Browsing by Subject "Cellular and Molecular Physiology"
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Item [3H] Ethynylbicycloorthobenzoate ([3H] EBOB) Binding in Native and Recombinant GABAA Receptors(2000-05-01) Yagle, Monica A.; Dillon, Glenn; Martin, Michael; de Fiebre, ChristopherYagle, Monica A., [3H] Ethynylbicycloorthobenzoate ([3H] EBOB) Binding in Native and Recombinant GABAA Receptors. Master of Science (Pharmacology), May 2000, 59 pp., 3 tables, 7 illustrations, bibliography, 75 titles. Modulation of the GABAA receptor has been studied with noncompetitive convulsant ligands such as tert-butylbicyclophosphorothionate (TBPS) and picrotoxin (PTX). EBOB is a more recently developed ligand that appears to bind in the same region of the channel at TBPS, but with a higher affinity. While only a few studies have examined the binding of EBOB to vertebrate brain tissue and insect preparations, none have examined potential subunit-dependent binding of EBOB. We have thus examined [3H] EBOB binding in rat cerebellum and HEK293 cells stably expressing human α1β2γ2, human α2β2γ2, and rat α6β2γ2 GABAA receptors. For comparison, [35S] TBPS binding was also examined in α1β2γ2 receptors. Saturation and Scatchard analyses revealed saturable [3H] EBOB binding at one site in all tissue preparations with Kd values ranging from 3 to 9nM. [3H] EBOB binding, like [35S] TBPS binding was inhibited by the CNS convulsants dieldrin, lindane, tert-butylbicyclophosphorothionate (TBOB), PTX, TBPS, and pentylenetetrazole (PTZ) at one site in a concentration dependent fashion. Affinities were in the high nM to low μM range for all compounds except PTZ (low mM range). GABA modulated [3H] EBOB binding in a biphasic manner in α1β2γ2 receptors with a 100-fold difference between stimulatory and inhibitory affinities. Inhibition of GABA-mediated current by TBOB in α1β2γ2 receptors resulted in a functional IC50 of 0.2 μM, in agreement with binding study results. Differences seen in binding between the different receptor subtypes examined suggest that some characteristics of EBOB binding are subunit dependent. In addition, we have shown that [3H] EBOB is a useful ligand in the study of recombinant GABAA receptors and that results obtained with [3H] EBOB are comparable to those obtained with [35S] TBPS.Item A Calcium-Dependent Nuclear Signaling Pathway Transcriptionally Silences Atrial Natriuretic Factor Gene Expression(1995-08-01) Zeng, Hong; Stephen R. Grant; Walter McConathy; Richard EasomZeng, Hong, A Calcium-Dependent Nuclear Signaling Pathway Transcriptionally Silences Atrial Natriuretic Factor Gene Expression. Master of Science (Biomedical Science), August, 1995, 85 pp., 2 tables, 20 illustrations, bibliography, 90 titles. A cultured myocardial cell model was used to examine a potential role of calcium-dependent protein kinases and phosphatases in regulating the induction of the atrial natriuretic factor (ANF) gene mediated through adrenoreceptor signaling. In primary culture, rat neonate cardiomyocytes supplemented with phenylephrine (PE) following transfection (24 h) with a full length ANF promoter-reporter construct, showed elevated levels of promoter activity when compared to transfected cardiomyocytes cultured in the absence of PE. Prazosin, a dedicated α1-antagonist, completely blocked the transcriptional induction mediated through PE stimulation. Two different calcium mobilizing agents, BAY K8644 and gramicidin D, significantly reduced PE-stimulated ANF promoter activity. The over-expression of co-transfected exogenous CaM kinase II isoforms resulted in transcriptional silencing of PE-induced promoter activity for cardiac ANF. Transfection of a constitutively active, mutant form of the calcium-dependent phosphatase 2B, calcineurin, gene also transcriptionally silenced ANF gene expression. Exposure of PE-induced cardiomyocytes to either FK-506-treated cells in the absence of PE exposure suggesting that transcriptional silencing may be mediated through a transcriptional repression mechanism. Taken together, these results suggest that the activation of a Ca2+-dependent nuclear signaling pathway mediated through either CaM kinase II or calcineurin leads to complete transcriptional silencing of the embryonic ANF gene expression.Item A Clinical Research Study Involving the Use of Erythropoietin in Perioperative Patients Undergoing Surgery for Gynecologic Cancer(2002-07-01) Larson, Sharon Beth; Richardson, Barbara; Martin, MichaelThe purpose of this internship practicum report is to analyze the pathophysiology and impact of anemia in low-income gynecologic cancer patients. The report also assesses the impact of erythropoietin on hemoglobin levels prior to gynecologic cancer surgery. This report is based on a clinical research study to determine whether or not erythropoietin will mitigate the suppression of bone marrow inherent to the gynecologic cancer population and alleviate some of the symptoms and side effects of the anemia.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 Alterations in mRNA Levels of Selected Gene Products During Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells(2001-08-01) Vopat, Kelly S.; Agarwal, Neeraj; Wordinger, Robert J.; Pang, Iok-HouVopat, K., Alterations in mRNA Levels of Selected Gene Products during Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells. Master of Science (Biomedical Science), August 2001. 54 pp., 2 tables, 10 illustrations, bibliography, 105 titles. In order to explore the mechanisms involved in the signal transduction pathways of ischemia-induced apoptosis of RGCs in glaucoma, an in vitro ischmia model of transformed rat retinal ganglion cells (RGC-5) was utilized. RGC-5 cells were exposed to hypoglycemia, hypoxia, and ischemia for six hours. Hypoxia and ischemia resulted in apoptosis of RGC-5 cells as determined by TUNEL assay. The bax mRNA levels increased significantly in cells exposed to hypoxia. The mRNA levels of hemoxygenase, c-fos HSP 70, and BDNF showed a trend of increase in both the hypoxic and ischemic conditions. These results demonstrate that retinal ganglion cells undergo apoptosis in hypoxic conditions likely via an increase in bax/bcl-2. The up-regulation of BDNF and some stress proteins may be part of a cellular rescue effort trying to overcome the damage created by hypoxic and ischemic stresses.Item Anatomical and Biochemical Characterization of the Porcine Spinal Arachnoid(1994-06-01) Taylor, Martin T.; Turner, James; Yorio, Thomas; Wordinger, Robert J.Taylor, Martin T., Anatomical and Biochemical Characterization of the Porcine Spinal Arachnoid. Doctor of Philosophy (Biomedical Sciences), June, 1994. Mast cell products modulate many biologic barrier systems. In the central nervous system (CNS) two such barriers are the blood-brain barrier (B-B-B) and its analogous cerebrospinal fluid-blood barrier (CSF-B-B). Published research has demonstrated that mast cell products increase the permeability of the BBB, but no comparable research has been described for the CSF-B-B. The main goal of this dissertation was to begin to assess the role of mast cell products on the chief component of the CSF-B-B, the arachnoid mater. Specifically, the hypothesis that mast cell products modulate arachnoid function through receptor mediated second messenger system regulation was postulated. Initially, the light and electron microscopic features of the porcine spinal meninges generally and the arachnoid mater specifically were characterized and found to be similar to those of other species and CNS regions. In addition, mast cells were found to be present in the meninges where their product could affect the arachnoid mater. To study the effects of the selected mast products on the arachnoid mater, arachnoid cells were isolated and cultured. Morphologic, immunohistochemical, and physiological studies confirmed the cultured cells were arachnid cells and that they were capable of developing attributes of a barrier membrane in vitro, (e.g. tight junctions, increased transcellular resistance). The effects of mast cell products on arachnoid cells were then assessed biochemically. Arachnoid cells were found to produce cyclic adenosine monophosphate (cAMP) in response to forskolin and prostaglandin D2 (PGD2). Histamine inhibited both forskolin and PGD2 stimulated production of cAMP. Additionally, arachnoid cells produced inositol phosphates (IP) in response to carbachol and histamine via muscarinic and H1-histamine receptors respectively. Since histamine and PGD2 are produced and released by activated mast cells, and since cAMP and IP levels are known to modulate cellular barrier systems, it is concluded that meningeal mast cells and their products may regulate or modulate permeability of the CSF-B-B. An understanding of the specific biochemical actions of mast cell products on the arachnoid may ultimately aid in the understanding of many physiologic and pathologic processes involving the arachnoid such as hydrocephalus, subarachnoid and subdural hemorrhages, cerebral edema, meningitis, and meningiomas.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 Automodification Reaction of PARP-1 Reversibly Regulates the DNA-Binding of NF-kB(2001-11-01) Chang, Woo-Jin; Alvarez, Rafael; Mathew, Porunelloor A.; Goldfarb, Ronald H.Chang, Woo-Jin, Automodification Reaction of PARP-1 Reversibly Regulates the DNA-Binding of NF-kB, Doctor of Philosophy (Microbiology and Immunology), November, 2001, 92 Pages, 20 figures, 3 schemes, and bibliography. Poly(ADP-ribose) polymerase (PARP-1, E.C. 2.4.2.30) is a constitutively expressed nuclear enzyme. It comprises about 1% of the total nuclear protein and in phylogenetically well conserved in most eukaryotes, with a notable exception in yeast. PARP-1 post transitionally modifies DNA-binding proteins by transferring the ADP-ribose moiety from BNAD+. Although the exact biological function of poly(ADP-ribosyl)ation has not been clearly elucidated, the process is thought to be involved in DNA repair, replication, and gene expression. Previous studies have indicated that PARP-1 participates in eukaryotic gene expression including the genes under the control of nuclear factor-kB (NF-kB). It has been demonstrated that PARP-1 deficient mice are more resistant to lipopolysaccharide-induced endotoxic shock than isogenic wild-type mice due to the inactivation of NP-kB in the mutants. In order to further analyze the interactions between PARP-1, NF-kB, and its consensus DNA in a cell-free system, we co-incubated recombinant PARP-1 protein and the p50-subunit of NF-kB (NF-kB-p50) in the absence of DNA strand-breaks. Electrophoretic mobility shift assays (EMSA) showed that sequence-specific DNA-binding of NF-kB-p50 was dependent on autopoly(ADP-ribosyl)ation of PARP-1. The NF-kB-p50 DNA-binding was inhibitied when PARP-1 was not auto-poly(ADP-ribosyl)ated either in the absence of BNAD+ or in the presence of 3-aminobenzamide, an enzymatic inhibitor of PARP-1. Coimmunoprecipation and immunoblot analysis demonstrated that NF-kB-p50 formed a heterodimer with PARP-1 when PARP-1 was not auto-poly(ADP-ribosyl)ated. In addition, poly(ADP-ribosyl)ation assays showed that NF-kB-p50 protein was not susceptible to poly(ADP-ribosyl)ation under normal incubation conditions. Those in vitro observations described above were confirmed by experiments utilizing HeLa nuclear extracts. EMSA showed that NF-kB DNA-binding was inhibited in 3-AB-pre-treated HeLa cells. To our knowledge, this is the first report demonstrating that auto-poly(ADP-ribosyl)ation reaction by PARP-1 reversibly regulates the function of a transcription factor by inhibiting the formation of heterodimer between PARP-1 and a transcription factor.Item Brain Derived Neurotrophic Factor Regulates Müller Cell Survival via MAPK and PI3K Pathways(2003-05-01) Taylor, Sara A.; Agarwal, Neeraj; Wordinger, Robert J.; Pang, Iok-HouTaylor, Sara A., Brain Derived Neurotrophic Factor Regulates Müller Cell Survival via MAPK and PI3K Pathways. Master of Science (Biomedical Sciences), January, 2003, 112 pp., 4 tables, 39 illustrations, bibliography, 68 titles. Purpose: Glutamate has been implicated in many pathologies affecting the Central Nervous System including those in the retina, but the exact nature of the role of glutamate in neuronal degeneration remains unclear. In the retina. Müller cells are resistant to glutamate insults that are normally toxic to other cells of the retina, however the molecular and biochemical mechanisms that control their death or survival are not well understood. We used a series of pharmacological inhibitors and molecular biology agents on cultured Müller cells to dissect two key signaling pathways normally involved in cell survival, the Mitogen Activated Protein Kinase – Extracellularly Regulated Kinase (MAPK(ERK) pathway and the Phosphatidylinositide 3 Kinase (PI3K) pathway. Since preliminary data in our laboratory showed that Müller cells upregulate their secretion of neurotrophins including Brain Derived Growth Factor (BDNF) in response to glutamate treatment, we also examined the effect of BDNF on the activation of these two signaling pathways. Methods: Early passaged Müller cells were treated with various concentrations (5 nM -50 μM) of inhibitions of the MAPK(ERK) pathway (GW5074, U0126, and PD98059) or with various concentrations (1-50 μM) of inhibitors of the PI3K pathway (LY294002 or Akt inhibitor) in the presence and absence of 50 ng/ml of BDNF for 24 hours. These experiments were repeated in Müller cells transfected with either NFκB or Bc12 DNA. Cell cultures were then analyzed for surviving cells with an MTS/PMS assay, a colorametric method for determining the number of viable cells in a proliferation assay. Results: The MAPK (ERK) inhibitors PD98059 and GW5074 both resulted in decrease in Müller cell survival. PD98059 did not decrease Müller cell survival until concentrations were high enough to suppress ERK2 phosphorylation. Müller cells transfected with NFκB or Bc12 DNA were able to resist treatment with concentrations of PD98059 that reduced cell number in untransfected cells. The PI3K inhibitor LY294002 also resulted in significant decreases in Müller cell survival in both untransfected cells and cells transfected with NFκB or Bc12 DNA. Treatment with an inhibitor farther down in the PI3K pathway, Akt inhibitor, did not significantly decrease Müller cell survival. Finally, BDNF was not able to increase cell survival in Müller cells treated with PD98059 or U0126, although it did increase the survival of cells treated wit GW5074. BDNF was also able to reverse the decrease in cell survival caused by LY294002 in both untransfected Müller cells or Müller cells transfected with NFκB or Bc12 DNA. Conclusions: Our data shows that Mitogen Activated Protein Kinase – Extracellularly Regulated Kinase (MAPK(ERK) and Phosphatidylinositide 3 Kinase (PI3K) are both essential for Müller cell survival. There is modulation between the pathways and they may interconnected far upstream at a protein previously associated with only the MAPK(ERK) pathway. These results are consistent with a role for both pathways in Müller cell survival.Item Cellular Mechanisms in the Ocular Actions of Endothelin(1996-12-01) White, Karen A.; Yorio, Thomas; Pang, Iok-Hou; Dobbs, RichardWhite, Karen A., Cellular Mechanisms in the Ocular Actions of Endothelin. Doctor of Philosophy (Biomedical Sciences/Pharmacology), December, 1996, 151 pp., 25 tables, 23 figures, references, 111 titles. Endothelins are a family of regulatory peptides which could have important implications in this regulation of aqueous humor outflow and intraocular pressure (IOP). The objectives of this dissertation were to investigate the cellular mechanism of endothelin (ET) receptor interactions in ocular tissues focusing on their effect on second messengers such as phospholipase C (PLC) and calcium, and their interactions with phospholipase A2 (PLA2) in ciliary muscle cells. The hypothesis was that in human ciliary muscle (HCM) cells, endothelin-1 (ET-1), via the ETA receptor and a pertussis toxin sensitive G-protein, activates PLC, which in turn stimulates calcium mobilization. Independent of this pathway, ET-1 also activates PLA2 and increases the release of prostaglandins. These two pathways provide a cellular second messenger balance that influences ciliary smooth muscle contraction. The current study demonstrated that ET-1 and endothelin-2 (ET-2) stimulate calcium mobilization in HCM cells via an ETA receptor subtype. It appears that the increase in intracellular calcium ([Ca2+]i) is the result of ET coupled to PLC via a pertussis toxin sensitive G-protein. A biphasic calcium response is elicited with ET stimulation consisting of a transient increase in [Ca2+]I which appears to be primarily due to release of intracellular stores, followed by a lower sustained phase which appears to be dependent on the influx of extracellular calcium. Endothelin-1 also appears to stimulate an increase in prostaglandin E2 (PGE2) formation through activation of PLA2. Furthermore, it appears that the effects of ET-1 on PLC and calcium are independent of the ET-1 effects on PGE2 production, such that the ET-1 induced increase in [Ca2+]I are coupled to the PLC signaling pathway, whereas increase in PGE2 production appears to be the result of an ETA receptor coupled PLA2. Whether there are different subtypes of ETA receptors or the receptor is coupled through different G-proteins is uncertain. Endothelin-1 and Big ET-1 immunoreactivity was also observed in both HCM and human nonpigmented ciliary epithelial (HNPE) cells. This is the first time that ET-1 and Big ET-1 immunoreactivity has been detected in the HCM cells, suggesting that these cells have the capability to synthesize both peptides. Furthermore, the increase in ET-1 and Big ET-1 immunoreactivity upon stimulation with TNF-α suggests that cytokines may be important regulators of ET synthesis and release. The findings of this research aid in the understanding of the mechanism of action whereby ETs regulate aqueous humor dynamics and IOP. Through a better understanding of the cellular actions of ET, insight is gained into the development of new ocular selective agents acting at the ET receptor.Item Changes in Mammalian Chromatin Structure as a Function of Protein-Poly(ADP-Ribosyl)ation by Endonuclease Digestion(2004-06-01) Perez-Lamigueiro, Maria A.; Alvarez, Rafael; Das, Hriday K.; Basu, AlakanandaPerez-Lamiguerio, Maria A., Changes in Mammalian Chromatin Structure as a Function of Protein-poly(ADP-ribosyl)ation by Endonuclease Digestion. Master of Science (Biochemistry and Molecular Biology), June 2004. 66 pages, 12 illustrations, Bibliography, 45 titles. Mammalian chromatin was exposed to either Deoxyribonuclease I or Micrococcal Nuclease digestion as a function of time of incubation and enzyme concentration. Endonuclease enzymatic reactions were stopped with EDTA. Samples were run in 1.5% agarose gels and the oligonucleosomal electrophoretic migration patterns compared. Endonuclease experiments were carried out with rat liver chromatin pre-incubated in the presence or absence of 200 μM βNAD+. A solution of 1.0 mM benzamide was used to stop enzymatic modification. The electrophoretic observations demonstrated a faster and increased degradation of chromatin when proteins were poly(ADP-ribosyl)ated prior to digestion. These results support the hypothesis that that the covalent poly(ADP-ribosyl)ation of chromatin proteins, particularly histones, induces a more relaxed structure, rendering chromatin more sensitive to endonuclease digestion.Item Characterization of a Novel Extracellular Superoxide Dismutase Allele Discovered in Mouse Models of Atherosclerosis(2004-07-01) Pierce, Anson; Dory, Lad; Easom, Robert; Basu, AlakanandaAnson Pierce, Characterization of a Novel Extracellular Superoxide Dismutase Allele Discovered in Mouse Models of Atherosclerosis. Doctor of Philosophy (Biochemistry and Molecular Biology), July 2004, 128 pp., 3 tables, 22 illustrations, references, 230 titles. Many diseases display some involvement with oxidative mechanisms and could potentially benefit from antioxidant therapy designed to restore the balance between reductive and oxidative factors. Data presented in this dissertation explore and establish the protective effect hyperbaric oxygen (HBO) has on the development of atherosclerosis, an oxidation-driven inflammatory disease mediated through low-density lipoproteins in the vasculature. Atherosclerosis in the apolipoprotein E-/- (apoE-/-) mouse is drastically reduced after 10 weeks of HBO treatment. Macrophages in HBO treated mice have an increased antioxidant capacity and reduced ability to generate oxidants. From this work, a new polymorphism of a key antioxidant enzyme, extracellular superoxide dismutase (ecSOD), is identified and characterized in mice. The new polymorphism is termed the “short” allele, and has the potential to alter the regulation of ecSOD mRNA and protein, as well as enzyme activity. Examination of its effect on the ecSOD phenotype in mice shows dramatic changes in enzyme levels and activity. In the plasma compartment ecSOD activity and mass are elevated, and indicate based on heparin injection studies that a change in ecSOD distribution results in tissues of mice expressing the short allele. Systematic examination of ecSOD in tissues of mice shows that its distribution is altered such that it is more accessible to heparin; this is most evident in the liver and kidney of mice expressing the short allele. The finding that HBO is protective against atherosclerosis highlights a potentially promising approach to treatment for this devastating disease, sheds light on the role oxidative processes play in atherosclerosis, and identifies potential targets for antioxidant therapy. This study also shows for the first time that two alleles for a major antioxidant enzyme exist in mice that display markedly different effects on the ecSOD phenotype, a finding that underlines the importance of genetic homogeneity in mouse models and adds to our knowledge concerning the role antioxidants play in human health and disease.Item Characterization of IL-17A-Secreting CD8 T Cells(2008-12-01) Lee, Suheung; Harlan Jones; Porunellor Mathew; Peter KoulenIL-17A-secreting CD4 T cells (Th17 cells) have been demonstrated to play pivotal roles in modulating immune responses during various types to infectious and autoimmune diseases. While 1L-17A secreting CD8 T cells have been detected in numerous disease models, much less is known about them. In this thesis, the differentiation conditions and effector functions of IL-17A-secreting CD8 T cells have been examined. In order to differentiate naïve CD8 T cells into IL-17A secretors, TGF-β, IL-6, and neutralization of IFN-γ are required as in Th17 cells. IL-17A-secreting CD8 T cells produce the effector cytokines, IL-17A, IL-17F and IL-22, but do not produce granzyme B, implicating the lack of cytotoxicity. Furthermore, IL-17A-secreting CD8 T cells can respond to exogenous cytokines without a cognate antigen, suggesting that they can act in an innate fashion. Collectively, IL-17A-secreting CD8 T cells possess the same effector functions as Th17 cells, and thus may play as significant roles in various diseases at Th17 cells.Item Characterization of Protein Kinase C in Cisplatin Sensitive and Resistant Human Cervical Cancer HeLa Cells(2000-12-01) Mohanty, Sanghamitra; Basu, Alakananda; Simecka, Jerry; Dimitrijevich, DanMohanty, S., Characterization of protein kinase C in cisplatin sensitive and resistant human cervical cancer HeLa cells. Master of Science (Microbiology and Immunology), December, 2000. 37 pp., 11 illustrations, bibliography, 27 titles. Signal transduction plays a crucial role in carcinogenesis. A defect in signaling, by evading cell death or promoting cell proliferation, may result in neoplastic transformation or protection of cells from the cytotoxicity of anticancer drugs. Therefore, in order to understand the complex mechanism of drug resistance, it is relevant to probe into the important signal transduction pathways. Protein kinase C, a key signal transducer, influences cisplatin sensitivity in many cell lines. We examined whether or not the PKC signal transduction pathway is affected during development of resistance to cisplatin by tumor cells. PKC activators increased cisplatin sensitivity in both parental and cisplatin-resistant cells. Western blot analysis showed a slight decrease in cPKCα and nPKCε, an evaluation in nPKCδ and no change in the abundance of PKCϚ in HeLa/CP cells compared to HeLa cells. Though TPA-induced translocation of PKC isoforms was identical in both cell lines, down regulation of PKCδ was defective in resistant cells. Therefore, a deregulation in PKCδ was associated with cisplatin resistance.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 Characterization of the Bradykinn Receptor in Human Corneal Epithelium(1997-08-01) Wiernas, Terry Kirkham; Michael W. Martin; Glenn Dillon; Michael ForsterWiernas, Terry Kirkham, Characterization of the Bradykinin Receptor in Human Corneal Epithelium. Doctor of Philosophy (Biomedical Sciences), August, 1997, 255 pp., 5 tables, 39 figures, references, 137 titles. Bradykinin (BK) is a well-established mediator of inflammation. High levels of BK in human tears following ocular allergic provocation led to the hypothesis that BK receptors may exist on the corneal epithelium and could play a role in corneal inflammation and/or wound healing, in addition to other functions. To test this hypothesis, human corneal epithelial cells were cultured and used to conduct a series of studies to evaluate and characterize the BK receptor. Due to the limited supply and high cost of primary human corneal epithelial (P-CEPI) cells, in addition to the fact that these cells do not divide and proliferate over more than a few passages, SV40 virus-immortalized human CEPI cells (CEPI-17-CL4) were used as a model system. Extensive studies confirmed that the immortalized cells faithfully represented the primary cells. This study demonstrated the presence of BK receptors on corneal epithelial cells for the first time. The receptors were characterized as the B2 subtype and were found to be represented by an apparent single binding site. Furthermore, stimulation of these receptors was found to elicit concentration-dependent increases in both inositol phosphates, via activation of phospholipase C, and intracellular calcium mobilization. The rank order affinity of BK and its analogs as determined by binding assays was found to correlate well with the rank order potency of BK and its analogs in evoking the latter functional responses, which were blocked by two B2-receptor selective antagonists. A significant, concentration-dependent stimulation of [3H]thymidine uptake in CEPI cell DNA was elicited by BK which suggests a potential mitogenic effect of BK and a role in corneal wound healing. BK did not significantly affect the release of three pro-inflammatory cytokines, prostaglandin E2 or matrix metalloproteinase-1, and seemed to have an inhibitory effect on the release of tumor necrosis factor α. In conclusion, these studies have confirmed that CEPI-17-CL4 cells represent a good in vitro model of human corneal epithelium and have contributed to a better understanding of the ocular effects of BK and characterization of its receptor within the cornea.Item Characterization of the Myo-inositol Efflux Pathway in Cultured Bovine Lens Epithelial Cells(1997-12-01) Reeves, Rustin E.; Victoria Rudick; Robert Easom; Thomas YorioReeves, Rustin E., Characterization of the Myo-inositol Efflux Pathway in Cultured Bovine Lens Epithelial Cells. Doctor of Philosophy (Biomedical Sciences), December, 1997, 173 pp., 1 table, 28 figures, bibliography, 94 titles. The basic cellular requirement of volume regulation utilizes many different channel and transport pathways working on concord to maintain a constant cell volume. Among these are distinct pathways spontaneously activated by changes in cell volume that modulate the gain or loss of certain organic osmolytes, such as myo-inositol (MI). The major goal of this dissertation is to characterize and identify the mechanism involved in the MI effux pathway and explore its relationship with intracellular polyol accumulation in cultured bovine lens epithelial cells (BLECs). Hypertonic exposure of BLECs causes an increase in MI uptake and aldose reductase enzymatic activity, two events which ultimately influence osmolyte efflux. A biphasic efflux pathway induced by rapid cell swelling (hypotonic-induction) was demonstrated in BLECS switched from hypertonice to physiologic medium. Also, intracellular polyol accumulation from galactose exposure resulted in enhanced activation of the MI efflux pathway (polyol-induction). Chloride channel inhibitors effectively blocked MI efflux suggesting a relationship between anion (chloride) movement and intracellular MI loss from cell to medium. Expression of a chloride channel regulatory protein, pICln, was demonstrated by Northern blot analysis in cultured BLECs. Hypertonic exposure upregulates the expression of pICln mRNA while hypotonicity downregulates expression. The volume-sensitivity for transcription of PICln mRNA in BLECs lends strong support for its role in both anion and osmolyte loss associated with the MI efflux pathway. The MI efflux pathway functions as a “relief value” in cell volume regulation by providing a conduit to alleviate intracellular osmotic stress. The mechanism which evolved to function under normal cellular circumstances in relief of excessive accumulation of intracellular osmolytes (i.e. polyols), may, by design, inadvertently promote the loss of essential intracellular volume and nonvolume regulatory organic solutes. Ironically, under certain pathological conditions, this mechanism, designed to protect the cell from intracellular osmotic stress, may instead be detrimental to the cell by promoting the excessive loss of osmolytes essential for normal cell function.Item Conformational Properties of Circulating and Recombinant Forms of Human Plasma Lecithin Cholesterol ACYL Transferase(1995-06-01) Sundarrajan, Geetha; Walter McConathySundarrajan, Geetha, Conformational properties of circulating and recombinant forms of human plasma LCAT Master of Science (Biomedical Sciences), June, 1995, 69pp., 3 tables, 18 figures, 47 references. The relationship between enzymatic activity and conformational properties of the circulating and recombinant forms of human plasma LCAT were examined in the native and denatured states. The two denaturing agents used in this study were guanidine hydrochloride and heat. These studies led to the following conclusions: (1) Although the alpha helical content of desialylated recombinant LCAT (d-LCAT) is comparable to that of the other two forms of the enzyme (p-LCAT and r-LCAT), the desialylation of LCAT is associated with an increase in the beta sheet and a decrease in beta turn content. (2) The presence of sialic acids, in addition, seems to influence the local environments of aromatic amino acid residues. (3) From the denaturation and renaturation studies with guanidine hydrochloride and heat, it appears that the N-glycan structures of p-PCAT and r-LCAT may contribute differentially to the conformational stability of the enzyme. (4) The alpha helical structure of LCAT may not be involved in maintaining the active conformation of the enzyme.