Browsing by Subject "Molecular Biology"
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Item A Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes Required for DNA Damage-Induced Mutagenesis(2008-07-01) Gong, Jinjun; Siede, Wolfram; Sheedlo, Harold; Reeves, RustinA Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes required for DNA Damage-Induced Mutagenesis. Jinjun Gong Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107. Summary. Deoxyribonucleic acid (DNA) damage is common in a cell’s lifetime. DNA can be damaged by endogenous factors such as reactive oxygen species (ROS) or exogenous agents such as ultraviolet (UV) or industrial chemicals. DNA damage will trigger cell responses including cell cycle arrest, transcription activation, DNA repair or apoptosis. In addition to various DNA repair mechanisms including damage reversal, base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end joining, translesion DNA synthesis is an important DNA damage tolerance pathway that can bypass the lesion on template DNA to finish the replication for cell survival but at the risk of potential mutation in the daughter cells. Accumulation of mutation may lead to cancer occurrence. Translesion DNA synthesis components are highly conserved from yeast to humans. Important players in trans-lesion synthesis pathway such as Rev1, Rev3 and Rev7 were first discovered in budding yeast. Saccharomyces cerevisiae. Homologues were found later in human cells. I used the Saccharomyces cerevisiae deletion mutant collection to do a systematic screen to search for novel genes required for DNA damage induced mutagenesis in yeast. After CAN1 forward mutation assay for the systematic screen and reverse mutation assay for further confirmation, two candidate genes SWI6 and DOA4 were detected. Deletion of SWI6 and DOA4 decreases mutagenesis of cells. At the molecular level, Swi6, a transcription cofactor, is involved in mutagenesis by regulating expression of REV7 at the mRNA and protein levels. Rev7 is a regulatory subunit of DNA polymerase zeta, which is essential for DNA damage induced mutagenesis as well as spontaneous mutagenesis. Rev7 is not UV inducible or cell cycle regulated. The regulation of Rev7 at the transcriptional level by Swi6 is essential. Future experimental approaches are planned to address the mechanism by which DOA4 is involved in mutagenesis.Item Age Related Changes in Rabbit Cornea: Permeability and Membrane Properties(1994-12-01) Tai-Lee, Ke; Clark, Abbot F.; Gracy, Robert W.; McConathy, Walter J.Ke, Tai-Lee, Age Related Changes in Rabbit Cornea: Permeability and Membrane Properties. Doctor of Philosophy (Biochemistry), December, 1994, 139 pp., 26 tables, 13 illustrations, bibliography, 117 titles. This investigation was designed to characterize age-related changes in corneal function and biochemical structure. The specific aims were to: 1) systematically assess changes in permeability to compounds of different molecular weights and lipophilicities, 2) examine differences in tissue binding by utilizing a theoretical transport model, and 3) evaluate the biochemical changes in lipid composition and distribution. Experiments to compare young (six weeks) versus old (three to four years) rabbit corneal permeability were carried out utilizing an in vitro diffusion model. Changes in corneal transmembrane resistance, permeability to various compounds, and metabolic capability were examined by various analytical techniques. In addition, a theoretical penetration model which took into account stromal binding was studied. Corneal lipid composition and distribution were assessed by HPLC and GC. in corneal transmembrane resistance, permeability to various compounds, and metabolic capability were examined by various analytical techniques. In addition, a theoretical penetration model which took into account stromal binding was studied. Corneal lipid composition and distribution were assessed by HPLC and GC. Permeabilities of selected compounds with different physicochemical properties were evaluated in young and old intact and denuded (wounded) rabbit corneas. With age, the membrane permeability significantly decreased in parallel with an increase in transmembrane resistance. Age-related changes in activities of esterase and phosphatase were also found. For some compounds, the aged corneas exhibited longer lag times in penetration studies. This suggested that the binding constant in the cornea from older animals was higher than in young animals. Maximum binding capacity from theoretical model calculations correlated well with experimental results in the young corneal stroma but correlation was less rigorous for old corneal stroma. Age-related changes in lipid composition and distribution in corneas were observed and provide indirect evidence for a decrease in membrane fluidity (decrease in the ratio of phosphatidylcholine/sphingomyelin) in the aged cornea. Results indicate that the aging process in the cornea is associated with changes in biochemical structural matrix including membrane lipid composition and physical properties such as fluidity (microviscosity). Functional correlations include changes in: 1) transmembrane resistance, 2) membrane permeability, 3) enzymatic activities (esterase and phosphatase), and 4) binding properties of the cornea. A possible mechanism for understanding and developing an intervention for age-related changes in the cornea is postulated.Item Analysis of Yeast Genes Influencing the Lethality of DNA Damage Related Checkpoint Mutants(2009-05-01) Kim, Eunmi; Siede, WolframThe purpose of this study was to determine the functions of Hug1 and Srl3. It has been reported that HUG1 or SRL3 deletion rescues the lethality of a DNA damage checkpoint gene deleted mutant, mec1Δ. It is known that the lethality of mec1Δ can be rescued by high dNTP levels. To elucidate the functions of these proteins, the phenotypes of hug1Δ and srl3Δuvs, as well as the transcript profile of hug1Δ were analyzed. Novel phenotypes of hug1Δ were uncovered: resistance to oxidative stress or heat shock, earlier arrest in G1/G0 phase, defect in hydroxyurea-induced filamentation, and slow growth inresponse to combined stresses of hydroxyurea and reduced dextrose content. These phenotypes correlate with a transcription profile that indicated altered stress responses in hug1Δ as compared to WT. We assumed that the reason for many constitutively expressed stress-related transcripts is a higher dNTP level in hug1Δ compared to WT. The similarities in the phenotypes of dif1Δ and sml1Δ to those of hug1Δ support the assumption. The phenotypes of dif1Δ and sml1Δ were studied since Dif1 and Sml1 are known inhibitors of ribonucleotide reductase activity. Furthermore, Dif1, Sml1, and Hug1 are considered proteins that evolved from the same ancestor protein. Initially, Srl3 was a protein of special interest because the commercially available srl3Δ mutant causes high spontaneous mutation rates and sensitivity to UV light. However, during the course of this study, it was found that the two phenotypes originated from a second, unrelated mutation in srl3Δ strain. Through complementation test and sequencing, this mutation was identified as a nonsense mutation of MMS2, a gene involved in post-replication repair.Item Anionic Ligand-Gated Ion Channels: The Convulsive Site and Mechanism of Action(2001-08-01) Dibas, Mohammed I.; Hriday Das; Thomas Yorio; Neeraj AgarwalDibas, Mohammed, Anionic Ligand-Gated Ion channels: The Convulsive Site And Mechanism of Action. Doctor of Philosophy (Biomedical Sciences), August 2001, pp153, 1 table, 24 illustrations, 76 titles. Picrotoxin, a CNS convulsant inhibits all anionic ligand gated ion channels. The mechanism and the binding site for picrotoxin and its related ligands are still undefined. The second transmembrane (TMII) domain of these ligand gated ion channels is found to play a key role in the mechanism of block by picrotoxin. It has been shown that the incorporation of a phenylalanine residue in place of threonine at position 6’ within the TMII domain of B2 subunit conferred high resistance toward picrotoxin in GABAA a3B2(T6’F)y2 receptors. Mediating their blocking effect through the PTX-site, PTZ, TBPS, and U-93631 lost their inhibitory effects due to the same mutation B2(T6’F). Interestingly, this mutation uncovered a low affinity, highly efficacious stimulatory site for PTZ. PTZ seems to mediate its stimulatory effect through a novel distinct site different from that for benzodiazepine. The effect of varying subunit configuration of GABAA receptors dramatically affected the ability of the mutation B2(T6’F) to abolish the inhibitory effect of picrotoxin. While picrotoxin failed to block the current induced by GABA in a3B2(T6’F)y2 receptors, picrotoxin partially blocked the current in a3B2(T6’F)y2 receptors. In B2(T6’F)y2 receptors, picrotoxin restores its full efficacy. When phenylalanine was incorporated at position 6’ in the a1 subunit, picrotoxin completely blocked the current induced by GABA in a1(T6’F)B2y2 receptors. The combined results showed that the ability of (T6’F) mutation to regulate the inhibitory mechanism of picrotoxin as dependent on the subunit configurations and at which subunit is mutated. In addition, picrotoxin is known to inhibit GABAA receptors in use-facilitated mechanism, while it inhibits the glycine receptor in a non-use facilitated fashion. The molecular determinant behind the use-facilitated mechanism was modulated by the nature of the amino acid at position 15’ within the second transmembrane domain. The mutation of serine 15’ to either glutamine or asparagine in the glycine a1 receptors converted picrotoxin from a non-use facilitated blocker to a use-facilitated one. The latter finding suggested that this residue might residue within the PTX binding site or play a key role in the transduction pathway for picrotoxin mechanism. The overall results further support the fact that TMII domain plays a key role in the picrotoxin mechanism.Item Anisotropy of Myosin and Actin in Contraction of Skeletal Muscle(2004-12-01) Shepard, Athena A.; Julian Borejdo; Thomas Burghardt; Ben HarrisShepard, Athena A., Anisotropy of Myosin and Actin in Contraction of Skeletal Muscle. Doctor of Philosophy (Molecular Biology and Immunology), December, 2004, 161 pp., 1 table, 42 illustrations, bibliography, 253 titles. Muscle contraction results from the interaction of myosin and actin proteins contained in the muscle sarcomere. During actomyosin interactions, myosin consumes ATP and imparts an impulsive force to actin resulting in sliding of myosin and actin filaments to produce work. These proteins constitute the elementary motor responsible for cellular motility. The overall goal of this research project was to elucidate the mechanism of the actomyosin interaction on a molecular level. Novel time-resolved optical microscopic techniques followed myosin and actin orientation changes during skeletal muscle contraction. Fluorescence anisotropy was used to study the real time orientation changes of myosin, actin, and nucleotide during a single cross bridge cycle beginning in a state of rigor. Rabbit psoas fibers were isolated on a microscopic slide and labeled with fluorescently labeled regulatory light chain to monitor orientation changes of the lever arm of myosin, with fluorescent phalloidin to monitor orientation changes of actin and/or with Alexa ADP to monitor ATP hydrolysis. Caged ATP was perfused into the fiber prior to analysis to allow a small population of cross-bridges to execute a single cross-bridge cycle. Flash photolysis with UV light during analysis converted caged ATP from an inactive from to an active from. Confocal and multi-photon imaging allowed illumination of a small population of fluorescently labeled cross-bridges to measure orientation changes over time. The conclusions of this dissertation are: 1) The regulatory light chain rotates during skeletal muscle contraction and the lever arm model is supported, 2) Release of ADP from S1 corresponds to a single rotation of the lever arm, 3) Actin rotates during skeletal muscle contraction, 4) The rotation of actin is passive, i.e. it rotates as a consequence of dissociation of S1 from actin. The results revealed orientation changes in key contractile proteins during muscle contraction in the non-disease state organism. By understanding the mechanism of muscle contraction in the healthy scenario, hopefully a better understanding of diseased states stemming from mutations in contractile proteins (Usher’s Syndrome, Snell’s Waltzer Disease, and certain familial hypertrophic cardiomyopathies) will be made available, leading to a better preventative measures or treatments to treat such diseases in the future.Item Characterization of Recombinant Lecithin: Cholesterol Acyltransferase, Secreted by a Human Lung Cell Line (1069-111) and by Pichia Pastoris Yeast Cells(2004-05-01) Tchedre, Kissaou T.; Caffrey, James L.; Harris, Ben G.; Wu, Ming-ChiTchedre, Kissaou T., Characterization of Recombinant Lecithin: Cholesterol Acyltransferase, Secreted by a Human Lung Cell Line (1069-111) and by Pichia pastoris Yeast Cells (Biomedical Sciences), May, 2004, Lecithin: cholesterol acyltransferase (LCAT) is a key enzyme in mammalian lipoprotein metabolism. Associated with the surface of high-density lipoproteins (HDL), LCAT contributes to the homeostasis of circulating free and esterified cholesterol via the reverse cholesterol transport pathway. The purpose of these studies was to characterize a recombinant form of LCAT, secreted by a human lung cell line (Beta gene 1069/111) and to evaluate a new expression system for LCAT using transformed Pichia pastoris cells. A human lung cell line (Beta gene 1069/111), transfected with pBIISK (Stratagene)+ vector was used as the source of recombinant (rLCAT) for the first stage of characterization studies. Human lung cells were expanded in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum for the expression of the recombinant LCAT. At 80 – 90% confluency, the medium was changed to a serum free preparation and the flasks were incubated for 48 hrs at 37°C to facilitate the secretion of the enzyme. Beta gene (1069/111) LCAT was purified from the conditioned medium using phenyl sepharose chromatography. The purified enzyme was characterized according to: carbohydrate composition, and enzyme kinetic parameters. The enzymatic characteristics, of the human lung cell line LCAT had similar Km and Vmax values to other LCAT preparations, isolated from other expression systems and human plasma. Deglycosylation reduced the molecular weight of the enzyme from about 67,000 to about 43,000 suggesting a carbohydrate component of 25-32% of the enzyme’s total mass. Detailed analysis of the carbohydrate structures revealed N-glycan structures in a complex pattern of sialylated and fucosylated tri and tetra-antennary glycosides (8). In addition to the Beta gene expression, a Pichia pastoris yeast expression system was also developed consisting of human LCAT cDNA cloned into pPICZαA vector along with a removable amino-terminal polyhistidine tag. The Pichia pastoris cells were transformed with a vector containing the LCAT gene cDNA and transformants were selected on agar plates containing zeocine (100μg/ml). Polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) were used to confirm the correct integration of the LCAT gene cDNA into the pPICZαA vector. The recombinant LCAT produced by the yeast cultures was purified by Talon affinity chromatography, taking advantage of the removable histidine tag. The enzymatic activity was determined using proteoliposome vesicles. The Yeast expression system yielded ~18 mg of enzyme protein/500 ml and thus may provide an appropriate enzyme source for characterization studies via NMR analysis and x-ray crystallography.Item Discovery and Characterization of Tetranucleotide Short Tandem Repeats in North American Bears (Ursids)(2015-05-01) Graham, Michelle L.; John V. PlanzAccurate individual identification is essential to wildlife crime investigations and conservation genetics. Current methodology utilizes dinucleotide short tandem repeats (STRs) that can be difficult to type accurately and have high mutation rates; however, tetranucleotide STRs have greater stability and allele diversity. The main objective of this study was to identify potential tetranucleotide STR loci and internal variants for the American black bear, brown bear, and polar bear. Barcoded genome libraries were prepared for each species from extracted and enzymatically fragmented DNA, size selected, quantified, enriched using biotinylated RNA baits to capture twelve common mammalian sequence motifs, and massively parallel sequenced. One potential locus was identified using the NextGENe® software and six potential loci were identified using algorithm mining.Item Discovery-Driven Proteomics to Advance the Evaluation of the Role of Lipids and Lipid Peroxidation-Related Carbonyl Stress in Biological Model Systems(2013-05-01) Talamantes, Tatjana D.; Laszlo ProkaiResearch has come a long way with the advancement of sequenced genomes in regards to a variety of organisms. In addition, the technology used to explore biological information encoded by these genome sequences has also been enhanced. However, the knowledge of the role that simple components play in biological functions and cellular processes of the organism, from a global perspective, remains limited. With the advent of proteomics, exploring pathways and networks influenced by cellular components such as lipids, has become possible in the context of systems biology. This dissertation has been divided into two parts. Both will focus on a discovery-driven approach to elucidate the diverse biological functions of lipids as well as stress and its consequences in two biological model systems using mass spectrometry-based proteomics. The first part investigates the broader role of phospholipids (specifically phosphatidylglycerol), essential components of biological membranes in prokaryotes and eukaryotes, in a genetically modified strain of cyanobacteria. Ultimately, understanding the impact of lipid-regulation in this simple organism is expected to reveal previously unknown lipid-signaling mechanisms, which could be further investigated in higher-order organisms. The second part is dedicated to the investigation of lipid-signaling mechanisms generated by electrophilic products of oxidative stress-induced lipid peroxidation, and its consequences in disturbing homeostasis in zebrafish embryos. Exploring free radical-mediated lipid peroxidation involved in cellular responses promises a comprehensive understanding of the role and/or contribution that electrophilic products of lipid peroxidation play in modulating oxidative stress-related signaling pathways potentially involved in various pathophysiological dysfunctions. Bioinformatics software successfully constructed networks from the proteins identified as being regulated by the induced carbonyl-stress including intra- and intercellular processes involved in eIF2 signaling, glycolysis, and remodeling of epithelial adherens junctions.Item Distribution of Poly(ADP-ribose) Glycohydrolase in Different Functional Domains of the Cell Nucleus(1996-08-01) Pacheco-Rodriguez, Gustavo; Rafael Alvarez; Robert Easom; Ming-Chi WuPacheco-Rodriguez, Gustavo, Distribution of Poly(ADP-ribose) Glycohydrolase in Different Functional Domains of the Cell Nucleus. Doctor of Philosophy (Biomedical Sciences), August, 1996, 147 pp, 3 tables, 44 illustrations, bibliography, 138 titles. In this study, the distributor poly(ADP-ribose) glycohydrolase (PARG) in different subdomains of the cell nucleus and the role of non-covalent interactions of poly(ADP-ribose) with nuclear proteins have been characterized. An assay that allows the simultaneous determination of specific non-covalent interactions of poly (ADP-ribose) with nuclear proteins as well as PARG activity by high resolution polyacrylamide gel electrophoresis was developed. This method was made possible by the enzymatic synthesis of (ADP-ribose)2-70 at 10 μM NAD+ with purified poly(ADP ribose) polymerase (PARP). Either purified or nuclear-associated PARG degraded poly(ADP-ribose) biphasically. Nuclei were fractioned into functional domains namely, chromatin, nuclear matrix and nuclear envelope. These domains were characterized biochemically by their protein composition and by electron microscopy. PARG activity was identified mainly with chromatin and the nuclear matrix. Interestingly, PARG activity was also associated with the nuclear envelope. Thus, the poly(ADP-ribosyl)ation pathway is regulated topologically. It was further determined that poly(ADP-ribose) interacts non-covalently with purified histone proteins or proteins in the nuclear environment. In addition, the nuclear matrix proteins also interacted non-covalently with poly(ADP-ribose). These non-covalent interactions appear to regulate the catabolism of poly(ADP-ribose) via a catabolite intermediate constituted of a [protein][poly(ADP-ribose)] complex. The affinity of the nuclear associated protein responsible for triggering the degradation of poly(ADP-ribose) correlates with the affinity of histone H4 for ADP-ribose chains of 20 residues or more. The findings of this research stresses that : a) poly (ADP-ribose) is catabolized by PARG in vivo; b) PARG is associated with chromatin, nuclear matrix and the nuclear envelope; c) the degradation of poly(ADP-ribose) is dependent on its non-covalent interactions with nuclear proteins; and d) histone H4 appears to be responsible for triggering the catabolism of poly(ADP-ribose).Item EGCG and Its Role in Prostate Cancer Angiogenesis(2005-05-01) Thomas, Rusha; Porunelloor Mathew; Ming-Chi Wu; Dan DimitrijevichThomas, Rusha, EGCG and its role in prostate cancer angiogenesis. Master of Science (Biochemistry and Molecular Biology), May 2005, 47 pages, 14 illustrations, reference list, 44 titles. Hypoxia inducible factor-1 (HIF-1)-mediated upregulation of vascular endothelial growth factor (VEGF) has been implicated in angiogenesis associated with malignancies. HIF-1 consists of a constitutively expressed HIF-1β subunit, and a hypoxia-inducible HIF-1α subunit. Hypoxic induction of HIF-1α correlates with increased transcriptional activation of its downstream target genes, including VEGF. Epidemiologic and laboratory studies indicate that green tea has cancer preventive activity which has been attributed to its polyphenol components, the major one being epigallocatechin gallate (EGCG). This study investigated the effect of EGCG on normoxic VEGF expression in PC-3ML human prostate cancer cells. In contrast to previous studies where EGCG inhibited VEGF expression in breast and colon cancer cell lines, our results demonstrated that EGCG has the ability to upregulate HIF-1α transcription factor via inhibition of prolyl hydroxylation and subsequent von Hippel-Lindau protein interaction. HIF-1α upregulation by EGCG led to increased VEGF promoter activity and protein expression.Item Estrogen Signaling Protects Mitochondrial Membrane Potential Integrity from Oxidative Stress in Lens Epithelial Cells(2008-05-01) Flynn, James Martin; Cammarata, Patrick R.; Wordinger, Robert J.; Dimitrijevich, S. DanFlynn, James Martin, Estrogen Signaling Protects Mitochondrial Membrane Potential Integrity from Oxidative Stress in Lens Epithelial Cells. Doctor of Philosophy, (Cell Biology and Genetics) May, 2008, 265 pages, 36 figures, bibliography, 190 titles. Loss of mitochondrial membrane potential has been determined to be one of the initiating factors in activation of apoptosis after cellular damage. Estrogen and estrogen analogues have been shown to enhance cell survival in numerous tissues through rapid pro-survival cell signaling. This study was focused on elucidating mechanisms through which estrogen protects the cells by preventing the activation of mitochondrial permeability transition pores and the subsequent loss of mitochondrial membrane potential. It is hypothesized that the anti-apoptotic mitochondrial protein BAD, once phosphorylated by estrogen activated upstream kinases, can prevent the formation of the permeability transition pre via direct interaction. To address this, lens epithelial cells were used as a model system for the examination of mitochondrial depolarization during periods of either oxidative or hyperglycemic stress. Estrogen attenuated the loss of impermeability of the mitochondrial membrane, thus maintaining the cells during acute periods of stress. It was discovered that a number of the estrogen receptor isoforms are expressed in lens epithelium, and that the wild-type estrogen receptor-β1 isoform is localized to the mitochondria in lens epithelial cultures derived from both human males and females. siRNA treatment against estrogen receptor-β determined that is a required component to elicit estrogen’s protective abilities against oxidative stress induced mitochondrial depolarization. Furthermore, administration of exogenous estrogen rapidly activated signaling pathways, particularly ERK, which were shown to have influence over the loss of mitochondrial membrane potential. Studies using both pharmacological inhibitors of MAPK signaling, as well as siRNA of ERK2 kinase demonstrate a correlation between the activation of ERK and the severity of response to oxidative stress. Investigation of downstream substrates of ERK revealed that the mitochondrial protein BAD is phosphorylated after the administration of estrogen, yet it is not required for the prevention of mitochondrial depolarization as originally hypothesized. In conclusion, these studies have confirmed a mitochondrial targeted mechanism activated by estrogen which is rapid, gender independent, estrogen receptor-β mediated signal transduction pathway. The targeting of mitochondrial function to reduce oxidative or hyperglycemic stress, thereby preventing activation of the permeability transition pore, defines a novel concept which will contribute to innovative regimens for prevention or treatment of mitochondrial pathology.Item Genetic Diversity of Easter Island (Rapanui) Population from Identifiler® Plus autosomal, Y-filer®, and Y-Plex™ 6 Y-STR Loci(2015-05-01) Guadian, Laura; Chakraborty, Ranajit; Budowle, Bruce; Hodge, Lisa M.This study investigated the genetic diversity of the Easter Island (Rapanui) population using data on 15 autosomal Short Tandem Repeats (STRs) typed with the commercial STR kits Identifiler® Plus and 23 Y-chromosome STRs typed using Y-filer (17 loci) and Y- PLEX™ 6 (6 loci). The analysis was conducted using genotype and haplotype data of 122 presumably unrelated individuals that included 48 males and 74 females. This study: (i) examined if Easter Island population had reduced genetic diversity in comparison with cosmopolitan populations such as Mainland Chilean, Polynesian, European, and African; (ii) compared genetic affinity of the Easter Island population with historically related cosmopolitan populations; and (iii) investigated the forensic utility of autosomal STRs and Y-STRs in the Easter Island population.Item Histamine Induced Changes in Phospholipase C Activity, Calcium Mobilization, and Contractility in Human Ciliary Muscle Cells(1996-06-01) Markwardt, Kerry L.; Michael W. Martin; Thomas Yorio; Eugene QuistMarkwardt, Kerry L., Histamine induced changes in phospholipase C activity, calcium mobilization, and contractility in human ciliary muscle cells. Doctor of Philosophy (Biomedical Sciences), June, 1996. Histamine has long been known to be an important mediator of inflammation and autocoid throughout the body. It has been shown to cause the contraction of many types of smooth muscle. Due to its known presence in many ocular structures and aqueous humor especially during inflammatory states, it was hypothesized that histamine could have an effect on intraocular pressure (IOP). This could occur if histamine triggered events which ultimately lead to contraction of the ciliary muscle, since it is established that contraction of the ciliary muscle affects aqueous humor outflow. Therefore, it was hypothesized in this study, the histamine causes increases in inositol phosphate production and intracellular calcium in human ciliary muscle cells which ultimately leads to contraction. To test this hypothesis, human ciliary muscle (CM) cells were cultured and used in various experiments to determine the effect of histamine on inositol phosphate production, intracellular calcium mobilization, and contractility. This study, for the first time in CM cells, showed that histamine, via an H1 receptor subtype, caused dose dependent increases in both inositol phosphates and intracellular calcium. Furthermore, it was shown that these histamine-induced events ultimately lead to contraction of the CM cells. Combining the results from all our studies, the data indicate that in human CM cells, histamine via an H1 receptor, activates phospholipase C which generates inositol phosphates such as inositol triphosphate (IP3). IP3 binds to an IP3 sensitive receptor on the endoplasmic reticulum causing the initial release of calcium which is sufficient to cause contraction of the CM cells. The intracellular release of calcium is also involved in activating a calcium channel which allows the influx of extracellular calcium into the cell. The results of these studies suggest that histamine could potentially have an IOP lowering effect in the eye due to contraction of the ciliary muscle. Overall, these studies contribute to a better understanding of the effect of histamine on a key IOP regulating tissue in the eye.Item Identification of Oxidized Proteins in Alzheimer's Disease(2002-08-01) Choi, Joungil; Gracy, Robert R.; Harris, B.; Lacko, Andras G.Joungil Choi, Identification of Oxidized Proteins in Alzheimer’s Disease. Doctor of Philosophy (Molecular Biology and Immunology). August, 2002. Pages-110. Tables 8. Figures 24. Oxidative modification of specific proteins is central to the pathology of Alzheimer’s disease (AD). The purpose of this study was to identify the oxidation-sensitive proteins in neuronal cells, fibroblasts from AD subjects, and in the blood of AD patients. In all cases, age-matched non-Alzheimer’s samples were used as controls. Proteomic methods were used to isolate and characterize the oxidized proteins. These included two-dimensional gel electrophoresis, immunolocalization of oxidized proteins and identification by MALDI-TOF mass spectroscopic methods. It was hypothesized that knowledge of these critical oxidation-sensitive proteins would shed light on the underlying mechanism of the disease. In addition, it was postulated that these proteins might prove to be biomarkers for early detection and monitoring the progress of the disease. The results show that two different oxidative stressors (H2O2 generated enzymatically, or the amyloid beta peptide, AB25-35) induce apoptotic cell death and oxidation of specific proteins (heat shock protein 60 and vimentin) in skin fibroblasts from AD subjects and in neuronal cells. In addition, the results indicate that susceptibility of these two proteins to oxidative stress is increased in fibroblasts from AD patients, compared to non-AD controls. Pretreatment with antioxidants (e.g., vitamin E or flavonoids) protect these proteins from oxidative damage. Both heat shock protein 60 and vimentin, have been suggested to function as antiapoptotic proteins. Thus, their oxidative damage could lead to the apoptotic neuronal cell death in Alzheimer’s disease. In the blood plasma of AD subjects, isoforms of fibrinogen gamma chain and alpha-1 antitrypsin were found to be oxidized. These proteins exhibited to a two- to six-fold greater specific oxidation index in plasma from AD subjects when compared to controls. Both of these proteins have been suggested to be implicated in oxidation-mediated damage of inflammation in the AD brain.Item Mechanism of Gramicidin D-Induced Insulin Secretion From BTC3 Cells(1995-08-01) Dibas, Adnan I.Dibas, Adnan I., Mechanism of Gramicidin D-Induced Insulin Secretion From BTC3 cells. Doctor of Philosophy (Biomedical Sciences), August, 1995, 190 pp., 5 tables, 38 illustrations, bibliography, 265 titles. Gramicidin D, a sodium ionophore, was discovered to be a potent insulin secretagogue in the B-cell line BTC3 cells. Gramicidin D (1 uM) induced a 3.28-fold increase in insulin release relative to control, and when studied in a dynamic cell-perifusion system, was biphasic. Insulin secretion was accompanied by effects of gramicidin D to increase intracellular concentrations of Na+([Na+]i) and Ca2+ ([Ca2+)i) in BTC3 cells as determined by dynamic single-cell video imaging techniques, gramicidin D had no effect on cellular pH. The mechanism of gramicidin D-induced increase in [Ca2+ and suggested to be mediated by a combination of membrane depolarization-induced activation of voltage-sensitive Ca2+ channels and the activation of a Na+/Ca2+ exchanger in the reverse mode. Gramicidin D-induced increase in [Ca2+]I in the first phase correlated temporally with a profound (5.56-fold) activation of multifunctional Ca2+/calmodulin-dependent protein kinase II. While these observations are consistent with the involvement of this enzyme in gramicidin D-induced insulin secretion, further observations suggested that the kinase may play only a modulatory role in insulin secretion. A similar activation of myosin light chain kinase was not detected. In contrast to BTC3 cells, gramicidin D failed to induce insulin secretion from pancreatic islets. BTC3 cells and pancreatic islets exhibited distinct responses to ouabain, an inhibitor of the Na+/K+ ATPase, with respect to [Ca2+]I and insulin secretion suggesting that different mechanisms controlling Na+ homeostasis exist in these B-cell preparations. Furthermore, Na+/K+ ATPase activity in BTC3 cell membranes was found to be approximately fifty percent that of primary B-cells. Gramicidin D was identified as a secretagogue in BTC3 cells with a novel mechanism of action. The ability of this ionophore to induce insulin secretion from these cells and not primary B-cells is thought to be a function of different mechanisms of Na+ homeostasis and documents a functional difference in this insulinoma cell line.Item Mechanistic Studies of the Sheep Liver 6-Phosphogluconate Dehydrogenase and cDNA Cloning(1996-07-01) Price, Nancy E.; Neeraj Agarwal; Robert Easom; Stephen R. GrantPrice, Nancy E., Mechanistic Studies of the Sheep Liver 6-Phosphogluconate Dehydrogenase and cDNA Cloning. Doctor of Philosophy (Biomedical Sciences), July, 1996, 124 pp., 5 tables, 28 Figures, 2 appendices, bibliography, 45 titles. A kinetic characterization of sheep liver 6-phosphogluconate dehydrogenase including product and dead-end inhibition patterns, primary deuterium isotope effects, and the pH dependence of kinetic parameters has been completed in order to determine the kinetic mechanism, and chemical mechanism of the enzyme. A rapid equilibrium random kinetic mechanism has been proposed, with product and dead-end inhibition patterns both being symmetric. Primary deuterium isotope effects were equal on V and V/K, confirming a rapid equilibrium mechanism, and indicate that hydride transfer is at least partially rate limiting in the overall reaction. The maximum velocity is pH dependent, decreasing at low and high pH with slopes of 1 and -1, respectively. The V/KNADP and V/K6PG also decrease at low and high pH with slopes of 1 and -1. The pH rate profiles are consistent with a general acid/general base mechanism where the catalytic residues are involved in binding. Reverse protonation states between the general acid and the general base is proposed where an unprotonated general base accepts a proton from the C-3 hydroxyl of 6PG concomitant with hydride transfer followed by decarboxylation of the resulting 3-keto intermediate to give an enediol which is protonated by the general acid to form ribulose-5-phosphate. The pH dependence of the pKi profile of the inhibitory analog 5-phosphoribonate decreases at low and high pH with slopes of 1, and -1 respectively, and suggests that intrinsic pKs are observed in the V/K profiles. The pKs of both the general base and general acid in the E:6PG complex appears to be perturbed such that the general base pK decreases slightly, and the pK of the general acid increases slightly, as a result of direct interaction with 6PG. Additionally, in preparation for site-directed mutagenesis, cDNA clones for sheep liver 6PHDH were obtained by RT-PCR.Item Molecular Basis for 2B4-CD48 Interactions(2001-08-01) Huynh, Van T.; Mathew, Porunelloor A.; Goldfarb, Ronald; Das, HridayHuynh, Van T., Molecular Basis for 2B4-CD48 Interactions. Master of Science, Molecular Biology and Immunology, August 2001, 93 pp., 3 tables, 19 illustrations, bibliography, 51 titles. Natural killer cells are lymphocytes that play a role against cancer and viral infections. 2B4 is a membrane glycoprotein expressed on natural killer cells. In the present study we characterized 2B4 from mice strains BALB/c, 129/svj and A.CA. Nucleotide and peptide analysis revealed that polymorphyic residues in 2B4 are located in the variable domain. My second project was to determine the amino acids involved in the binding between 2B4 and CD48. Twelve mutations were made in human 2B4 to disrupt their interaction. In the last part of the study, an attempt has been made to elucidate the role of tyrosine and threonine amino acids found in the novel tyrosine motifs (TxYxxI/V) that reside in the cytoplasmic domain.Item Molecular Cloning, Expression, and Regulation of the Na+/Myo-Inosiotl Cotransporter Gene(1996-08-01) Zhou, Cheng; Chaitin, Michael; Easom, Richard; Garner, MargaretZhou, Cheng, Molecular Cloning, Expression, and Regulation of the NA+/Myo-Inositol Cotransporter Gene. Doctor of Philosophy (Biomedical Sciences), August 1996. Mammalian cells respond to osmotic stress by accumulation of high concentrations of intracellular osmolytes. Osmotic-induced accumulation of the osmolyte, myo-inositol (MI), is achieved through activation of the NA+/MI cotransporter. Hypertonic stress results in elevated NA+/MI cotransporter mRNA abundance and transcription rate, and increased transporter activity. The goals of this dissertation are to establish the osmoregulation of the NA+/MI cotransporter gene expression in lens cells, and to investigate the transcriptional regulation of the NA+/MI cotransporter gene. Expression of the Na+/MI cotransporter in cultured bovine lens epithelial cells (BLECs) was demonstrated by RT-PCR amplification and Northern blot analysis. Hypertonic stress resulted in induction of the NA+/MI contransporter mRNA abundance in cultured BLECs. The induction patterns of the NA+/MI cotransporter and aldose reductase mRNA abundance by hypertonic stress indicated that osmoregulation of MI and sorbitol accumulations were regulated in concert. Accumulation of MI is an early-onset protective system, which is suppressed by the elevated sorbitol, the late-onset protective system. 5’-RACE analysis indicated that multiple transcription start sites were utilized in controlling of the expression of the NA+/MI cotransporter. Osmotic stress resulted in preferential utilization of a hypertonic promoter a. The bovine NA+/MI cotransporter gene was cloned and analyzed. The regulation of the Na+/MI cotransporter expression was investigated by transient transfection assays using promoter-luciferase constructs. Although multiple promoters were functional in cultured BLECs, only the hypertonic promoter a was osmotically responsive. Characterization of this osmotic-responsive element(s) between -536 to -300 bp upstream of the hypertonic transcription start site a. The studies presented in this dissertation refined the osmoregulation of the Na+/MI cotransporter gene expression. Hypertonicity induces MI accumulation by activation of an osmotic-responsive promoter. The consequences of the activation of this promoter lead to more cotransporter mRNA, more cotransporter protein, and higher transporter activity, resulting in accumulation of a higher concentration of intracellular Mi.Item Molecular Regulation of Interferon Gamma in 2B4-Activated Natural Killer Cells: Functional Role in Tumor Rejection(2001-11-01) Johnson, Lori Ann; Mathew, Porunellor A.; Goldfarb, Ronald H.; Dimitrijevich, S. DanNatural killer cells are a third population of lymphocytes, distinct from T and B cells. NK cells are non-MHC-restricted cytotoxic effector cells which are effective against intracellular pathogens, virally-infected cells and tumor cells. 2B4 is a natural killer cell receptor originally identified in the mouse as a surface molecule involved in non-MHC-restricted killing and enhancement of IFN-γ secretion. The human and rat homologues of 2B4 have recently been cloned in our laboratory. Interferon gamma (IFN-γ) is a cytokine with potent anti-viral and anti-proliferative effects. In addition, this cytokine acts as a global immune regulator by regulating gene expression and serving to attract other immune cells. In this work, we establish the function of human 2B4 in a NK cell line, YT. We have shown that human 2B4 activation induces cytolytic function and enhances IFN-γ release in YT cells. Additionally we show that 2B4’s regulation of IFN-γ occurs at the transcriptional level, both through mRNA stability and increased promoter activity. We also demonstrate that several regions in the IFN-γ promoter respond to 2B4 activation and IFN-γ both separately and together in the rejection of metastatictumor cells in C57B7/6 mice. Our results confirm that both 2B4 and IFN-γ are critical in the rejection of metastatic tumor cells. Through the use of activating monoclonal antibodies, our studies indicate that 2B4’s anti-tumor activity is through IFN-γ as well as through cytolytic function of NK cells.Item Molecular Regulation of Wound Contraction and Scar Formation Using a Three-Dimensional Connective Tissue Model(2002-12-01) Kern, Jami RaDel; Thomas Yorio; Robert W. Gracy; Peter B. RavenKern, Jami RaDel, Molecular Regulation of Wound Contraction and Scar Formation Using a Three-Dimensional Connective Tissue Model. Doctor of Philosophy (Biomedical Sciences, Biochemistry and Molecular Biology), December 2002, 156 pp., 1 table, 27 illustrations, references, 112 titles. The focus of these studies was to characterize a novel connective tissue model for use in experiments examining possible contraction initiators in the wound healing process, i.e. endothelin-1 (ET-1). Through these studies, it has been shown that use of a telomerized dermal fibroblast cell line addresses the concerns relating to variations due to heterogeneity of normal human cells cultured in vitro, without creating a cancerous cell line or interfering with normal phenotypic changes. In addition, the incorporation of telomerized cells into our TE, which does not spontaneously contract (US Patent #6471958), provides a unique model to study the contraction and scar formation process. Using the TE populated with hTERT fibroblasts, an innovative technique was developed to identify the initiation of tissue contraction using an optical fiber interferometry system. The process allows observation of contraction within five minutes of stimulus addition and also enables continuous data capture over a period of several hours. The greatest strength of this system is its sensitivity, since optic interferometer allows measurement of displacement (contraction) to the tens of nanometers. Along those lines, the current studies have identified ET-1 as a potential early initiator in wound healing and suggest a novel pathway through which it functions. This proposed mechanism includes both direct effects of ET-1 through the Rho-associated kinase pathway and indirect effects potentiated by TGF-β. Future studies addressing whether TGF-β converges on the Rho-associated kinase pathway or acts independently through other signaling mechanisms should be initiated. The discovery of early initiators of tissue contraction is essential in the identification of potential therapeutic targets in the quest to reduce prolonged and severe tissue contracture and scaring.