Biochemistry, Biophysics, and Structural Biology Commons^™

16s Ribosomal Dna Sequencing Confirms The Synonymy Of Vibrio Harveyi And V. Carchariae, Eric J. Gauger, Marta Gomez-Chiarri

Fisheries, Animal and Veterinary Sciences Faculty Publications

Seventeen bacterial strains previously identified as Vibrio harveyi (Baumann et al. 1981) or V. carchariae (Grimes et al. 1984) and the type strains of V. harveyi, V. carchariae and V. campbellii were analyzed by 16S ribosomal DNA (rDNA) sequencing. Four clusters were identified in a phylogenetic analysis performed by comparing a 746 base pair fragment of the 16S rDNA and previously published sequences of other closely related Vibrio species. The type strains of V. harveyi and V. carchariae and about half of the strains identified as V. harveyi or V. carchariae formed a single, wellsupported cluster designed as ‘bona …

Mechanism For Membrane Electroporation Irreversibility Under High-Intensity, Ultrashort Electrical Pulse Conditions, R. P. Joshi, K. H. Schoenbach

Bioelectrics Publications

An improved electroporation model is used to address membrane irreversibility under ultrashort electric pulse conditions. It is shown that membranes can survive a strong electric pulse and recover provided the pore distribution has a relatively large spread. If, however, the population consists predominantly of larger radii pores, then irreversibility can result. Physically, such a distribution could arise if pores at adjacent sites coalesce. The requirement of close proximity among the pore sites is more easily satisfied in smaller organelles than in outer cell membranes. Model predictions are in keeping with recent observations of cell damage to intracellular organelles (e.g., mitochondria), …

Influence Of Protein Tyrosine Kinases On Cell Volume Change-Induced Taurine Release, Herminia Pasantes-Morales, Rodrigo Franco

School of Veterinary and Biomedical Sciences: Faculty Publications

Taurine efflux occurs in association with cell swelling in both hyposmotic and isosmotic conditions and during cell shrinkage in apoptotic death. Release occurs through a leak pathway, is largely Ca²⁺-independent and is sensitive to Cl^– channel blockers. Taurine efflux elicited by hyposmolarity is reduced or suppressed by tyrosine kinase blockers and increased by tyrosine phosphatase inhibitors. The specific kinases involved are still unknown and may be different in the various cell types. Non-receptor and scr-related protein kinases have been identified in some cells as elements that directly phosphorylate the taurine efflux pathway. Possible tyrosine kinase targets are …

Methods And Compositions For Protection Against Bovine Viral Diseases, Subramaniam Srikumaran

School of Veterinary and Biomedical Sciences: Faculty Publications

The present invention relates to methods and compositions for eliciting an immune response against bovine viral epitopes. The methods comprise combining at least one heat shock protein with at least one bovine viral epitope to form a purified epitope/heat shock protein complex and administration of an immune system stimulating amount of the purified epitope/heat shock protein complex. The compositions comprise, a purified epitope/heat shock protein complex comprising at least one bovine viral epitope complexed with at least one heat shock protein, and a pharmaceutically acceptable carrier, diluent or excipient.

Positive Regulation Of Pka On Human Gonadotropin-Releasing Hormone (Hgnrh) Gene Expression In Human Placental Jeg-3 Cells, Zhaoyang Wen

Chemistry & Biochemistry Theses & Dissertations

Using the human placental choriocarcinoma JEG-3 cell line as an in vitro human placental model, we studied the mechanisms of the PKA positive regulation of the hGnRH gene expression in the human placenta. Studies in JEG3 cells showed that through the PKA catalytic subunit a, human GnRH upstream promoter activity was stimulated by PKA signaling pathway in a cAMP dependent mechanism. The sequence between —202 (Afl II) and —554 (BamH I) base pair in the human GnRH upstream promoter region appeared to be responsible for the PKA positive regulation of the gene expression. Furthermore, Western blot analysis demonstrated the involvement …

Evidence For Two Mechanisms Of Amino Acid Osmolyte Release From Hippocampal Slices, R. Franco, M. E. Torres-Marquez, H. Pasantes-Morales

School of Veterinary and Biomedical Sciences: Faculty Publications

A 30% decrease in osmolarity stimulated ³H-taurine, ³H-GABA and glutamate (followed as ³H-D-aspartate) efflux from rat hippocampal slices. ³H-taurine efflux was activated rapidly but inactivated slowly. It was decreased markedly by 100 μM 5-nitro-(3-phenylpropylamino) benzoic acid (NPPB) and 600 μM niflumic acid and inhibited strongly by tyrphostins AG18, AG879 and AG112 (25–100 μM), suggesting a tyrosine kinase-mediated mechanism. Hyposmolarity activated the mitogen-activated protein kinases (MAPK) extracellular-signal-related kinase-1/2 (ERK1/ERK2) and p38, but blockade of this reaction did not affect ³H-taurine efflux. Hyposmosis also activated phosphatidyl-inositide 3-kinase (PI3K) and its prevention by wortmannin (100 nM) essentially abolished …

Nuclear Export Of 60s Ribosomal Subunits Depends On Xpo1p And Requires A Nuclear Export Sequence-Containing Factor, Nmd3p, That Associates With The Large Subunit Protein Rpl10p, Olivier Gadal, Daniela Strau, Jacques Kessl, Bernard Trumpower

Dartmouth Scholarship

Nuclear export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified. Using a large subunit reporter (Rpl25p-eGFP), we have isolated several temperature-sensitive ribosomal export (rix) mutants. One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits. We find that thermosensitive nmd3 mutants are impaired in large subunit export. Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is exported by the nuclear export receptor Xpo1p. Moreover, we show that export of 60S subunits is Xpo1p dependent. We …

Genomic Diversity Of Pseudomonas Aeruginosa In Cystic Fibrosis, Nathaniel Edwards Head

Theses, Dissertations and Capstones

Pseudomonas aeruginosa is a common environmental microorganism. However, it has the genetic capacity to cause diseases in patients with compromised host defense systems. Cystic fibrosis (CF) is one of the major hereditary diseases among Caucasian populations. CF patients are born with a defective chloride channel that is responsible for maintaining fluid and electrolyte balance across the lumen of the lung. This imbalance leads to the production of an abnormal dehydrated viscous mucous, prohibiting the patient from normally clearing the respiratory airway. Chronic pulmonary infections with P. aeruginosa are the major causes of high morbidity and mortality in CF. Two of …

Characterization And Regulation Of Skn-Directed Autoimmune Skin Lesions, Pamela Jane Staton

Theses, Dissertations and Capstones

In the Skn model of autoimmunity, pathogenic T cells sensitized to Skn epidermal antigens (Skn-immune spleen cells or SIS cells) elicited disease in the form of skin lesions when adoptively transferred into shaved immunoincompetent syngeneic adult recipients (‘lesion-forming animals’) while concomitant injection of immunoregulatory normal spleen cells at twice the concentration (2 x normal spleen cells or 2XNS cells) of SIS cells significantly lessened lesion development (‘lesion-controlling animals’) by Day 7 post cell transfer. In a time-course RT-PCR cytokine analysis of skin from lesion-forming and lesion-controlling animals, IL-7 mRNA was significantly elevated in lesion-controlling skin on Day 7 while no …

Characterization Of The Biological Activities Of Recombinant Fusion Protein Green Fluorescent Protein/Human Zona Pellucida Protein 3 (Gfp/Hzp3), Zhiyong Lin

Theses and Dissertations in Biomedical Sciences

Despite numerous reports indicating the successful production of bioactive recombinant ZP3, no report has shown the rhZP3 having direct binding activity with human sperm. Recombinant ZP3 generated from our previous study displayed binding activity with human sperm through indirect evidence from hemizona assay (HZA).

This present study focused on the production of recombinant ZP3 with direct binding activity with human sperm. Through the application of a pEGFP expression vector, fusion protein GFP/ZP3 was successfully generated and expressed. The expression of GFP/ZP3 was evidenced by RT-PCR and western blot. The fusion protein was partially purified by Ni-NTA affinity column from cell …

Amino Acid Osmolytes In Regulatory Volume Decrease And Isovolumetric Regulation In Brain Cells: Contribution And Mechanisms, Herminia Pasantes-Morales, Rodrigo Franco, M. Eugenia Torres-Marquez, Karla Hernandez-Fonseca, Arturo Ortega

School of Veterinary and Biomedical Sciences: Faculty Publications

Brain adaptation to hyposmolarity is accomplished by loss of both electrolytes and organic osmolytes, including amino acids, polyalcohols and methylamines. In brain in vivo, the organic osmolytes account for about 35% of the total solute loss. This review focus on the role of amino acids in cell volume regulation, in conditions of sudden hyposmosis, when cells respond by active regulatory volume decrease (RVD) or after gradual exposure to hyposmotic solutions, a condition where cell volume remains unchanged, named isovolumetric regulation (IVR). The amino acid efflux pathway during RVD is passive and is similar in many respects to the volume-activated anion …

Induction Of Apoptosis In Human Prostate Cancer Cells By Resveratrol, Gary Zulfikar Morris

Chemistry & Biochemistry Theses & Dissertations

Recently attention has been brought to trans-resveratrol's {TR) anticancer activity, as determined through a number of cultured cancer cell models. This activity was attributed to TR behaving as an estrogen, and the orientation of TR' s hydroxyl groups. Based on this work it was of interest to determine whether TR would also be toxic in prostate cancer cells; if toxic, did TR induce necrosis or apoptosis in the cells; was it toxic through hormone mediated pathways; and were TR's hydroxyl groups responsible for its biological activity. To this end, cellular viability was assessed in two different prostate cancer cell …

Intracellular Transport, Assembly, And Degradation Of Wild-Type And Disease-Linked Mutant Gap Junction Proteins, Judy K. Vanslyke, Suzanne M. Deschênes, Linda S. Musil

Biology Faculty Publications

More than 130 different mutations in the gap junction integral plasma membrane protein connexin32 (Cx32) have been linked to the human peripheral neuropathy X-linked Charcot–Marie–Tooth disease (CMTX). How these various mutants are processed by the cell and the mechanism(s) by which they cause CMTX are unknown. To address these issues, we have studied the intracellular transport, assembly, and degradation of three CMTX-linked Cx32 mutants stably expressed in PC12 cells. Each mutant had a distinct fate: E208K Cx32 appeared to be retained in the endoplasmic reticulum (ER), whereas both the E186K and R142W mutants were transported to perinuclear compartments from which …

Efflux Of Osmolyte Amino Acids During Isovolumic Regulation In Hippocampal Slices, Rodrigo Franco, Octavio Quesada, Herminia Pasantes-Morales

School of Veterinary and Biomedical Sciences: Faculty Publications

The efflux of potassium (K⁺) and amino acids from hippocampal slices was measured after sudden exposure to 10% (270 mOsm), 25% (225 mOsm) or 50% (150 mOsm) hyposmotic solutions or after gradual decrease (22.5 mOsm/min) in external osmolarity. In slices suddenly exposed to 50% hyposmotic solutions, swelling was followed by partial (74%) cell volume recovery, suggesting regulatory volume decrease (RVD). With gradual hyposmotic changes, no increase in cell water content was observed even when the solution at the end of the experiment was 50% hyposmotic, showing the occurrence of isovolumic regulation (IVR). The gradual decrease in osmolarity elicited …

Response Properties Of Tibial Campaniform Sensilla On The Cockroach Leg In Restrained Preparations And Freely Moving Animals, Angela L. Ridgel

Theses, Dissertations and Capstones

The ability to detect changes in forces is important for effective use of a leg in posture and locomotion. This thesis examines how forces are detected in the legs of cockroaches by tibial campaniform sensilla. Campaniform sensilla are mechanoreceptors that encode forces through ovoid cuticular caps embedded in the exoskeleton. The tibial sensilla are unique in that they consist of two subgroups with mutually perpendicular cap orientations.

We characterized the responses of the tibial receptors in restrained preparations by applying forces to the leg at controlled magnitudes and rates. The tibial sensilla, as a group, were sensitive to increasing and …

Eph Receptors And Ephrins, Masaru Nakamoto

Biology Faculty Publications

The Eph receptors are the largest known family of receptor tyrosine kinases. The Eph receptors and theirmembrane-attached ligands, ephrins, show diverse expression patterns during development. Recent studies havedemonstrated that Eph receptors and ephrins play important roles in many developmental processes, includingneuronal network formation, the patterning of the neural tube and the paraxial mesoderm, the guidance of cellmigration, and vascular formation. In the nervous system, Eph receptors and ephrins have been shown to act aspositional labels to establish topographic projections. They also play a key role in pathway ®nding by axons andneural crest cells. The crucial roles of Eph receptors and …

Isovolumic Regulation In Nervous Tissue: A Novel Mechanism Of Cell Volume Regulation, O. Quesada, R. Franco, K. Hernandez-Fonseca, K. Tuz

School of Veterinary and Biomedical Sciences: Faculty Publications

Cell volume regulation is a property present in most animal cell lineages that allows them to recover their original volume after events of swelling or shrinkage. Such events can be caused by changes in external osmolarity or to osmotic gradients generated during normal cell functioning.4,6 The mechanism of cell volume regulation involves transmembrane fluxes of osmotically active solutes in the necessary direction to counteract the net gain or loss of intracellular water.9 The process through which cells recover their normal volume after swelling is named Regulatory Volume Decrease (RVD). This consists of the efflux of inorganic osmolytes, such as K …

Human Gonadotropin-Releasing Hormone (Hgnrh) Gene Expression And Hormone Regulation In Human Placental Jeg-3 Cells, Heming Zheng

Theses and Dissertations in Biomedical Sciences

Using the human placental choriocarcinoma JEG-3 cell line as an in vitro human placental model, I studied the mechanisms of the tissue-specific expression and steroid hormone regulation of the hGnRH gene in the human placenta. The results showed that all of the previously identified four elements are required for the full activity of the hGnRH upstream promoter in JEG-3 cells, while the element 4 (FP4, −987/−968) is the most important. Studies performed with 5′ end deletion of this region confirmed these observations. Further, supershift assay using Oct-1 antibody demonstrated the involvement of Oct-1 in the FP4 DNA-protein interaction in JEG-3 …

Use Of Extractable Lipofuscin For Age Determination Of Blue Crab Callinectes Sapidus, Se-Jong Ju, David H. Secor, H. Rodger Harvey

OES Faculty Publications

The blue crab Callinectes sapidus is an economically and ecologically important species in many temperate estuaries, yet stock assessments have been limited to length-based methods for demographic analyses. We evaluated the potential of age pigments (lipofuscins) sequestered in neural tissue of eye-stalks and brains to estimate the age of blue crabs collected from Chesapeake Bay and Chincoteague Bay. The rate of lipofuscin accumulation was determined using crabs of known age reared in the laboratory. Age pigments were extracted from neural tissues (eye-stalk or brain), quantified, and normalized to protein content to allow comparisons across tissue types and crab sizes. Field-collected …

Optimal Replication Activity Of Vesicular Stomatitis Virus Rna Polymerase Requires Phosphorylation Of A Residue(S) At Carboxy-Terminal Domain Ii Of Its Accessory Subunit, Phosphoprotein P, Leroy N. Hwang, Nathan Englund, Tapas Das, Amiya K. Florida, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

The phosphoprotein, P, of vesicular stomatitis virus (VSV) is a key subunit of the viral RNA-dependent RNA polymerase complex. The protein is phosphorylated at multiple sites in two different domains. We recently showed that specific serine and threonine residues within the amino-terminal acidic domain I of P protein must be phosphorylated for in vivo transcription activity, but not for replication activity, of the polymerase complex. To examine the role of phosphorylation of the carboxy-terminal domain II residues of the P protein in transcription and replication, we have used a panel of mutant P proteins in which the phosphate acceptor sites …

Tissue-Specific Expression And Steroid Hormone Regulation Of Human Gonadotropin -Releasing Hormone (Hgnrh) Gene In Placental Cells (Jeg -3 Cells), Zhengguang Chen

Theses and Dissertations in Biomedical Sciences

Using JEG-3 cells as an in vitro model, I investigated the mechanisms behind the tissue-specific expression and steroid hormone regulation of the hGnRH gene in the human placenta. The hGnRH upstream promoter was found to be functionally active in JEG-3 cells. The DNA sequence responsible for functioning of the upstream promoter in JEG-3 cells is narrowed to a region between –1048 bp and –730 bp. This DNA fragment contains four elements, which can bind with nuclear extract from JEG-3 cells (but not from GT1-7 cells).

Estradiol (E2) represses the hGnRH upstream promoter activity in JEG-3 cells. This inhibition is receptor-mediated, …

Nucleotide Sequences For Detection Of Serpulina Hyodysenteriae, Gerald E. Duhamel, Robert Elder

School of Veterinary and Biomedical Sciences: Faculty Publications

The invention provides a method for detecting the presence of Serpulina hyodysenteriae in a biological Sample, an oligonucleotide primer and an S. hyodysenteriae-specific oligonucleotide probe useful in that method, and an article of manufacture that contains the primers and/or probe. Also provided are an about 2.3-kb DNA fragment derived from genomic DNA of S. hyodysenteriae and encoding for an about 56 kDa polypeptide, a recombinant expression vector containing the DNA fragment, the 56 kDa polypeptide and a monoclonal antibody reactive with the peptide, and a method of assaying for antibodies reactive with the 56 kDa peptide.

Interaction Of Laminin Beta2 With Eps8 And Desmin In Muscle Cells, Luke Xueliang Cui

Theses, Dissertations and Capstones

Laminin β2 is localized to the synaptic basal lamina (BL) in muscle and glomerular BL in kidney. To find interacting proteins, a mouse kidney cDNA library was screened with domain I of rat laminin β2 using the yeast two-hybrid system. Fifteen positive clones were found. The DNA sequence of three each of these clones corresponded to the phosphoprotein Eps8 (epidermal growth factor receptor pathway substrate clone 8, which is tyrosine phosphorylated after EGF stimulation) and the intermediate filament (IF) protein keratin. To determine if this interaction occurred in muscle cells, C2 cells transfected with the rat …

The Role Of Retinoids And Protein Kinase C In Vascular Smooth Muscle Cell Proliferation And Function, Shaoshan Wang

Theses, Dissertations and Capstones

The structural and functional changes in the arterial wall of humans and animals accompany the development of hypertension and atherosclerosis. Hence, great interest exists in the mechanisms that govern vascular smooth muscle cell contraction and proliferation. Rat aortic rings maintained in organ culture show a significant loss of contractility; whereas, vascular smooth muscle cells in culture quickly dedifferentiate into a secretory phenotype with a marked capacity for proliferation. The addition of all-trans retinoic acid (RA) and retinol to the plasma-containing medium preserved the contractile response of cultured vessels. Removal of endothelium prior to culture abolished these effects. All-trans retinoic acid …

Clarification Of The Hybrid Origin Of Carex X Deamii Herm. (Cyperaceae) Based On Macro And Micro Morphological Characters, Paul B. Marcum

Theses, Dissertations and Capstones

The origin of Carex x deamii Herm. has been in question ever since it was named to science in 1938. Collections of the hybrid have always been associated with C. shortiana Dewey and either C. typhina Michx. or C. squarrosa L.. C. typhina and C. squarrosa are closely related taxa (Section Squarrosae) and often are morphologically similar. Because of this similarity, determining the correct parental species to the hybrid has been extremely difficult. It is known that the hybrid is sterile and only reproduces asexually. Pollen was analyzed to ascertain the viability of all four taxa. This study utilizes both …

Plasmd Bearing A Cdna Copy Of The Genome Of Bovine Viral Diarrhea Virus, Chimeric Derivatives Thereof, And Method Of Producing An Infectious Bovine Wral Darrheavirus Using Sad Plasmid, Ruben O. Donis, Ventzislav B. Vassilev

School of Veterinary and Biomedical Sciences: Faculty Publications

A plasmid bearing a cDNA copy of the genome of bovine viral diarrhea virus (BVDV), chimeric derivatives of the plasmid and a method of producing an infectious bovine viral diarrhea virus using the plasmid are disclosed. The invention relates to a plasmid DNA molecule that replicates easily in E. coli and contains a sufficient portion of the genome of BVDV, cloned as cDNA, to be a suitable template to produce RNA in vitro which, upon transfection into bovine cells, gives rise to infectious BVDV. The BVDV created by the process of the invention can be engineered for use as a …

In Situ Hybridization For The Detection And Localization Of Swine Chlamydia Trachomatis, C. Chae, D.-S. Cheon, D. Kwon, O. Kim, B. Kim, J. Suh, D. G. Rogers, K. D. E. Everett, A. A. Anderson

School of Veterinary and Biomedical Sciences: Faculty Publications

Gnotobiotic piglets were inoculated intralaryngeally with swine Chlamydia trachomatis strain R33 or orally with swine C. trachmatis strain R27. Archived formalin-fixed, paraffin-embedded tissues from piglets euthanatized 4–7 days postinoculation were examined by in situ hybridization for C. trachomatis nucleic acid using a nonradioactive digoxigenin-labeled DNA probes that targeted specific ribosomal RNA or omp1 mRNA molecules of the swine C. trachomatis strains. Positive hybridization signals were detected in bronchial epithelial cells, bronchiolar epithelial cells, pneumocytes, alveolar and interstitial macrophages, and jejunal and ileal enterocytes. Chlamydia-infected cells had a strong signal that was confined to the intracytoplasmic inclusions. Positive hybridization signals were …

Leukemia Inhibitory Factor (Lif): Murine Preimplantation Embryo Development, Implantation Rates, And Skeletal Development, Michael Hayes Mitchell

Theses and Dissertations in Biomedical Sciences

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine which demonstrates perplexing physiological effects. It has been demonstrated that LIF is essential for implantation in mice. Little is known relating to the manner by which LIF effects pre-implantation and post-implantation development. The objectives of this project were to determine the effects LIF on pre-implantation development, to determine the effects that it may have on implantation rates, successful pregnancy rates, and resorption rates, and to determine the effects that LIF has on the skeletal development of mice. For the embryo transfer experiments, embryos were exposed to test compounds in the transfer medium …

Molecular Staging Of Colorectal Cancer: K-Ras Mutation Analysis Of Lymph Nodes Upstages Dukes’ B2 Patients, Jennifer S. Thebo

Dissertations

The successful treatment of colorectal cancer depends upon the ability of staging systems to identify patients who are at risk for recurrence. The currently used systems fail in a significant number of patients. Multiple attempts have been made to improve upon these systems in order to provide better treatment for at-risk patients.

A variety of oncogenes and tumor suppressor genes have been implicated in colorectal cancer. Mutations in one of these, K-ras, occur in 40-60% of colorectal carcinomas. However, the relationship between specific mutations and clinical outcome is unclear.

The purpose of this study was to determine whether or not …

Fission Yeast Mitotic Regulator Dsk1 Is An Sr Protein-Specific Kinase, Zhaohua Irene Tang, Mitsuhiro Yanagida, Ren-Jang Lin

WM Keck Science Faculty Papers

Intricate interplay may exist between pre-mRNA splicing and the cell division cycle, and fission yeast Dsk1 appears to play a role in such a connection. Previous genetic analyses have implicated Dsk1 in the regulation of chromosome segregation at the metaphase/anaphase transition. Yet, its protein sequence suggests that Dsk1 may function as a kinase specific for SR proteins, a family of pre-mRNA splicing factors containing arginine-serine repeats. Using an in vitro system with purified components, we showed that Dsk1 phosphorylated human and yeast SR proteins with high specificity. The Dsk1-phosphorylated SF2/ASF protein was recognized strongly by a monoclonal antibody (mAb104) known …