Microbiology Commons^™

Acute Effects Of Whole-Body Proton Irradiation On The Immune System Of The C57bl/6 Mouse, Eric H. Kajioka

Loma Linda University Electronic Theses, Dissertations & Projects

The acute effects of proton whole-body irradiation (WBI) on leukocytes, lymphocytes, and hematological parameters in the spleen and blood of C57BI/6 mice were examined and compared to the effects of photon (gamma) WBI derived from a 60cobalt (60Co) source. Adult, female C57BL/6 mice were exposed to a single dose (3 Gy, 0.4 Gy/min dose rate) of either proton WBI at the Bragg peak, proton WBI at the entry plateau, or Co WBI, and sacrifice intervals were at 1,4, 7, and 10 days post- WBI. Flow cytometry analysis of the spleen and peripheral blood showed depression in cell counts for all …

Dna-Dna Checkerboard Hybridization To Identify Bacteria In Human Plaque Samples, Jasan Lee Zimmerman

Loma Linda University Electronic Theses, Dissertations & Projects

Previous research has shown that using DNA probes to identify the presence of specific bacteria in human plaque samples is superior to the commonly used anaerobic cultural procedures. Some periodontal pathogens are either uncultivable or very difficult to grow, such as Prevotella intermedia, P. nigrescens, and Eubacterium brachy. Digoxygenin probes were prepared for 15 oral bacteria. These probes were used with a checkerboard DNA-DNA hybridization scheme to analyze whether human plaque samples contained any of the 15 bacterial species. Our results have shown that this method is successful in identifying bacterial DNA present in the subgingival patient plaque …

Engineering Secreted Proteins For Gene Transfer And Dna Vaccination, Jingxue Liu

Loma Linda University Electronic Theses, Dissertations & Projects

In recent years gene therapy has become a promising way of alleviating incurable human ailments, its concept emerging as the ultimate therapy for many infectious and genetic diseases. Two important aspects of the development of successful gene therapy protocols are the ability to monitor gene transfer readily, and the establishment of new protocols for treating specific diseases. In this work, Renilla luciferase and human glutamic acid decarboxylase (GAD) 65 were engineered for secretion to address some aspects of these issues.

Secreted reporter proteins are promising tools to study gene transfer and expression in a non-destructive manner, and bioluminescent proteins are …

Analysis Of Protein And Rna Interactions Of The Hiv-1 Rev Protein, John Francis

Loma Linda University Electronic Theses, Dissertations & Projects

The HIV-1 Rev protein enters the nucleus via it’s nuclear localization sequence/RNA binding domain and interacts with a ≈234 nt region of viral RNA, termed the Rev Response Element (RRE), which is located in the env region of unspliced and singly-spliced HIV-1 mRNA. The Rev nuclear export sequence (NES) then mediates translocation of the viral transcript to the cytoplasm, allowing for translation of viral structural genes and active virion formation. We report identification of two yeast nucleopore proteins, NUP49 and NUP100, that have been found to interact with Rev in vivo using the yeast two-hybrid system. NUP49 and NUP100 contain …

Influence Of The Ccr2-V64i Polymorphism On Human Immunodeficiency Virus Type 1 Coreceptor Activity And On Chemokine Receptor Function Of Ccr2b, Ccr3, Ccr5, And Cxcr4, Benhur Lee, Benjamin J. Doranz, Shalini Rana, Yanji Yi, Mario Mellado, Jose M. R. Frade, Carlos Martinez-A., Stephen J. O'Brien, Michael Dean, Ronald G. Collman, Robert W. Doms

Biology Faculty Articles

The chemokine receptors CCR5 and CXCR4 are used by human immunodeficiency virus type 1 (HIV-1) in conjunction with CD4 to infect cells. In addition, some virus strains can use alternative chemokine receptors, including CCR2b and CCR3, for infection. A polymorphism in CCR2 (CCR2-V64I) is associated with a 2- to 4-year delay in the progression to AIDS. To investigate the mechanism of this protective effect, we studied the expression of CCR2b and CCR2b-V64I, their chemokine and HIV-1 coreceptor activities, and their effects on the expression and receptor activities of the major HIV-1 coreceptors. CCR2b and CCR2b-V64I were expressed at …

Characterization Of Metal-Regulated Genes In Pseudomonas Fluorescens, Melanie Pearson

Honors Theses

One side effect of today's industrialized world is increased levels of heavy metals in the environment. Many of these metals are necessary for biological function as trace elements, but at higher concentrations are toxic. Other metals, such as cadmium, are not beneficial at any level, and have only deleterious effects on living organisms. Cadmium is primarily thought to interfere with normal biological function of proteins. Human exposure to cadmium appears to primarily damage the kidneys, but may also affect the liver, lungs, immune system, and central nervous system. Bacteria, however, have developed several methods for handling toxic heavy metals: cation …

Igf-Ii Expression In Human Tumor Tissues And Human Igf-Ii Ribozyme Action, Zhaodong Xu

Loma Linda University Electronic Theses, Dissertations & Projects

Insulin-like growth factors (IGFs) are potent mitogens for a variety of cancer cells in vitro. In breast, prostate and neuroblastoma cancer cells, it has been suggested that IGF-II plays a paracrine/autocrine role. However, information on cell-type -specific IGF-II expression in vivo is limited. In situ hybridization and immunohistochemistry were carried out to determine the cell type expressing IGF-II in different tumor tissues. Both IGF-II mRNA and protein were localized to malignant cells, and expression in the stroma was minimal in all the tumors. The data are consistent with the hypothesis that cancer cell growth is regulated by IGF-II, and therefore …

Plant-Delivered Cholera Toxin B Subunit As An Immunomodulatory Molecule, Takeshi Arakawa

Loma Linda University Electronic Theses, Dissertations & Projects

The B subunit of cholera toxin (CTB) functions as an effective mucosal carrier molecule of chemically or genetically conjugated antigens. Orally delivered CTB or CTB fusion protein binds to G_M1-ganglioside receptor molecules located on cells in the gutassociated lymphoid tissues, the site of immunological induction. We have generated transgenic plants synthesizing CTB conjugated with vaccine antigens for food plant-based prevention of cholera and rotavirus toxin-induced diarrhea and for autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM).

Mice fed with transgenic potato tubers synthesizing 0.3% of CTB as total tuber protein generated serum and mucosal antibodies with toxin neutralizing …

Multiple Members Of A Third Subfamily Of P-Type Atpases Identified By Genomic Sequences And Ests, Margaret S. Halleck, Deepti Pradhan, Christie Blackman, Charlotte A. Berkes, Partrick Williamson, Robert A. Schlegel

Biology Faculty Publications

The Saccharomyces cerevisiae genome contains five P-type ATPases divergent from both of the well-known subfamilies of these membrane ion transporters. This newly recognized third subfamily can be further divided into four classes of genes with nearly equal relatedness to each other. Genes of this new subfamily are also present and expressed in multicellular organisms such as Caenorhabditis elegans and mammals; some, but not all, can be assigned to the classes identified in yeast. Different classes of genes and different genes within a class are expressed differentially in tissues of the mouse. The recently cloned gene for the mammalian aminophospholipid translocase …

Study Of The Hiv-1 Gene Regulatory Proteins Tat And Rev By The Two-Hybrid System In Yeast, David Allen Elkins

Loma Linda University Electronic Theses, Dissertations & Projects

Tat and Rev, small proteins encoded by the Human Immunodeficiency Virus type 1 (HIV-1), regulate its pattern of gene expression within infected cells. Tat increases transcription from the integrated pro virus by approximately a thousand-fold; Rev effects a shift in the pattern of splicing of the viral mRNA. Both are likely to interact with cellular proteins in executing their respective functions. Tat has additional activities (including neurotoxicity and inhibition of lymphocyte activation) which may be extraneous to its viral functions.

In order to identify cellular proteins interacting with Tat and Rev, the genes for both were cloned into plasmid vectors …

Genomic Diversity In Staphylococcus Aureus, Bret Max Barton

Loma Linda University Electronic Theses, Dissertations & Projects

Staphylococcus aureus chromosomal DNA from 125 methicillin-resistant and 10 methicillin-sensitive clinical isolates was digested with rare-cutting restriction endonucleases and subjected to pulsed-field gel electrophoresis (PFGE). Thirty-nine distinct genomic macrorestriction patterns (GPs) were identified using SmaI; 29 of these patterns are from methicillin-resistant S. aureus (MRSA).

A dendrogram showing percent similarity among the patterns was constructed which revealed the considerable genomic diversity of the collection even though the isolates had been obtained during outbreaks. The majority of the MRSA formed one broad group with two outbreak subclasses. However, 8 of the 29 MRSA GPs (28%) were diverse.

Digestion with Csp …

Diseased Thyrocytes Have Aberrant Communication And Tnf Receptor Secretion, Mary Labue

Loma Linda University Electronic Theses, Dissertations & Projects

Autoimmune diseases are a world-wide health concern and are an intense area of investigation. However, one area of research has been largely ignored. This involves the contribution of the target tissue to the disease process. We hypothesized that cell-cell communication is disrupted by lymphocytic infiltration and advancing fibrosis and this disruption results in altered target cell responses to immune components produced at the site of inflammation, including Tumor Necrosis Factor-alpha (TNF- α).

We have used thyroiditis as our model of autoimmunity and have documented the development of spontaneous thyroiditis in MRL-lpr/lpr mice. We have found aberrant connexin expression in …

Genetic And Phylogenetic Divergence Of Feline Immunodeficiency Virus In The Puma (Puma Concolor), Margaret A. Carpenter, Eric W. Brown, Melanie Culver, Warren E. Johnson, Jill Pecon-Slattery, Dulce Brousset, Stephen J. O'Brien

Biology Faculty Articles

Feline immunodeficiency virus (FIV) is a lentivirus which causes an AIDS-like disease in domestic cats (Felis catus). A number of other felid species, including the puma (Puma concolor), carry a virus closely related to domestic cat FIV. Serological testing revealed the presence of antibodies to FIV in 22% of 434 samples from throughout the geographic range of the puma. FIV-Pco pol gene sequences isolated from pumas revealed extensive sequence diversity, greater than has been documented in the domestic cat. The puma sequences formed two highly divergent groups, analogous to the clades which have been defined for …

Expression Of The Human Milk Protein, Beta-Casein : In Transgenic Potato Plants, Wendy Roberts

Loma Linda University Electronic Theses, Dissertations & Projects

Mother’s milk is recommended by the nutritional and pediatric communities as the best food for suckling infants. For non breast-fed infants, bovine milk-based formulas have been regarded as a suitable substitute. In the early 1900's, physicians found that foreign proteins in cow milk were responsible for gastrointestinal disease and even digestive collapse of the newborn fed a cow milk-based diet¹⁶. The allergenicity of cow milk for the human infant appears to be caused predominantly by p-casein¹⁷. In this research, the cDNA encoding human p-casein was introduced into potato cells under the control of the bidirectional, auxin-inducible …

Active Inhibition Of T Cell Proliferation By Monocytes, Dean Anthony Lee

Loma Linda University Electronic Theses, Dissertations & Projects

Monoclonal antibodies (mAb) to MHC class II molecules (anti-class II mAb) have been well described in their ability to inhibit T cell activation. The most current model is that anti-class II mAb interfere directly with the T cell receptor-major histocompatibility complex crosslinking which is necessary for T cell activation. The anti-CD3 mAb, OKT3, is an MHC class II molecule-independent mitogen which causes T cell activation by direct ligation of the TCR-CD3 complex. OKT3 and a panel of anti-class II mAb were used to dissect the individual roles played by these two receptors in activation and inhibition of peripheral blood mononuclear …

Developmental Regulation Of The Murine Cd4 Gene, Rhonda Kay Hansen

Loma Linda University Electronic Theses, Dissertations & Projects

CD4 is a membrane bound glycoprotein that plays an important role in T cell development and activation. An extensive analysis of the CD4 locus identified potential transcriptional control elements located 5' of the promoter and within the first intron. Since the molecular mechanism for the control of CD4 expression during T cell development had not been determined, it is the focus of this dissertation. A series of recombinant DNA constructs were made to determine the contribution of putative transcriptional control elements in regard to CD4 expression, and to identify elements being used to control CD4 expression during T cell development. …

Regulation Of The Insulin-Like Growth Factor System By Retinoic Acid In Human Osteoblast Cells, Yuehua Zhou

Loma Linda University Electronic Theses, Dissertations & Projects

Retinoic acid (RA) is an important regulator of growth and differentiation in many cell types, including bone. However, its effects on human osteoblast cell growth and differentiation have not been well studied. Therefore, I investigated the effect of RA on proliferation and differentiation of normal human bone cells (HBCs) and human osteosarcoma, SaOS-2 cells. RA decreased baseline as well as serum-stimulated proliferation in normal HBCs. To determine the effect of RA on differentiation, expression of several osteoblastic differentiation markers were studied. RA decreased type I procollagen mRNA levels and l,25(OH)₂D₃-stimulated osteocalcin and alkaline phosphatase (ALP) mRNA …

A Model Of Cytomegalovirus Association With The Development Of Heart Graft Atherosclerosis, Evan Skowronski

Loma Linda University Electronic Theses, Dissertations & Projects

The development of graft atherosclerosis in heart transplant recipients has been associated with the development of post-transplant cytomegalovirus infection. The mechanism of this involvement, however, remains unclear. I undertook a study to determine the role of cytomegalovirus in the development of graft atherosclerosis. First, a rapid method of detecting cytomegalovirus based on capillary polymerase chain reaction and gel electrophoresis was employed to determine the presence of cytomegalovirus in a wide variety of clinical samples. Detection of cytomegalovirus is a lengthy procedure under normal conditions, and timely detection of cytomegalovirus in transplant recipients may allow intervention early enough to block the …

Pathogenicity Of Murine Cytomegalovirus Mutants, Victoria Jean Cavanaugh

Theses and Dissertations in Biomedical Sciences

The purpose of this study was to identified nonessential murine cytomegalovirus (MCMV) genes involved in pathogenesis in vivo. Our approach to identifyjng these genes consisted of constructing MCMV mutants, and then analyzing these mutants in vitro and in vivo. Recombinant viruses (RV) expressing the β-glucuronidase marker gene were constructed by site-directed insertion and deletion mutagenesis of the MCMV Hind III-J and -I regions of the viral genome. Mutations were targeted to this region of the MCMV genome because the corresponding region of the human CMV genome is nonessential and is involved in down-regulating major histocompatibility complex (MHC) class I expression …

Enhancer Trap Technique: A Novel Tool For Identification And Developmental Characterization Of Genes Of Drosophila, Amit Singh

Biology Faculty Publications

The classical technique of mutational screen for identification of genes controlling early development has now approached saturation. A new era in genetic identification and developmental characterization of genes in Drosophila has commenced with the advent of the enhancer trap technique. This technique involves mobilization of a P-lacZ vector to diverse chromosomal locations in the fruit fly genome to bring it under the regulation of developmentally expressed genes or their enhancer elements. The technique offers a strikingly elegant method of gaining entry into fruit fly genes.

An Improved Method For Chemical Devitellinization Of X-Gal Stained Drosophila Embryos, Amit Singh, Madhuri Kango-Singh, P. Sinha

Biology Faculty Publications

In Drosophila developmental biological studies, X-gal staining is commonly employed to study the spatio-temporal expression of the lacZ reporter gene in the transformed flies or their embryos. Study of the lacZ pattern in embryos often suffers from the lack of an efficient and high yieldirrg technique for devitellinization of X-gal stained embryos. Devitellinization techniques employed during antibody staining, in situ hybridization or embryonic cuticular preparations generally do not give satisfactory results when used for similar purpose in X-gal stained embryos. This results in the flaky appearance of the blue stain. We present here an improved chemical devitellinization technique which gives …

Media Components Influence Viral Gene Expression Assays In Human Fetal Astrocyte Cultures, Micheline Mccarthy, Charles Wood, Larisa Fedoseyeva, Scott R. Whittemore

Nebraska Center for Virology: Faculty Publications

In vitro neurovirological studies of viral infectivity or viral gene expression may be confounded by the mulHple neural cell types and/or fibrob last contamination present in early passage cultures prepared from dissociated human central nervous system (eNS) tissue. We have developed highly enriched astrocyte cultures for neurovirological study by culturing in a serum-free defined medium, 816, supplemented with basic fibroblast growth factor (FGF-2). Subculture in this medium selects against fibroblast proliferation and favors sustained proliferation of a highly enriched glial fibrillary acidic protein (GFAP)-positive cell population. These astrocytes support productive replication of cytomegalovirus (CMV) and transient expression of transfected CMVand …

Transcriptional Regulation Of Insulin-Like Growth Factor : Binding Protein-4 By Protein Kinase A And Protein Kinase C Signal Transduction Pathways In Human Bone Cells, Kuk-Wha Lee

Loma Linda University Electronic Theses, Dissertations & Projects

The Insulin-like Growth Factor (IGF) system is an important local regulator of osteoblast proliferation, the primary determinant of bone formation. Six IGF Binding Proteins (IGFBPs) either inhibit or enhance IGF actions. Previous studies have shown that IGFBP-4 is an important negative regulator of osteoblast cell proliferation and that agents such as PTH which increase intracellular cAMP significantly increase expression of IGFBP-4 at the protein and mRNA levels. The underlying molecular mechanism which accounted for IGFBP-4 expression had not been determined and was the focus of my dissertation. Agents which increase intracellular cAMP rapidly increased IGFBP-4 mRNA levels. Nuclear run-off experiments …

A Lion Lentivirus Related To Feline Immunodeficiency Virus: Epidemiologic And Phylogenetic Aspects, Eric W. Brown, Naoya Yuhki, Craig Packer, Stephen J. O'Brien

Biology Faculty Articles

Feline immunodeficiency virus (FIV) is a novel lentivirus that is genetically homologous and functionally analogous to the human AIDS viruses, human immunodeficiency virus types 1 and 2. FIV causes immunosuppression in domestic cats by destroying the CD4 T-lymphocyte subsets in infected hosts. A serological survey of over 400 free-ranging African and Asian lions (Panthera leo) for antibodies to FIV revealed endemic lentivirus prevalence with an incidence of seropositivity as high as 90%o. A lion lentivirus (FIV-Ple) was isolated by infection of lion lymphocytes in vitro. Seroconversion was documented in two Serengeti lions, and discordance of mother-cub serological status …

Phylogenetic Associations Of Human And Simian T-Cell Leukemia/Lymphotropic Virus Type I Strains: Evidence For Interspecies Transmission, Igor J. Koralnik, Enzo Boeri, W. Carl Saxinger, Anita Lo Monico, Jake Fullen, Antoine Gessain, Hong-Guang Guo, Robert C. Gallo, Phillip Markham, Vaniambadi Kalyanaraman, Vanessa Hirsch, Jonathan Allan, Krishna Murthy, Patricia Alford, Jill Pecon-Slattery, Stephen J. O'Brien, Genoveffa Ranchini

Biology Faculty Articles

Homologous env sequences from 17 human T-leukemia/lymphotropic virus type I (HTLV-I) strains from throughout the world and from 25 simian T-leukemia/lymphotropic virus type I (STLV-I) strains from 12 simian species in Asia and Africa were analyzed in a phylogenetic context as an approach to resolving the natural history of these related retroviruses. STLV-I exhibited greater overall sequence variation between strains (1 to 18% compared with 0 to 9% for HTLV-I), supporting the simian origin of the modern viruses in all species. Three HTLV-I phylogenetic clusters or clades (cosmopolitan, Zaire, and Melanesia) were resolved with phenetic, parsimony, and likelihood analytical procedures. …

Phylogenetic Analysis Of Segment 10 From African Horsesickness Virus And Cognate Genes From Other Orbiviruses, Rafael O. De Sá, Marla Zellner, Marvin J. Grubman

Biology Faculty Publications

Utilizing the reverse transcriptase-polymerase chain reaction (RT-PCR) procedure, we have synthesized full-length copies of segment 10 from African horsesickness virus (AHSV) serotypes 1,4 and 8. The genes were cloned, sequenced and compared with the sequence of the cognate gene from AHSV serotypes 3 and 9. Sequences were analyzed to assess evolutionary relationships among serotypes using cladistics. Based on this analysis the data support a close relationship between serotypes 4 and 9 and between serotypes 1 and 8 and a closer relationship of serotype 3 to the 4 and 9 group.

Development And Application Of A Thermistor Current Meter, Carl M. Way, Albert J. Burky, Christine Miller-Way

Biology Faculty Publications

This report provides details for the construction of a hot-bead thermistor current meter which is capable of measuring water velocities on a millimeter spatial scale and for the construction of a compact and accurate calibration system. Hot-bead thermistor current meters can be built with response times of 200 ms capable of measuring velocities between 0.1 and 80 cm s-1. The construction of a sturdy probe for application in lotic systems such as high gradient Hawaiian streams was achieved by the use of heavy-duty acrylic tubing, small stainless steel gas-chromatography tubing, and flexible Tygon spaghetti tubing. An acrylic handle anchors the …

Book Review: The Baculovirus Expression System: A Laboratory Guide (1992) King, L. A. & Possee, R. D., David D. Dunigan

Nebraska Center for Virology: Faculty Publications

The power of molecular biology is unleashed with the ability to clone and sequence genes, and then express these genes in heterologous systems. This sets the stage for the full analysis of proteins that are otherwise difficult to isolate and/or purify, especially when present at very low copy number per cell or when isolated from relatively precious materials. Overexpression of protein is now possible in a number of systems including prokaryotes (e.g., E. coli) and various eukaryotes (yeast, insects, and plants). The issue then becomes, which system (1) most closely reflects the homologous expression with respect to posttranslational modifications, …

Worldwide Prevalence Of Lentivirus Infection In Wild Feline Species: Epidemiologic And Phylogenetic Aspects, Robert A. Olmstead, Raymond Langley, Melody E. Roelke, Robert M. Goeken, Diane Adger-Johnson, Julie P. Goff, John P. Albert, Craig Packer, M. Karen Laurenson, Tim M. Caro, Lue Scheepers, David E. Wildt, Mitchell Bush, Janice S. Martenson, Stephen J. O'Brien

Biology Faculty Articles

The natural occurrence of lentiviruses closely related to feline immunodeficiency virus (FIV) in nondomestic felid species is shown here to be worldwide. Cross-reactive antibodies to FIV were common in several free-ranging populations of large cats, including East African lions and cheetahs of the Serengeti ecosystem and in puma (also called cougar or mountain lion) populations throughout North America. Infectious puma lentivirus (PLV) was isolated from several Florida panthers, a severely endangered relict puma subspecies inhabiting the Big Cypress Swamp and Everglades ecosystems in southern Florida. Phylogenetic analysis of PLV genomic sequences from disparate geographic isolates revealed appreciable divergence from domestic …

Genetics And Applications Of Nisin Production In Lactococcus Lactis Subsp. Lactis And Conjugal Exchange Of This Trait, Jeffery R. Broadbent

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Chapter I reviews current literature on gene transfer systems in lactic acid bacteria, how genetically altered microorganisms for food are presently regulated, and how nisin is used as a food preservative.

Chapter II investigates previous reports which linked genes for nisin biosynthesis and sucrose utilization (Nip⁺Suc⁺) to plasmid DNA in two well characterized L. lactis subsp. lactis strains. Plasmid curing studies, conjugations, and DNA-DNA hybridizations indicated that these genes were encoded by chromosomal loci in all Nip⁺Suc⁺ strains examined. Similar results were noted in nisin-sucrose transconjugants of L. lactis subsp. cremoris and S. …