Medical Sciences Commons^™

A Mechanistic Study Of The Protective Effects Of S-Adenosyl-L-Methionine Against Hepatotoxicity Of Acetaminophen, Marcus V. Terneus Jr.

Theses, Dissertations and Capstones

Hepatic toxicity is known to be associated with excessive doses of the over-the-counter analgesic, acetaminophen (APAP). APAP overdose is the leading cause of drug-induced liver failure in the United States. APAP hepatotoxicity is dependent on the biotransformation of APAP by cytochrome P450 to the toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). APAP, when taken in excessive doses, can lead to severe liver damage with the potential to progress to liver failure. Despite substantial efforts in past studies, the mechanism by which APAP induces such damaging effects is not completely understood. Recent discoveries suggest that glutathione (GSH) depletion, protein alkylation and reactive metabolite …

Protein Kinase C Activity In Mouse Eggs Regulates Gamete Membrane Interaction, Hiroto Akabane

Theses, Dissertations and Capstones

Every mammalian life develops from one cell after fertilization of an egg by the sperm. The molecular pathways governing this event are still poorly understood. Numerous reports indicate that mammalian eggs highly express various protein kinase C (PKC) isoforms. Accordingly, we hypothesize that PKC activity in the egg plays an important role during egg-sperm membrane binding and fusion. In this study, we tested our hypothesis in mouse gametes using two types of PKC inhibitors (calphostin c and staurosporine) and the typical PKC activator, phorbol ester 12-tetradecanoylphorbol-13 acetate (PMA). After treatment with the individual drug, eggs were inseminated with sperm. The …

Thimerosal-Induced Neuritoxicity: Apoptosis Occurs Through A Mitochondrial-Mediated Pathway Via The Jnk Signaling Pathway, Michelle L. Herdman

Theses, Dissertations and Capstones

Thimerosal is an organic mercurial containing an ethylmercury moiety attached to the sulfur atom of thiosalicylate. Since the 1930s, thimerosal has been used as an antiseptic and a preservative in a wide variety of products, including medicinal preparations administered to children and pregnant women. Past exposures to mercurials have indicated that mercury is a neurotoxin, and can also affect the kidney, skin, eyes, and immune system. Additionally, fetuses exposed to mercurials are more susceptible to toxicity because the nervous system is continuously developing. However, despite its widespread use, thimerosal was only studied on a limited basis until the end of …

Regulation Of Biofilm Formation Of Pseudomonas Aeruginosa, Nathaniel Edwards Head

Theses, Dissertations and Capstones

Cystic fibrosis (CF) is the most common, autosomal recessive lethal genetic disease in the Caucasian population, resulting from a malfunctioned cystic fibrosis transmembrane conductance regulator (CFTR) and leading to bacterial lung infections. P. aeruginosa, an opportunistic pathogen, establishes a chronic infection in CF with a phenotype of overproduction of an exopolysaccharide (alginate) due to host-directed mutagenesis. While free-floating planktonic bacteria can be properly cleared from the CF lung, P. aeruginosa, along with alginate production, establishes an infection in the form of a biofilm which supports its survival in nature and in vivo. As a result, genomic structure, …

Characterization Of The Dna-Binding Properties Of Silent Information Regulator 3 Protein, Cotteka Nichisha Johnson

Theses, Dissertations and Capstones

The eukaryotic genome is organized into distinct domains with discrete regulatory potential, influenced by the variation in local chromatin structure. An example of chromatin-mediated consequences on gene expression is mating type determination of yeast Saccharomyces cerevisiae, which depends on silencing of specific gene cassettes. Silencing in yeast involves key proteins, amongst which the Silent Information Regulator (Sir) proteins are essential. Sir1p, Sir2p, and Sir4p are recruited to nucleate silencing events, while Sir3p serves as the pivotal factor in spreading the silent state. This investigation characterizes the DNA binding properties of Sir3p using DNA fragments with varying degrees of inherent flexibility, …

Diverse Roles Of Pkcα In Vascular Smooth Muscle Contraction, Ava Caudill Dykes

Theses, Dissertations and Capstones

A complete understanding of the mechanisms underlying smooth muscle contractility has proven elusive due to the many interacting factors thought to influence contractile behavior in this muscle type. One such factor, protein kinase Cα (PKCα), is proposed to play a central role in both the activation and modulation of smooth muscle contraction. PKCα is activated through a G-protein coupled receptor and is subsequently translocated to its site of action. Although the mechanisms regulating PKCα site specific translocation are not completely understood, it is thought that this process is essential for PKCα specificity of action within the cell. In chapter II …

Therapeutic Electromagnetic Field (Temf) And Gamma Irradiation On Human Breast Cancer Xenograft Growth, Angiogenesis And Metastasis, Ivan L. Cameron, Luzhe Sun, Nicholas Short, W. Elaine Hardman, C. Douglas Williams

Biochemistry and Microbiology

Background

The effects of a rectified semi-sinewave signal (15 mT amplitude, 120 pulses per second, EMF Therapeutics, Inc.) (TEMF) alone and in combination with gamma irradiation (IR) therapy in nude mice bearing a human MDA MB231 breast cancer xenograft were tested. Green fluorescence protein transfected cancer cells were injected into the mammary fat pad of young female mice. Six weeks later, mice were randomly divided into four treatment groups: untreated controls; 10 minute daily TEMF; 200 cGy of IR every other day (total 800 cGy); IR plus daily TEMF. Some mice in each group were euthanized 24 hours after the …

Endothelial Cell Pseudopods And Angiogenesis Of Breast Cancer Tumors, Ivan L. Cameron, Nicholas Short, Luzhe Sun, W. Elaine Hardman

Biochemistry and Microbiology

Background

A neoplastic tumor cannot grow beyond a millimeter or so in diameter without recruitment of endothelial cells and new blood vessels to supply nutrition and oxygen for tumor cell survival. This study was designed to investigate formation of new blood vessels within a human growing breast cancer tumor model (MDA MB231 in mammary fat pad of nude female mouse). Once the tumor grew to 35 mm³, it developed a well-vascularized capsule. Histological sections of tumors greater than 35 mm³were stained with PAS, with CD-31 antibody (an endothelial cell maker), or with hypoxia inducible factor 1α …

Dietary Omega-3 Fatty Acids And Ionizing Irradiation On Human Breast Cancer Xenograft Growth And Angiogenesis, W. Elaine Hardman, Luzhe Sun, Nicholas Short, Ivan L. Cameron

Biochemistry and Microbiology

Background

The effects of an omega-3 (n-3) fatty acid enriched diet alone and in combination with gamma irradiation (IR) therapy in nude mice bearing a human MDA-MB231 breast cancer xenograft were tested. The cancer cells were injected into the mammary fat pad of young female mice. Six weeks later, mice were randomly divided into two diet groups: 1) mice with 10% corn oil (rich in omega 6 fatty acids) in their food, 2) mice consuming a 10% fat diet that was enriched in n-3 fatty acids. After two weeks on the diet, treatment with 200 cGy of IR every second …

Defective B Cell Responses In The Absence Of Sh2d1a, Massimo Morra, Robert A. Barrington, Ana C. Abadia-Molina, Susumo Okamoto, Aimee Julien, Charles A. Gullo Phd, Anuj Kalsy, Matthew J. Edwards, Gang Chen, Rosanne Spolski, Warren J. Leonard, Brigitte T. Huber, Persephone Borrow, Christine A. Biron A. Biron, Abhay R. Satoskar, Michael C. Carroll, Cox Terhorst

Biochemistry and Microbiology

More than half of patients with X-linked lympho-proliferative disease, which is caused by a defect in the intracellular adapter protein SH2D1A, suffer from an extreme susceptibility to Epstein-Barr virus. One-third of these patients, however, develop dysgammaglobulenemia without an episode of severe mononucleosis. Here we show that in SH2D1A-/- mice, both primary and secondary responses of all Ig subclasses are severely impaired in response to specific antigens. Because germinal centers were absent in SH2D1A-/- mice upon primary immunization, and because SH2D1A was detectable in wt germinal center B cells, we examined whether SH2D1A-/- B cell functions were impaired. Using the adoptive …

Effects Of Aging On Pressure-Induced Mapk Activation In The Rat Aorta, Kevin M. Rice

Theses, Dissertations and Capstones

With age, the cardiovascular system experiences substantial alterations in cellular morphology and function. The factors regulating these changes are unknown; however, the mitogen activated protein kinase (MAPK) pathways have emerged as critical components for mediating numerous cellular responses including control of cell growth, differentiation and adaptation. Here we compare the expression, basal activation and the ability of increased pressure to activate the MAPK pathways in adult (6 month old), aged (30 month old) and very aged (36 month old) Fischer 344 x Brown Norway F1 Hybrid rats. Histochemical analysis demonstrated an age-related increase in tunica media thickness of approximately 11% …

Mechanisms Of Growth Hormone Enhancement Of Excitatory Synaptic Transmission In Hippocampus, Ghada Saad Zaglool Ahmed Mahmoud

Theses, Dissertations and Capstones

Growth hormone (GH) deficiency is associated with impaired learning and memory. One possible target for GH effects on memory is the hippocampus, a brain region containing GH receptors (GHRs). To determine if GH acutely alters hippocampal function, recombinant human GH (rhGH) was applied to in vitro rat hippocampal brain slices. Extracellular recordings were used to assess effects of GH on the field EPSP (fEPSP) and long-term potentiation (LTP) of the fEPSP. GHR expression was measured in GH-treated and control rat hippocampal slices using RT-PCR. The GH signaling pathway was investigated by studying the effect of GH on the fEPSP after …

Discovering The Barriers To Health-Promoting Lifestyles Among Appalachian Veterans With Uncontrolled Hypertension, Tara L. Porter

Theses, Dissertations and Capstones

Introduction: The purpose of this study was to discover the barriers to health promoting lifestyles among the Appalachian veteran population with uncontrolled hypertension.

Design: A quantitative research study was conducted over five months using a non-experimental, descriptive research design. Pender’s Health Promotion Model served as the study’s framework. Subjects were recruited from a primary care setting through advertisement within the hospital. A convenience sample of thirty-two subjects was obtained.

Method: The Health Promotion Lifestyle Profile (HPLP) II survey assessed for barriers to controlled hypertension in the following areas: health-promoting lifestyle, health responsibility, stress management, spiritual growth, interpersonal relationships, nutrition, and …

Effects Of Tamoxifen On Mitochondrial Nos Activity: Alteration In The Intramitochondrial Ca2+ Homeostasis, Sandeep S. Joshi

Theses, Dissertations and Capstones

Tamoxifen (Tam) is an anticancer drug that induces oxidative stress and apoptosis via mitochondria- and nitric oxide (NO)-dependent pathways. Here, we report that therapeutic concentrations of Tam stimulate the mitochondrial NO synthase (mtNOS) activity of isolated rat liver mitochondria by increasing the intramitochondrial ionized Ca²⁺ concentration ([Ca²⁺]_m). Tam decreases transmembrane potential (∆ψ) due to increased [Ca²⁺]_m that neutralizes the negative charges of the inner mitochondrial membrane. Thus, the present study reports a novel mechanism for the widely used anti- caner drug, Tam.

Effect Of Dna Base Modification On Polymerase Chain Reaction Efficiency And Fidelity, Jan A. Sikorsky

Theses, Dissertations and Capstones

Polymerase stop assays, used to quantify DNA damage, assume single lesions are sufficient to block thermostable DNA polymerase progression. To explore this assumption, 90 base oligonucleotides containing normal or modified DNA bases were amplified using real-time PCR. Data implied that the PCR efficiency was influenced to differing degrees depending on which base lesion was present on the input oligonucleotide; specifically, while reactions with templates containing a single 8-oxo-7,8-dihydro-2í-deoxyguanosine (8-oxodG) were not noticeably altered, the presence of a single 8-oxo-7,8-dihydro-2í-deoxyadenosine, an abasic site, or a cis-syn thymidine dimer (TT dimer) dramatically delayed amplification. In addition, the presence of two tandem 8-oxodGs …

The Cell Surface Receptor Slam Controls T Cell And Macrophage Functions, Ninghai Wang, Abhay Satoskar, William Faubion, Susumu Okamoto, Stefan Feske, Charles A. Gullo Phd, Kareem Clarke, Miriam Rodriguez Sosa, Arlene H. Sharpe, Cox Terhorst

Biochemistry and Microbiology

Signaling lymphocyte activation molecule (SLAM), a glycoprotein expressed on activated lymphocytes and antigen-presenting cells, has been shown to be a coregulator of antigen-driven T cell responses and is one of the two receptors for measles virus. Here we show that T cell receptor–induced interleukin (IL)-4 secretion by SLAM−/− CD4+ cells is down-regulated, whereas interferon γ production by CD4+ T cells is only slightly up-regulated. Although SLAM controls production of IL-12, tumor necrosis factor, and nitric oxide in response to lipopolysaccharide (LPS) by macrophages, SLAM does not regulate phagocytosis and responses to peptidoglycan or CpG. Thus, SLAM acts as a coreceptor …

The Genetic And Molecular Characterization Of The Polycystic Kidney Disease-Causing Mouse Gene Bicc1, Sarah J. Price

Theses, Dissertations and Capstones

Polycystic kidney disease (PKD) is one of the most common hereditary diseases and is characterized by progressive cyst formation, substantial renal enlargement, and frequently, progression to end-stage renal disease. One way to learn more about the etiology of this disease is to study mouse models that imitate the human situation. The juvenile congenital polycystic kidney disease (jcpk) gene on mouse Chromosome 10 has been found to cause a severe, early onset form of PKD when inherited in an autosomal recessive manner (Flaherty et al., 1995). Previous genetic studies mapped the jcpk locus to a 1 cM region on mouse Chromosome …

Roles Of Pkc Isozymes In Retinoic Acid-Induced B16 Mouse Melanoma Cell Growth Inhibition And Differentiation, Jianming Han

Theses, Dissertations and Capstones

Protein Kinase C (PKC) has been implicated in many cellular responses. Of the 11 isotypes in this family, seven are expressed in B16 mouse melanoma cells − PKCα, PKCδ, PKCε, PKCθ, PKCλ, PKCζ, and PKCµ.

Treatment of B16 cells with retinoic acid (RA) results in growth inhibition and differentiation. Simultaneously, RA induces a 6-8 fold increase of PKC α protein level, but only about 2-fold increase of PKC enzyme activity. The over- expression of wild type PKCα in B16 cells mimicked the action of RA. The over- expression of enzymatically inactive PKCα, either open or closed conformation, also inhibited B16 …

Docosahexaenoic Acid Modulates Class I Major Histocompatibility Complex Protein Function, Kaleb Marie Hypes

Theses, Dissertations and Capstones

Fatty acids influence immune responses, but how this occurs at the molecular level is not understood. Class I major histocompatibility complex molecules (MHC I), transmembrane proteins required for antigen presentation to T cell receptors, each consist of a 45kDa α chain noncovalently associated with a 12-kDa β₂-microglobulin (β₂m) molecule. Docosahexaenoic acid’s (DHA) effect on the binding of β₂m and anti-MHC I monoclonal antibodies (Mab) was measured. EL4 and RMA-S cells were cultured 48 hours with 0-45µM DHA or oleic acid. Murine to human β₂m exchange and Mab binding were assessed by fluorescence …

Cytoanalysis Of Pancreatic B-Cells: Using An Avian Model, Mammalian Tissue Culture And Implications Of Antisense Oligonucleotides Transfection, Ayman Salah-El-Deen Amer

Theses, Dissertations and Capstones

Calbindin-D_28k (CaBP_28K) is a vitamin D-dependent calcium-binding protein that may alter intracellular calcium ion levels, [Ca²⁺]i. This dissertation describes experiments done to gain an understanding of the potential role of CaBP_28k in pancreatic B-cells in control of insulin secretion. The localization of CaBP_28k and insulin in chicken pancreas are shown in Chapter 1. CaBP_28k expression was found to be highest in ventral and dorsal lobes and lowest in splenic lobe. Insulin concentrations were distributed similarly among these lobes. Confocal microscopic studies demonstrated colocalization of insulin and CaBP_28k in Bcells. These findings …

Renal Basolateral Transport Of Glucuronides And Other Organic Anions In Rat In Vitro Models, Sarah L. Miles

Theses, Dissertations and Capstones

Glucuronidation is a common Phase II biotransformation reaction that increases the hydrophilicity, and thus elimination, of toxins, xenobiotics, and endogenous compounds. Previous studies suggest that the kidney can secrete glucuronide conjugates, but the renal transport mechanisms for glucuronide secretion have not been determined. Based on the chemical nature of glucuronide metabolites, it is hypothesized that organic anion transporter (OAT) proteins along the basolateral membrane of the renal proximal tubule promote renal accumulation of glucuronide conjugates. The purpose of this study was to develop a rat renal proximal tubule model which demonstrates OAT activity and by which the contribution of OAT …

Waddington’S Widget: Hsp90 And The Inheritance Of Acquired Characters, Douglas M. Ruden, Mark D. Garfinkel, Vincent E. Sollars, Xiangyi Lu

Biochemistry and Microbiology

Conrad Waddington published an influential model for evolution in his 1942 paper, Canalization of Development and Inheritance of Acquired Characters. In this classic, albeit controversial, paper, he proposed that an unknown mechanism exists that conceals phenotypic variation until the organism is stressed. Recent studies have proposed that the highly conserved chaperone Hsp90 could function as a “capacitor,” or an “adaptively inducible canalizer,” that masks silent phenotypic variation of either genetic or epigenetic origin. This review will discuss evidence for, and arguments against, the role of Hsp90 as a capacitor for morphological evolution, and as a key component of what …

High Dietary Level Of Synthetic Vitamin E On Lipid Peroxidation, Membrane Fatty Acid Composition And Cytotoxicity In Breast Cancer Xenograft And In Mouse Host Tissue, Ivan L. Cameron, Jesus Munoz, Christopher J. Barnes, W. Elaine Hardman

Biochemistry and Microbiology

Background

d-α-tocopherol is a naturally occurring form of vitamin E not previously known to have antitumor activity. Synthetic vitamin E (sE) is a commonly used dietary supplement consisting of a mixture of d-α-tocopherol and 7 equimolar stereoisomers. To test for antilipid peroxidation and for antitumor activity of sE supplementation, two groups of nude mice bearing a MDA-MB 231 human breast cancer tumor were fed an AIN-76 diet, one with and one without an additional 2000 IU/kg dry food (equivalent to 900 mg of all-rac-α-tocopherol or sE). This provided an intake of about 200 mg/kg body weight per day. The mice …

Detection Of Forces And Body Load In Standing And Walking In The American Cockroach, J. Adam Noah

Theses, Dissertations and Capstones

Sense organs in the legs that detect body weight are an important component in the regulation of posture and locomotion. This thesis seeks to gain an understanding of how body weight is detected by sense organs of the legs, and determine how this information influences muscle activities in standing and walking. The first study tested the ability of tibial campaniform sensilla (receptors that monitor forces in the cockroach leg) to encode variations in body load using magnets attached to the thorax. Recordings of sensory activities in freely standing animals showed that proximal tibial sensilla (oriented perpendicular to the leg long …

The Neural Connectivity Of The Torus Semicircularis In The Cave Dwelling Form Of Astyanax Fasciatus, Timothy B. Angel

Theses, Dissertations and Capstones

Afferent projections of the torus semicircularis were traced in the blind cave form of Astyanax fasciatus using HRP tract-tracing techniques, and the results were compared to those found in previous studies of closely related sighted teleosts.

Following unilateral injections of tracer to the nucleus centralis of the torus semicircularis, retrogradely labeled cells were found in two octaval nuclei of the medulla. The descending octavolateralis nucleus was labeled bilaterally and the anterior octaval nucleus was labeled primarily on the ipsilateral side. Both the descending and anterior octavolateralis nuclei are known to receive auditory input from CN VIII in sighted fish. In …

The Characterization Of Two Differentially Expressed Plasma Proteins In Obese Versus Lean Rats In Two Rodent Models Of Obesity, Roger D. Boggs

Theses, Dissertations and Capstones

Zucker fa and La/N faf rats are widely studied models of genetic obesity and its complications. These two rodent models of obesity were utilized to search for a circulating protein marker for obesity. Plasma samples from both of these models of obesity were collected and analyzed via SDS-PAGE analysis. Two proteins were found which demonstrate differential expression between lean and obese rats. Both proteins demonstrated increased expression in the obese rats compared to the lean. One differentially expressed protein migrated on SDS-PAGE gels at 116 KD while the second migrated at 22 KD compared to molecular weight markers. The 22 …

A Mechanistic Study In The Nephrotoxicity Of P-Aminophenol, R. Christopher Harmon

Theses, Dissertations and Capstones

The acetaminophen metabolite, p-aminophenol (PAP), is a known nephrotoxicant. The current study is directed at understanding the mechanism of this nephrotoxicity. Renal cortical slices isolated from F344 rats were incubated with 0 – 0.5 mM PAP with or without pretreatments. Renal slices exposed to PAP showed toxicity as measured by increased lactate dehydrogenase leakage (LDH), decreased pyruvate-stimulated gluconeogenesis, and decreased total adenine nucleotides. There was, however, an increase in ATP as percent of total, implying maintained mitochondrial function. These data show that PAP is directly toxic to renal cortical slices. Pretreatment with EDTA or EGTA provided protection from PAP- induced …

The Role Of Ap-1 Transcription Complex In Retinoic Acid-Dependent B16 Melanoma Cell Growth Arrest And Differentiation, Ying Huang

Theses, Dissertations and Capstones

Our laboratory has been studying the effects of all-trans retinoic acid (atRA) on the growth and differentiation of the B16 mouse melanoma cells. RA inhibits both anchorage-dependent and –independent growth and stimulates melanin production in B16 cells, which are accompanied by an increase in AP-1 activity. To further investigate the RA-induced AP-1 activity, I established clones of B16 stably expressing an AP-1-luciferase reporter gene. Treatment of these clones with phorbol dibutyrate (PDB), a known activator of AP-1, rapidly increased AP-1 activity which reached its maximum level at 2-4 h and returned to baseline level by 24 h. In contrast, RA …

Regulation Of Retinoic Acid Receptor Α By Protein Kinase C In B16 Mouse Melanoma Cells, Goran Boskovic, Dinakar Desai, Richard M. Niles

Biochemistry and Microbiology

We have previously found that retinoic acid stimulates the expression of protein kinase Cα (PKC) in B16 mouse melanoma cells. Because it has been reported that PKC can phosphorylate retinoic acid receptor (RAR) and alter its function, we determined whether changes in the level and/or activity of PKC could affect the expression or function of the RAR in B16 melanoma. Using in vivophosphorylation and band shift techniques, we could not demonstrate that altering PKC activity and/or protein level changed thein vivo phosphorylation of RARα. However activation of PKC resulted in increased RARα protein. Increased receptor protein correlated with …

Role Of Lipid Peroxidation And Antioxidant Enzymes In Omega 3 Fatty Acids Induced Suppression Of Breast Cancer Xenograft Growth In Mice, W. Elaine Hardman, Jesus Munoz Jr., Ivan L. Cameron

Biochemistry and Microbiology

Background:

Supplementing mice with high levels of dietary n-3 polyunsaturated fatty acids (PUFAs) increases the n-3 PUFAs in cell membranes, increases the susceptibility of the cells for lipid peroxidation (LPO) and decreases the growth rate of mammary and other tumors. However, the results of an earlier study indicated that a factor in addition to LPO was involved in the reduction in tumor growth in n-3 PUFAs fed mice. Athymic mice bearing MDA-MB-231 human breast carcinoma xenografts, were fed fish oil concentrate (FOC) or control diets, with and without supplemental Vitamin E (2000 IU /kg diet) and were sacrificed both before …