of CCR4-positive T cells within the pancreatic infiltrates and inhibited the development of insulitis and diabetes. Furthermore, …

A Comparison Of The Low Mode And Monte Carlo Conformational Search Methods, Carol A. Parish, Rosina Lombardi, Kent Sinclair, Emelyn Smith, Alla Goldberg, Melissa Rappleye, Myrianne Dure

Chemistry Faculty Publications

The Low Mode (LM) and Monte Carlo (MC) conformational search methods were compared on three diverse molecular systems; (4R, 5S, 6S, 7R)-hexahydro-5,6-dihydroxy-1,3,4,7-tetrakis(phenylmethyl)-2H-1,3-diazapin-2-one (1), 2-methoxy-2-phenyl-2-triflouromethyl-N-α-methyl benzyl propanamide (2) and a trimeric 39-membered polyazamacrolide (3). We find that either method, or a combination of the methods, is equally efficient at searching the conformational space of the smaller molecular systems while a 50:50 hybrid of Low Mode and Monte Carlo is most efficient at searching the space of the larger molecular system.

Electrically Mediated Delivery Of Vector Plasmid Dna Elicits An Antitumor Effect, L. Heller, D. Coppola

Bioelectrics Publications

In vivo electroporation is an efficient means of increasing plasmid DNA delivery to normal tissues, such as skin and muscle, as well as directly to tumors. In the experiments described here, plasmid DNA was delivered by in vivo electroporation to B16 mouse melanomas using two very different pulsing protocols. Reporter expression increased 21- or 42-fold, respectively with electroporation over injection alone. The growth of experimental melanomas with an approximate diameter of 4 mm on the day of treatment was monitored after electroporation delivery of reporter plasmid DNA. Remarkably, short-term complete regressions using one of these pulsing protocols occurred in up …

Meiotic Cohesion Requires Accumulation Of Ord On Chromosomes Before Condensation, Eric M. Balicky, Matthew W. Endres, Cary Lai, Sharon E. Bickel

Dartmouth Scholarship

Cohesion between sister chromatids is a prerequisite for accurate chromosome segregation during mitosis and meiosis. To allow chromosome condensation during prophase, the connections that hold sister chromatids together must be maintained but still permit extensive chromatin compaction. In Drosophila, null mutations in the orientation disruptor (ord) gene lead to meiotic nondisjunction in males and females because cohesion is absent by the time that sister kinetochores make stable microtubule attachments. We provide evidence that ORD is concentrated within the extrachromosomal domains of the nuclei ofDrosophila primary spermatocytes during early G2, but accumulates on the meiotic chromosomes by …

Involvement Of Platelet-Derived Growth Factor, Pdgf Receptor Signaling, Focal Adhesion Kinase And Src In Pressure-Induced Vascular Smooth Muscle Hypertrophy, Darian Clark Rice

Theses and Dissertations in Biomedical Sciences

Elevated blood pressure is associated with varying degrees of arterial remodeling. The mechanisms by which extracellular mechanical stress is converted into intracellular alterations in signal transduction and gene expression have yet to be fully elucidated. Our goal was to investigate the early events in the vascular smooth muscle response to acute hypertension and to identify mediators involved in long-term hypertensive remodeling.

In the acute phase of hypertension we targeted cell surface integrin and growth factor receptors thought to be mechanically sensitive. The signaling molecules FAK, Src and ERK-MAPK are known to be triggered by integrin engagement and growth factor receptor …

Construction Of Rhas And Rhar Overexpression Plasmids And The Effects Of Crp Mutants On E.Coli L-Rhamnose Gene Expression, Amber Bromenshenkel, Susan Egan

Honors Capstones

Capstone submitted as a graduation requirement for the BSU Honors Program.

Comparison Of Tnf-A Levels Produced By Macrophages Treated With Brucella Abortus And Escherichia Coli Lps, Tajie Harris

Honors Capstones

Capstone submitted as a graduation requirement for the BSU Honors Program.

Prostate Specific Membrane Antigen (Psma): Immunoassay Development And Characterization Of Transcriptional Regulation, Zhen Xiao

Theses and Dissertations in Biomedical Sciences

Prostate cancer (PCA) is the most common cancer and the second leading cause of death among American men. The high mortality is greatly attributed to the lack of early detection tools and effective treatment for metastasis and relapses. Biomarkers that can discriminate benign from malignant tumor and signal the development of androgen independent and metastatic tumor are needed. A biomarker designated prostate specific membrane antigen (PSMA) has the potential to fulfill this need. The objective of this study is to develop a clinically useful immunoassay for quantitation of serum PSMA and to study the molecular mechanism underlying the upregulation of …

Cell Modeling, Matthew L. Campbell

Theses and Dissertations

The Air Force is currently developing new products that incorporate a variety of chemicals which may come in contact with product users. To define which chemicals are dangerous to the user, toxicity studies have been performed. However, analysis of toxicity ultimately requires models of the exposed cellular systems. This thesis provides an introduction of how to model and analyze small and large cellular systems. Understanding the underlying behavior of small models and their relation to large systems will lead to a better understanding of how the Air Force should construct intracellular models to assist in future toxicology studies. Developing analysis …

Genomic Analysis Of Homotypic Vacuole Fusion, E Scott Seeley, Masashi Kato, Nathan Margolis, William Wickner, Gary Eitzen

Dartmouth Scholarship

Yeast vacuoles undergo fission and homotypic fusion, yielding one to three vacuoles per cell at steady state. Defects in vacuole fusion result in vacuole fragmentation. We have screened 4828 yeast strains, each with a deletion of a nonessential gene, for vacuole morphology defects. Fragmented vacuoles were found in strains deleted for genes encoding known fusion catalysts as well as 19 enzymes of lipid metabolism, 4 SNAREs, 12 GTPases and GTPase effectors, 9 additional known vacuole protein-sorting genes, 16 protein kinases, 2 phosphatases, 11 cytoskeletal proteins, and 28 genes of unknown function. Vacuole fusion and vacuole protein sorting are catalyzed by …

Erv14p Directs A Transmembrane Secretory Protein Into Copii-Coated Transport Vesicles, Jacqueline Powers, Charles Barlowe

Dartmouth Scholarship

Erv14p is a conserved integral membrane protein that traffics in COPII-coated vesicles and localizes to the early secretory pathway in yeast. Deletion of ERV14 causes a defect in polarized growth because Axl2p, a transmembrane secretory protein, accumulates in the endoplasmic reticulum and is not delivered to its site of function on the cell surface. Herein, we show that Erv14p is required for selection of Axl2p into COPII vesicles and for efficient formation of these vesicles. Erv14p binds to subunits of the COPII coat and binding depends on conserved residues in a cytoplasmically exposed loop domain of Erv14p. When mutations are …

Actin Dynamics Regulate Myosin Assembly In Muscle Cells, John Dylan Cook

Inquiry: The University of Arkansas Undergraduate Research Journal

During muscle cell (i.e. myocyte )development, organization of actin and myosin into the contractile unit (i.e. sarcomere) is required for proper muscle contraction. By disrupting the processes of muscle cell development, I am able to see which steps play important roles in proper maturation of myocytes. Elucidation of the key pathways in muscle development could lead to a better understanding of human cardiac hypertrophies and muscle myopathies. Since actin filament formation precedes myosin organization, I am using actin assembly inhibitors to determine if actin filaments are a necessary prerequisite for myosin organization. It is hypothesized that disruption of actin will …

Fluorescence In Situ Hybridization On Sperm Using Alkaline Denaturation, L. J. Aridgides, M. Stacey, L. Brihn, D. Scott, C. Osgood

Bioelectrics Publications

Fluorescence in situ hybridization (FISH) permits the detection of unique DNA sequences of target DNA within single cells. FISH allows the identifica­tion of DNA sequences within individ­ual cells and readily identifies genetic changes within a mixed population of cells. However, problems arise in main­taining nuclear morphology, particular­ly following harsh denaturation proce­dures where cells are heated to 80°C, causing the nuclear boundary to be­come blurred or the cell to completely burst. In an attempt to preserve cellular morphology and high hybridization ef­ficiency, particularly in fresh tissue samples, we evaluated a FISH protocol using alkaline denaturation in place of heat.

Stat6 Mediates Interleukin-4 Growth Inhibition In Human Breast Cancer Cells, Jennifer L. Gooch, B. Christy, D. Yee

PCOM Scholarly Works

In addition to acting as a hematopoietic growth factor, interleukin-4 (IL-4) inhibits growth of some transformed cells in vitro and in vivo. In this study, we show that insulin receptor substrate (IRS)-1, IRS-2, and signal transducer and activator of transcription 6 (STAT6) are phosphorylated following IL-4 treatment in MCF-7 breast cancer cells. STAT6 DNA binding is enhanced by IL-4 treatment. STAT6 activation occurs even after IRS-1 depletion, suggesting the two pathways are independent. To examine the role of STAT6 in IL-4-mediated growth inhibition and apoptosis, a full-length STAT6 cDNA was transfected into MCF-7 cells. Transient overexpression of STAT6 resulted in …

Characterization Of An Acyl-Coa Thioesterase That Functions As A Major Regulator Of Peroxisomal Lipid Metabolism, Mary Hunt, Karianne Solaas, Bengt F. Kase, Stefan E H Alexson

Articles

Peroxisomes function in b-oxidation of very long- and long-chain fatty acids, dicarboxylic fatty acids, bile acid intermediates, prostaglandins, leukotrienes, thromboxanes, pristanic acid and xenobiotic carboxylic acids. These lipids are mainly chain-shortened for excretion as the carboxylic acids or transported to mitochondria for further metabolism. Several of these carboxylic acids are slowly oxidized and may therefore sequester coenzyme A (CoASH). To prevent CoASH sequestration and to facilitate excretion of chain-shortened carboxylic acids, acyl-CoA thioesterases, which catalyze the hydrolysis of acyl-CoAs to the free acid and CoASH, may play important roles. We have here cloned and characterized a peroxisomal acyl-CoA thioesterase from …

Flexibility Of Biv Tar-Tat: Models Of Peptide Binding, M Hsieh, E D. Collins, T Blomquist, Brooke Lustig

Faculty Publications, Chemistry

No abstract provided.

Presented Antigen From Damaged Pancreatic Beta Cells Activates Autoreactive T Cells In Virus-Mediated Autoimmune Diabetes., Marc S. Horwitz, Alex Ilic, Cody Fine, Enrique Rodriguez, Nora Sarvetnick

Journal Articles: Regenerative Medicine

The induction of autoimmunity by viruses has been attributed to numerous mechanisms. In mice, coxsackievirus B4 (CB4) induces insulin-dependent diabetes mellitus (IDDM) resembling the final step of disease progression in humans. The immune response following the viral insult clearly precipitates IDDM. However, the molecular pathway between viral infection and the subsequent activation of T cells specific for islet antigen has not been elucidated. These T cells could become activated through exposure to sequestered antigens released by damaged beta cells, or they could have responded to factors secreted by the inflammatory response itself. To distinguish between these possibilities, we treated mice …

Map Kinases In Cynomolgus Monkey Sperm Hyperactivation, Emily Nicole Haynes

Biological Sciences Theses & Dissertations

Mammalian sperm exhibit characteristic motility patterns, termed hyperactivated (HA) motility, associated with completion of capacitation. In cynomolgus monkey (Macaca fascicularis) sperm, this HA motility is dependent in vitro upon the addition of exogenous cyclic nucleotide mediators, caffeine and dibutyryl-cyclic adenosine monophosphate (dbcAMP). Previous reports have shown protein tyrosine phosphorylation to be an integral component of this caffeine- and cAMP-stimulated HA motility. This study investigated the involvement of the mitogen-activated protein (MAP) kinase-signaling cascade. Semen specimens were collected in Talp-HEPES medium from proven breeders via electroejaculation. After washing, sperm were incubated in the presence and absence of the MAP …

Sir3-Dependent Assembly Of Supramolecular Chromatin Structures In Vitro, Philippe T. Georgel, Madeleine A. Palacios Debeer, Gregory Pietz, Catherine A. Fox, Jeffrey C. Hansen

Biological Sciences Faculty Research

Baculovirus-expressed recombinant Sir3p (rSir3p) has been purified to near homogeneity, and its binding to naked DNA, mononucleosomes, and nucleosomal arrays has been characterized in vitro. At stoichiometric levels rSir3p interacts with intact nucleosomal arrays, mononucleosomes, and naked DNA, as evidenced by formation of supershifted species on native agarose gels. Proteolytic removal of the core histone tail domains inhibits but does not completely abolish rSir3p binding to nucleosomal arrays. The linker DNA in the supershifted complexes remains freely accessible to restriction endonuclease digestion, suggesting that both the tail domains and nucleosomal DNA contribute to rSir3p–chromatin interactions. Together these data indicate …

Regulation Of The Hcmv Ul98 Promoter By Cooperation Of The Cyclic Amp Response Element And Gamma Interferon Response Element, Tabmitha Patricia Young Jervey

Theses and Dissertations in Biomedical Sciences

The HCMV UL98 early alkaline exonuclease gene promoter was examined to determine the DNA sequences as well as viral and/or cellular proteins functional in the regulation of this early gene. To assess promoter activation, UL98 promoter sequences were first cloned upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with expression plasmids which express the HCMV major immediate early (IE) proteins 1E72 and 1E86. To more specifically determine the importance of individual cis-acting elements in UL98 promoter activation, the promoter region underwent mutagenesis to delete or alter sequences. The variant promoters were again cloned into a reporter-CAT construct and …