Electronic Theses and Dissertations

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Mechanisms of methylenedianiline toxicity to rat biliary epithelial cells
(2001-05-06) Vincente Santa Cruz; Mary F. Kanz, Ph.D.; Ronald A Faris, Ph.D.; Mary Treinen Moslen; Kenneth S. Ramos, Ph.D.; Guillermo A. Altenberg, M.D., Ph.D.; Gerald A Campbell, M.D., Ph.D.
Methylenedianiline (4,4’-diaminodiphenylmethane, DAPM), a compound used to produce polyurethanes and epoxy resins, rapidly injures biliary epithelial cells (BEC) of rats in vivo. DAPM also increases biliary inorganic phosphate and glucose, suggesting that DAPM loosens hepatic tight junctions. Early ultrastructural alterations in BEC mitochondria, however, suggested a possible role of mitochondrial dysfunction in the ability of BEC to maintain glucose transport and tight junction integrity. The working hypothesis for this dissertation project was that DAPM alters tight junction integrity and/or glucose absorption by mechanisms involving mitochondrial injury. The proposed aims were to: 1). Assess tight junction integrity in vivo using a “sleeping” rat model and radioactive, non-electrolyte tracers. 2). Develop an in vitro model of DAPM injury using primary cultures of polarized rat BEC. 3). Determine if BEC tight junction integrity is altered by DAPM/ metabolite(s) in vitro using both electrophysiological and conventional tracer methods. 4). Determine if DAPM/ metabolite(s) induce mitochondrial dysfunction in BEC by assessing cellular ATP levels and mitochondrial membrane potentials. 5). Determine if BEC glucose uptake is altered following exposure to analog, alpha-methyl-D-glucopyranoside. Numerous in vivo and in vitro methods were combined to develop a model of polarized rat BEC monolayers that were assessed for changes in TJ integrity, glucose uptake and mitochondrial function after exposure to bile from untreated rats, rats treated with vehicle, or rats treated with DAPM. This model confirmed prior studies that demonstrated increased hepatobiliary tight junction permeability following DAPM treatment in vivo. Tight junctions between BEC in vitro showed both increased “leakiness” and decreased ion selectivity following exposure to bile from DAPM-treaded rats (DAPM-Bile). Furthermore, this model indicated that BEC ATP levels and mitochondrial membrane potentials were altered prior to any changes in tight junction integrity and glucose absorption. These data support the working hypothesis that mitochondria are the initial site of DAPM injury in BEC and that mitochondrial dysfunction may indirectly impair BEC glucose uptake and tight junction integrity.
Molecular characterization of cation-coupled transporters: the H+-coupled Mg2+-citrate transporter, CitM, and the Na+/sulfate cotransporter, hNaSi-1
(2003-01-28) Hongyan Li; Ana M. Pajor; Steven C. King; Steven A. Weinman; Luis Reuss; Joel P. Gallagher
In this dissertation, two cation-coupled transporters were characterized at the molecular level. The CitM transporter from Bacillus subtilis was functionally expressed and characterized in E.coli cells. The human NaSi-1 transporter (hNaSi-1) and mutants were functionally expressed in Xenopus oocytes. Antibodies against hNaSi-1 were used to investigate tissue distribution and N-glycosylation. The roles of two conserved serine residues in the transport function of hNaSi-1 were investigated using site-directed mutagenesis and radiotracer assay. \r\n\r\n CitM belongs to a distinct gene family of secondary active transporters that includes the homologous citrate transporter CitH. In this dissertation, the Km of CitM for the complex of Mg2+-citrate was about 300 mM in the presence of saturating Mg2+ concentrations. CitM has a high substrate specificity for citrate. Other tested di- and tricarboxylic acids did not significantly inhibit citrate uptakes in the presence of Mg2+. However, CitM accepts complexes of citrate with metal ions other than Mg2+. The transport was inhibited in more alkaline but not in acidic transport buffer and also inhibited by ionophores that affect the transmembrane proton gradient, including FCCP, TCC and nigericin, suggesting a proton-coupled transport. Valinomycin did not affect the uptake by CitM, supporting an electroneutral transport model in which one proton is coupled to the uptake of one complex of (Mg2+-citrate)1-. \r\n\r\nThe low affinity Na+/sulfate cotransporter, hNaSi-1, belongs to a specific gene family of Na+-coupled transporters that includes the high affinity hSUT-1 and the Na+-coupled dicarboxylate (NaDC) transporters. Antibodies directed against a peptide of hNaSi-1 recognized the native protein in renal membranes as well as the recombinant protein expressed in Xenopus oocytes. There is a single N-glycosylation site, Asn-591, located at the extracellular C-terminus in hNaSi-1. Site-directed mutagenesis studies of Ser-260, Ser-288 and the surrounding amino acid residues of hNaSi-1 suggested that these residues are functionally required for hNaSi-1. MTSET inhibition on sulfate uptakes by the four mutants surrounding Ser-260, T257C, T259C, T261C and L263C, was dependent on the cation and substrate used. Since the presence of sodium and sulfate triggers conformational changes during the transport cycle of hNaSi-1, the cation and substrate dependence of MTSET inhibition suggest that these four substituted cysteines move during the transport cycle. Since the four mutated residues are located in TMD-5, this transmembrane domain is also likely to participate in the conformational movement during the transport cycle of hNaSi-1. \r\n
Parasite interactions with dendritic cells and macrophages: Implications for cutaneous Leishmaniasis caused by Leishmania amazonensis
(2003-03-26) Hai Qi; Lynn Soong; Vivian L. Braciale; Rolf Konig; Joseph M. Vinetz; David M. Mosser; Barbara L. Doughty
Protozoan Leishmania is an important human pathogen that affects millions people worldwide. Investigation of experimental Leishmania infection in mice has been instrumental to our understanding of interactions between the parasite and the host immune system. Previous studies have established the model of Th1-Th2 paradigm: gamma interferon (IFN-g)-secreting Th1 cells protect the host from developing progressive diseases, while interleukin (IL)-4-producing Th2 cells drive the disease pathogenesis. Focused on L. amazonensis infection in mice, this dissertation study is mainly intended to understand the cellular mechanism underlying the generation of parasite-specific Th2 cells and to ascertain the role for IFN-g in parasite-macrophage interactions. We showed that L. amazonensis parasites infected and activated dendritic cells (DCs), a population of phagocytic antigen-presenting cells specialized in activating naïve T cells. We found that DCs from susceptible or resistant mice differentially responded to amastigotes in CD40-dependent cytokine production and that amastigote-infected DCs favor Th2 priming in susceptible but not resistant mice. IFN-g is believed to be crucial for activating macrophages to kill intracellular parasites such as L. major. However, we found that L. amazonensis amastigotes but not promastigotes could not only survive but also replicate better in IFN-g-activated macrophages. The promastigote was evidently killed in IFN-g-activated macrophages. On the other hand, macrophages activated with IFN-g and LPS were able to limit intracellular amastigote replication. When tested in vivo, endogenous IFN-g apparently exerted minimal effects on the course of amastigote infection. It is likely that IFN-g plays a bidirectional role during L. amazonensis infection: when optimally coupled with other factors, it can activate macrophages to control parasite infection; while in the absence of such synergy, it would promote amastigote propagation by itself. Collectively, results presented in this dissertation have pointed to the unique ability of L. amazonensis amastigotes to modulate host immune system to the advantage of their own survival.
Effects of light-emitting diode photostimulation on burn wound healing
(2003-06-23) Mimi Leong; Lisa J. Gould; Massoud Motamedi; Linda G. Phillips; Hal K. Hawkins; Gregory Asimakis
Annually, more than 1.2 million persons in the United States require medical care for burns. Healing of deep burn wounds requires restored perfusion and neoangiogenesis to reestablish blood flow and limit ischemic damage.\r\nWe propose that LED photostimulation, by inducing macrophage proliferation and secretion of pro-angiogenic factors, will restore perfusion by increasing angiogenesis. \r\nAn in vitro inflammatory model and in vivo rodent thermal injury model were treated with LED at 670nm, 730nm, 880nm, or combination-670nm/730nm/880nm. Conditioned media were analyzed for VEGF and NO. Excised burn wounds underwent measurement of surface area, tensile strength, VEGF, nitrites, and immunohistochemical markers (iNOS, VEGF, cyclooxygenase-2, Factor VIII, ED-1) on days 3, 7, and 14.\r\nBoth in vitro and in vivo findings demonstrate that LED therapy has vulnerary effects on angiogenesis, by affecting macrophage production of VEGF and NO. These effects are wavelength and fluence-dependent.\r\n
Microvascular endothelial response to cocaethylene exposure: morphological and molecular observations
(2004-11-02) Danyel Hermes Tacker; Anthony O. Okorodudu, Ph.D.; Robert H. Glew, Ph.D.; Norbert K. Herzog, Ph.D.; M. Tarek Elghetany, M.D.; Kathryn A. Cunningham, Ph.D.; Hal K. Hawkins, M.D., Ph.D.
Cocaethylene (CE) is an active metabolite of cocaine and ethanol and is a toxicant of physiological relevance due to the high rate of cocaine and ethanol co-exposure (~80%) in cocaine abusers. It has prolonged action and increased potency on known physiological targets relative to the effect of cocaine. Since pathology in cocaine abusers is typically chronic and systemic, and CE persists in the body three to five times longer than cocaine, a link between CE and systemic disease in cocaine abusers was proposed. Consequently, this dissertation contains the studies that were used to test the hypothesis that the microvascular endothelium is a target tissue that is central in the pathogenic mechanism of cocaine-associated systemic disease, and that endothelial injury after CE exposure would result in dysregulation and altered barrier function due to changes in intracellular second messengers and signaling. To test this hypothesis, an in vitro model of CE exposure in human dermal microvascular endothelial cells (HMEC-1) was developed. Four Aims were designed to compartmentalize various components of the endothelial response to CE. The Aims included an array of methods to address cellular toxicity and dysfunction, including classical cytotoxicity and viability assays (Aim One), microscopic and electrical analyses of monolayer integrity (Aim Two), molecular analysis of second messengers, signaling molecule phosphorylation, and transcription factor DNA binding activity (Aims Three and Four). Aim One experiments demonstrated a lack of overt endothelial cytotoxicity caused by CE. Aim Two morphological analysis of endothelial intercellular borders and barrier integrity showed that CE exposure in the endothelial monolayers resulted in increased permeability, and hence a decrease in barrier integrity. These changes were observed temporally with alterations in cytosolic and total cellular free calcium ion (Aim Three), inositol 1,4,5 trisphosphate, and phosphorylated p38 mitogen-activated protein kinase concentrations, as well as changes in DNA binding activity and dimer composition of nuclear factor-kappaB (Aim Four). The observed changes suggest a distinct alteration of endothelial cell and monolayer function consistent with increased vascular permeability in vivo. Potential pathological outcomes of such effects include inflammation, vasculitis, systemic disease, and organ failure.
Respect for research subjects: Reality or rhetoric?
(2004-11-03) Amy Lynn McGuire; William J. Winslade, JD, PhD; Wayne R. Patterson, PhD; Michele A. Carter, PhD, RN; Jonathan D. Moreno, PhD; Harold Y. Vanderpool, PhD, ThM; Chester R. Burns, MD, PhD
The National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research (National Commission) identified respect for persons as one of three fundamental ethical principles for research in its Belmont Report in 1979. Since then, the moral obligation to respect research subjects has been interpreted primarily in terms of respect for individual autonomy. It is my thesis that respect for research subjects requires more than simply respecting subjects’ autonomy. This dissertation examines the ethics of respect for research subjects from a historical, conceptual, and policy perspective. The purpose of this dissertation is to rehabilitate the concept of respect in research ethics and policy and to begin a meaningful dialogue about the ethics of respect for research subjects. \r\n I begin with a historical analysis of research ethics, focusing on the history of disrespect for research subjects. I argue that the National Commission was responding to this history of disrespect when it identified respect for persons as a guiding ethical principle for research. Despite the effort of several commissioners to develop a more robust notion of respect, I contend that the language used in the National Commission’s Belmont Report left an impoverished impression of what is required to respect research subjects. This has permeated the subsequent bioethics literature and has misinformed the ethics and policy of research.\r\n This dissertation calls for reflective dialogue about the ethical duty of respect in the context of human subjects research. I seek to initiate the conversation by developing a multidimensional account of respect as an overarching normative categorty for research. I use the work of several prominent bioethicists who have struggled to gain a deeper appreciation of what it means to respect others as a foundation for a broader conceptualization of respect for research subjects. Finally, I examine several policy implications of taking respect seriously in the context of human subjects research and propose three recommendations for how a more robust interpretation of the ethical duty of respect can inform research ethics and policy.
Structural studies of the YedU stress protein
(2004-11-12) Yonghong Zhao; Robert O. Fox; Vincent J. Hilser; Luis Reuss; Hiram F. Gilbert; David W. Bolen
The knowledge of stress proteins is important for understanding stress response, pathology of a broad set of diseases, and the development of therapeutics. The Escherichia coli YedU stress protein, also known as Hsp31, is highly induced upon heat shock. To obtain a better understanding for the possible molecular function of the YedU stress protein, it was expressed, purified, and crystallized. The crystal structure of YedU was determined at 2.2 Å resolution in a multiple isomorphous replacement (MIR) experiment. \r\nYedU monomer has an alpha/beta/alpha sandwich domain and a second smaller domain. Between the sandwich domain and the second smaller domain, there is a putative catalytic triad composed of Cys184, His185, and Asp213. A metal-binding site was identified, where a zinc(II) ion is coordinated by a 2-His-1-carboxylate motif composed of His85, Glu90, and His122. The possible functions of the metal-binding site and the Cys184-His185-Asp213 triad are discussed. \r\nIt was reported that YedU has chaperone activity in vitro. YedU forms dimers in solution and the dimer interface was identified. The molecular surface of the YedU homodimer exhibits a number of solvent-exposed hydrophobic patches that are reminiscent of substrate-binding sites of molecular chaperones. To investigate the role of a central hydrophobic patch in substrate binding, four mutants were made, each replacing a hydrophobic residue with a charged residue. Compared with the wild type YedU protein, the chaperone activity of these mutants was only slightly reduced, suggesting that these residues alone do not play a dominant role in substrate binding at high temperatures.
Humna fetal neural stem cells: Proliferation and differentiation in response to growth factors and role in locomotor recovery after spinal cord contusion injury
(2005-01-12) Yevgeniya Igorevna Tarasenko; Ping Wu, M.D., Ph.D.; Kathryn A. Cunningham, Ph.D.; Golda Kevetter Leonard, Ph.D.; Giulio Taglialatela, Ph.D.; Clive N. Svendsen, Ph.D.; Claire E. Hulsebosch, Ph.D.
Human fetal neural stem cells (hNSCs) may be useful for developing a cell-based therapy to treat spinal cord injury (SCI). In these studies we examined the effects of epigenetic mitogens on proliferation and differentiation of hNSCs in vitro and the outcome of hNSC grafting into contusion injured rat spinal cords in vivo.\r\nCells were cultured in seven regimens with basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and leukemia inhibitory factor (LIF), either alone or in combinations. We found that a combination of bFGF, EGF and LIF expanded hNSCs more efficiently than any other treatment. Differentiation patterns of hNSCs expanded under different conditions were also analyzed. Cells expanded under different mitogen regimens varied in their phenotypic differentiation patterns and also in their response to a priming treatment with a combination of bFGF, heparin and laminin (FHL). Particularly, significant generation of cholinergic cells was observed only in hNSCs expanded with EGF/bFGF or EGF/bFGF/LIF, but not with other treatment regimens. \r\nSubsequently, we examined the effect of temporal transplantation of hNSCs into contusion injured rat spinal cords. FHL-primed or unprimed hNSCs were grafted into the epicenter of injured spinal cords on either the same day, three or nine days after a moderate contusion injury. Histological analyses of the spinal cord revealed that stem cells survived three months post engraftment only in animals that received grafts at 9-day post injury. The survival rates of such cells were significantly lower than those grafted into the intact cord. Both primed and unprimed hNSCs differentiated into neurons; however, only primed cells gave rise to cholinergic neurons. Functional assessment based on the BBB score and exploratory activity three months after grafting showed that hindlimb function and/or trunk stability improved significantly in only the group that received primed hNSC transplants on the ninth day post contusion. \r\nOur results indicate that 1) hNSCs are highly plastic with their proliferation and differentiation potential dependent upon different growth factor treatments; and 2) in vitro stem cells priming is beneficial to achieve the desired differentiating phenotypes in vivo and help to attenuate locomotor deficits after SCI. \r\n
Individual sensitivity to novelty and (+)-3,4-methylenedioxymethamphetamine: Roles for serotonin and GABA neurotransmission
(2005-01-27) Julie Danielle Ross; Kathryn A. Cunningham; Thomas A. Kent; Terry E. Robinson; Mary L. Thomas; Cheryl S. Watson
Drug addiction continues to be a problem in our society, and better understanding of the neuroanatomical and neurochemical alterations that delineate the switch between causal drug use and compulsive drug addiction is needed. Characterizing what makes one individual more vulnerable to the development of compulsive drug-taking behaviors may hold the key to this complex phenomenon. Because individual differences in humans exist to the subjective effects of 3,4-methylenedioxymethamphetamine (MDMA) and these differences are rooted, in part, in individual sensitivity to the drug effects, we utilized two animal models of increased sensitivity in the current studies. First, in a sensitization animal model we examined the mechanisms of increased sensitivity to (+)-MDMA and found a critical role for serotonin (5-HT) neurotransmission, in particular the 5-HT2A receptor (5-HT2AR) in the nucleus accumbens (NAc) and prefrontal cortex (PFC). We then carried this finding into a model of individual difference in which animals are separated based on their differential locomotor response to a novel environment into high responder rats (HR) and low responder rats (LR). In addition to an increased sensitivity to (+)-MDMA, we uncovered basal differences in the 5-HT system between HR and LR rats, an increased level of expression of the 5-HT2AR in the NAc of HR rats in particular. Additionally, we examined the brain structures activated secondary to novelty in HR vs. LR rats and the phenotype-specific behavioral changes after repeated exposure to the environment. Our findings revealed a strong influence of GABA neurotransmission that may underlie the differences between HR vs. LR behavioral phenotypes. These findings lend support to the idea that the neural systems underlying drug-induced and stress-induced behaviors overlap and may help to understand how individual sensitivity to both (+)-MDMA and novelty may confer an increased vulnerability to the development of compulsive drug-taking behavior.
Serotonin 5-HT_2C receptors: Role in (+)-MDMA sensitization and distribution in the ventral tegmental area
(2005-03-09) Marcy Jo Bubar; Kathryn A. Cunningham, Ph.D.; T. Celeste Napier, Ph.D.; Mary L. Thomas, Ph.D.; Joel P. Gallagher, Ph.D.; Golda A. Kevetter-Leonard, Ph.D.
Serotonin (5-HT) released consequent to acute (+)-3,4-methylenedioxy-methamphetamine [(+)-MDMA; \"ecstasy\"] administration stimulates 5-HT_2C receptors (5-HT_2CR) to exert inhibitory influence on (+)-MDMA-induced behaviors. Thus, changes in 5-HT_2CR responsiveness upon repeated intermittent exposure to (+)-MDMA may contribute to the development and/or expression of behavioral sensitization. We tested the hypothesis that intermittent exposure to (+)-MDMA or the 5-HT_2CR agonist MK 212 results in enhanced (+)-MDMA-evoked locomotor activity (\"behavioral sensitization\") concurrent with decreased functional responsiveness of the 5-HT_2CR. Male Sprague-Dawley rats pretreated with saline, (+)-MDMA, or MK 212 for 7 days revealed that (+)-MDMA or MK 212 pretreatment results in transient tolerance to MK 212-induced hypomotility, indicating loss of 5-HT_2CR responsiveness, that coincides with enhanced (+)-MDMA-evoked hyperactivity at an early (24 h) withdrawal time-point. This suggests a role for 5-HT_2CR in the induction and early expression of (+)-MDMA sensitization. While behavioral sensitization in (+)-MDMA-pretreated rats was transient and paralleled the time-course of diminished 5-HT_2CR responsiveness, MK 212-pretreated rats displayed persistent (> 2 wks) enhancement of (+)-MDMA-evoked hyperactivity despite recovery of 5-HT_2CR responsiveness. The loss of 5-HT_2CR responsiveness at 24h withdrawal was not linked to reduced 5-HT_2CR protein expression in the ventral tegmental area (VTA), nucleus accumbens (NAc), or prefrontal cortex in either (+)-MDMA- or MK 212-pretreated rats. However, an up-regulation of 5-HT_2CR protein expression was observed in the VTA at 2 wks withdrawal in MK 212-pretreated rats, which may contribute to the persistence of (+)-MDMA-evoked hyperactivity. The ability of 5-HT_2CR to limit the expression of (+)-MDMA-evoked hyperactivity is attributable to the inhibitory influence of 5-HT_2CR upon VTA dopamine (DA) neuron firing and DA release in the NAc. This effect may be mediated indirectly via depolarization of GABA neurons. However, we revealed (via double-label immunofluorescence and retrograde tracing) that 5-HT_2CR are located on both GABA and DA neurons in the VTA, a subset of which project to the NAc. Thus, the potential for a direct stimulatory effect of 5-HT_2CR upon DA mesocorticoaccumbens pathway activation also exists. This may predominate under certain conditions, such as in response to repeated 5-HT_2CR stimulation, as a result of modifications in 5-HT_2CR responsiveness.
The relationship between nitric oxide synthase (NOS) and cyclooxygenase (COX) in the control of cervical ripening and parturition
(2005-03-14) Stephen Gureasko Marx; Robert E. Garfield, Ph.D., Supervisor; Yurij Vedernikov, M.D., Ph.D.; Thomas Collins, Ph.D.; Randall Given, Ph.D.; Gwendoly V. Childs, Ph.D.; George Saade, M.D.
Stephen G. Marx\r\nThe University of Texas Medical Branch at Galveston, April 2005\r\n\r\nSupervisor: Robert E. Garfield\r\n\r\nThe purpose of these studies is to examine if there is relationship between iNOS and COX-2 in the control of cervical ripening and parturition. Cervices were obtained from estrus and timed pregnant Sprague-Dawley rats (n = 4-10 per group) under normal conditions; or after treating with LPS (100ƒÝg i.p.), Onapristone (3mg/rat), progesterone (2.5 mg, twice daily), L-NAME (50mg/day), or SNP (0.3mg/rat). Collagen changes were measured and visualized with the picrosirius polarization method. Expression of iNOS and COX-2 mRNA was determined using RT-PCR. Immunohistochemistry (IHS) was performed for localization of the iNOS and COX-2 enzymes (significance: P<0.05). Picrosirius polarization showed a decrease in the organization and birefringence of the cervical collagen from the non-pregnant state through pregnancy and is supported by changes in the luminosity (P<0.001). The iNOS and COX-2 enzymes were mainly localized in the cervical muscle with labeling also in the vascular smooth muscle and epithelium. Under normal term pregnant conditions, iNOS mRNA levels decrease as COX-2 mRNA levels increased demonstrating an inverse correlation (Spearman r = -0.497; P = 0.00295). Onapristone stimulated preterm labor and/or birth causing a parallel increase in iNOS and COX-2 mRNA demonstrating a positive correlation (Spearman r = 0.456; P = 0.03). Progesterone prolonged pregnancy stimulating a decrease in the iNOS and COX-2 (P=0.036) mRNA. In comparing term to preterm laboring conditions, there is a significant increase in the iNOS mRNA (P=0.004) but not the COX-2 mRNA. LPS enhanced the iNOS mRNA (P<0.001) but had no effect on the COX-2 mRNA. L-NAME had no effect on the COX-2 or iNOS mRNA. SNP decreased the COX-2 and iNOS with the decrease in the iNOS being significant (P=0.007). In conclusion, under normal term pregnant conditions iNOS and COX-2 play an important role in regulating cervical ripening and parturition but the pathways appear to act independently of one another in regulating iNOS and COX-2 expression at the mRNA level. Under preterm laboring conditions, when NO is up regulated and/or over expressed, there may be an interaction between the NO and PG pathways in the control of cervical ripening and parturition. \r\n\r\n
Identification and Characterization of Effectors/Binding Molecules for the Small GTPase Rab15
(2005-03-18) David Jay Strick; Lisa A. Elferink, Ph.D.; Pomila Singh, Ph.D.; Ping Wu, M.D. Ph.D.; Nancy K. Wills, Ph.D.; Mary L. Thomas, Ph.D.; Gregg T. Nagle, Ph.D.; Brian J. Knoll, Ph.D.
Endocytic trafficking is a key mechanism for regulating receptor availability on\r\nthe plasma membrane as well as receptor degradation. Clathrin-dependent endocytosis\r\ninvolves receptor internalization into early endosomes. Here internalized receptors are\r\nsorted for degradation in lysosomes, direct recycling back to the cell surface or indirect\r\nrecycling via a second recycling compartment called the pericentriolar recycling\r\nendosome. Rab GTPases regulate specific membrane trafficking steps including vesicle\r\nbudding, vesicle transport and fusion with downstream target compartments. Rab\r\nfunction is mediated by the cyclical binding and hydrolysis of GTP, which in turn\r\nregulates the recruitment of downstream effector molecules directly involved in\r\nmembrane transport steps. This dissertation focuses on the endocytic GTPase Rab15.\r\nRab15 localizes to early and pericentriolar recycling endosomes, and differentially\r\nregulates receptor transport at these distinct organelles. For example, over expression of\r\nGTP-bound Rab15 inhibits internalization of the Transferrin Receptor and inhibits\r\nhomotypic endosome fusion in vitro. Conversely, over expression of Rab15-GDP\r\ndifferentially stimulates Transferrin receptor recycling from the early endosome and\r\npericentriolar recycling endosome respectively. Rab15 may differentially regulate\r\nreceptor trafficking through these distinct endocytic compartments by binding\r\ncompartment specific effectors. To test this hypothesis, I performed yeast two-hybrid\r\nscreens to identify and characterize Rab15 binding partners. This dissertation is the\r\nfunctional characterization of three Rab15 binding proteins; Mammalian Suppressor of\r\nSec4, Rab15 Effector Protein and Rab15 Binding Protein. Using molecular, biochemical\r\nand imaging approaches, I demonstrated that interactions between Rab15 and Mss4\r\nmodulate the inhibitory effect of Rab15-GTP on receptor entry into early endosomes.\r\nThe second binding partner, Rab15 Effector Protein, localized specifically to the\r\npericentriolar recycling endosome where it regulated Transferrin receptor recycling back\r\nto the cell surface. Finally, Rab15 Binding Protein is a neural specific protein of\r\nunknown function, suggesting an important regulatory function for Rab15 in neural\r\nreceptor trafficking. These results confirm that Rab15 is a bi-functional GTPase, which\r\ndifferentially regulates receptor trafficking through early and pericentriolar recycling\r\nendosomes, by binding specific effector proteins. Moreover, identification of putative\r\nRab15 effector molecules further defines the endocytic pathway, thus providing valuable\r\ninformation for the characterization of trafficking-related diseases and potential drug\r\ntargets in the future.
Analysis of nuclear transport signals of the human apurinic/apurimidinic endonuclease (APE1/REF1)
(2005-04-22) Elias Bernard Jackson Jr.; Sankar Mitra; John Papaconstantinou; Istvan Boldogh; Guillermo Altenberg; Ben Van Houten
The nuclear localization signal (NLS) in human apurinic/apyrimidinic endonuclease (APE1), a key protein in both DNA base excision repair and transcriptional regulation, has not been analyzed in detail. We examined the role of specific residues in nuclear translocation of APE1, using green fluorescent protein (EGFP) fused to APE1 as a reporter. Nuclear localization (NL) of ectopic APE1 was abrogated for the mutant lacking 20 N-terminal aa residues (ND20). Fusion of these 20 residues directed nuclear localization of EGFP. While an APE1 mutant lacking N-terminal 7 residues (ND7 APE1) showed normal nuclear localization, ND7 APE1 with E12A/D13A double mutation resulted in drastic decrease of nuclear localization, indicating that E12 and D13 are critical components of the NLS. \r\n On the other hand, nuclear localization of the full-length APE1 containing the E12A/D13A mutations suggests that the putative NLS and residues 8-13 contribute independently to nuclear import. Nuclear accumulation of the ND7 APE1(E12A/D13A), but not EGFP alone, after treatment with leptomycin B or after oxidative stress suggests the presence of a previously unidentified nuclear export signal in APE1. Together, these results indicate that the mechanism of nuclear localization of APE1 is complex and regulated via import and export.\r\n In addition increase DNA damage occurs in astronauts during space flight. Humans in space are exposed both to radiation and microgravity. It is clear that the increased exposure to radiation influences DNA damage however it is not clear as to the role that microgravity plays in this increase in damage. \r\n Our investigation on the effects of microgravity on the nuclear translocation of DNA repair enzyme APE1 has revealed that microgravity interferes with the normal trafficking of APE1. \r\n
IL-1 activation of NF-kB contributes to perinatal hypoxia/ischemia induced cell death
(2005-06-01) Xiaoming Hu; J. R. Perez-Polo,; Olivera B. Nesic-Taylor; Norbert K. Herzog; Marjorie R. Grafe; Karin W. High; David K Rassin
IL-1 activity has been implicated in perinatal hypoxia/ischemia (HI) brain damage without the underlying mechanisms being characterized. We used a 7-day-old rat model to elucidate the role of NF-kB in HI stimulation of IL-1 signaling. HI was induced by permanent ligation of the left carotid artery followed by 90 minutes of hypoxia (7.8% O2). We observed increased cell death and caspase 3 activity in the hippocampus and the cortex 3 to 48h post-HI. IL-1beta protein expression increased began at 3h after HI and lasted until 24h post-HI in the hippocampus and 12h post-HI in the cortex. Intracerebroventricular injection of 2mg IL-1 receptor antagonist (IL-1Ra) 2h post-HI significantly reduced cell death and caspase 3 activity. EMSA analyses for NF-kB activity showed increased p65/p50 DNA-binding activity at 24h post-HI. Western blot analyses and immunofluorescent staining showed significant nuclear translocation of p65. Protein expression levels of iNOS and COX2, known to be transcriptionally regulated by NF-kB, also increased at 24h post-HI. All these changes were reversed by IL-1Ra blockade of IL-1, consistent with IL-1 triggering of inflammatory apoptotic outcomes via NF-kB transcriptional activation. The observed increase in cytoplasmic phosphorylated IkBa and nuclear translocation of Bcl-3, was also attenuated by IL-1Ra blockade, suggesting that HI-induced IL-1 activation of NF-kB is via both degradation of IkBalpha and nuclear translocation of Bcl-3. \r\nInhibition of NF-kB via decoys containing NF-kB binding consensus sequences present in IgG-kB promoter showed specific inhibition of p65/p50 binding activity, while Bcl-x decoys specifically inhibited c-Rel/p50 (p52) binding activity. RPAs showed that IgG-kB decoys significantly decreased IL-1alpha, TNF-alpha and TNF-beta mRNA levels compared to minimal changes after Bcl-x or scrambled decoy treatment, indicating a specific IgG-kB decoy effect on inflammation post-HI. Microarray data indicated that 1) Genes that were significantly downregulated by IgG-kB decoys were not affected by Bcl-x decoys and vice versa, another piece of evidence for selective effects of different decoys. 2) A large number of cell death/survival related genes were affected by IgG-kB decoys, confounding the final outcomes. Our results suggest that IgG-kB decoys selectively inhibit inflammatory responses to HI. However, careful design of decoy sequences is essential to acquire selective effects on cell death.
Understanding the function of ICOS/ICOSL costimulation in experimental autoimmune myasthenia gravis
(2005-06-15) Benjamin Gregory Scott; Premkumar Christadoss, M.D.; Vivian Braciale, Ph.D.; Rolf Konig; John Papaconstantinou, Ph.D.; Angela Vincent, M.B., B.S.
The inducible costimulatory molecule (ICOS) is a relatively new member of the CD28 family of costimulatory molecules. For the first time, we have characterized the role of ICOS/ICOSL costimulation in experimental autoimmune myasthenia gravis (EAMG), a model of human MG. Following acetylcholine receptor (AChR) immunization, ICOS gene-deficient mice were resistant to the development of EAMG due to faulty germinal center formation, decreased levels of anti-AChR IgG of all isotypes tested, and a lack of IgG and complement binding to the neuromuscular junction (NMJ). Compared to control lymphocytes, lymphocytes from AChR-immunized ICOS-deficient mice proliferated poorly and produced significantly less IFN-gamma and IL-10 following in vitro stimulation with AChR or the immunodominant AChR alpha-subunit peptide 146-162. In vivo, the lack of ICOS costimulation led to diminished B cell and plasma cell expansion, whereas the number of CD4+ T helper cells was increased. Collectively, these results indicate that lymphocyte costimulation through the ICOS/ICOSL pathway is a vital component of the adaptive immune response to AChR in EAMG.\r\n
Regulation of BACE1 promoter activity by nuclear factor-kappaB\r\n
(2005-06-30) Krystyn Zimmer Bourne; J. Regino Perez-Polo; Steffen Rossner; Norbert K. Herzog; Kelly T. Dineley; Cheryl S. Watson; B. Mark Evers
The brains of Alzheimer’s disease (AD) patients display cerebrovascular and parenchymal deposits of beta-amyloid peptides, which are derived by proteolytic processing of the amyloid precursor protein (APP). The beta-site APP-cleaving enzyme 1 (BACE1) is required for the generation of beta-amyloid peptides. The NF-kappaB binding DNA consensus sequence in the BACE1 promoter upstream of the gene’s transcription start site suggests a role for NF-kappaB in the expression of neuronal brain BACE1. Failure of activation of NF-kappaB responses to stress in the aged and disregulation of NF-kappaB in the AD brain may result in part in altered NF-kappaB regulation of BACE1 and alterations in cell specific BACE1 transcription and beta-amyloid protein processing.\r\n We identified a number of putative NF-kappaB transcription factor binding sites on the rat BACE1 promoter. The effects of NF-kappaB binding to the “primary” NF-kappaB binding site of the BACE1 promoter were stimulatory for activated astrocytic cells and repressive for neuronal cells. Age-associated perturbations of NF-kappaB activation may result in increasingly aberrant regulation of beta-amyloid processing by BACE1 via changes in the cellular levels of different NF-kappaB protein subunits and cumulative increases in astrocytically-derived beta-amyloid. We confirmed the observation that in PC12 cells the overall activity of NF-kappaB and BACE1 was significantly different depending on the apoptotic initiators: H2O2 or beta-amyloid. Our results are consistent with feedback mechanisms involving beta-amyloid exposure overriding NF-kappaB regulation of BACE1 over time, a source of negative feedback, consistent with observed neuropathologies. It is also likely that a series of transcription factor binding events determine which NF-kappaB binding sites are operant and this may explain the observed cell specificity of BACE1 regulation. Our observation that BACE1 expression was affected by both soluble and aggregated insulin, coupled with our previous observations that both aggregated Abeta1-42 and Abeta42-1 affected BACE1 expression, strongly suggest that the process of protein aggregation displayed by proteins sharing specific structural characteristics (i.e. zinc stabilized hexamers) may have pathological significance. It is tempting to hypothesize that insulin, and/or other similarly structured proteins, have effects on BACE1 activity that may play a role in the establishment and/or progression of Alzheimer’s disease. \r\n
Regional and temporal differential regulation of the N-methyl-D-aspartate receptor by phencyclidine during development
(2005-07-06) Noelle Catherine Anastasio; Kenneth M. Johnson, PhD.; Kathryn Cunningham, PhD.; Giulio Taglialatela, PhD.; Geoffrey T. Swanson
Disruptions in glutamatergic neurotransmission may play a role in the pathogenesis of schizophrenia. The purpose of this study was to determine phencyclidine (PCP)-induced changes in the NMDA receptor subunit composition and the relationship of these changes to the deficits in pre-pulse inhibition (PPI) caused by PCP treatment. Postnatal rats were treated with atypical or typical antipsychotics or selective dopamine or serotonin receptor antagonists prior to acute or sub-chronic PCP. This study provides evidence that two distinct mechanisms underlie effects of acute and sub-chronic PCP on NMDA receptor subunit up-regulation. Furthermore, we discovered that D1, D2, and 5-HT2A receptors play a pivotal role in sub-chronic PCP-induced up-regulation of NR1 and NR2A. Finally, we were able to correlate changes in NMDA receptor subunits to the behavioral effects of PCP in this animal model of schizophrenia.
Characterization of serum-induced CYP1A1 expression and activity in mouse embryo fibroblasts
(2005-07-07) Carmen-Veronica Naira Obianwu; Cornelis Elferink Ph.D.; Xiaodong Cheng Ph.D.; Jonathan B. Ward Ph.D.
The AhR is a ligand-activated transcription factor that mediates the toxic effects of environmental contaminants that include TCDD. Using a TCDD dose-response treatment in MEFs, we observed a super induction of CYP1A1 with newborn calf serum (NCS) in the presence (10nM/15nM) of TCDD. In addition to NCS, fetal bovine serum (FBS) also has the capability to yield a CYP1A1 super induction. These results suggest that components within the sera affect the activity of the AhR and consequent CYP1A1 expression. To pursue this idea, characterization of the serum factors were investigated. The findings indicated that serum factor(s) in both sera are heat sensitive at 50◦C, withstand removal from charcoal stripping sera and are ¡Ý 10,000 kDa in size. Using RT-PCR, we found that NCS factors only, could super induce CYP1A1 at the gene level. Moreover, MEFs are the only cells observed in this study that are susceptible to CYP1A1 super induction.
“Differential regulation of PTHrP gene expression by 1,25(OH)2D3 in prostate cancer cell lines”
(2005-07-08) Veronica Alejandra Tovar Sepulveda; Miriam Falzon, Ph.D.; Nancy L. Weigel, Ph.D.; Melvin S. Soloff, Ph.D.; Mary L. Thomas, Ph.D.; Cheryl S. Watson, Ph.D.; Cary W. Cooper, Ph.D.
Parathyroid hormone-related protein (PTHrP) is expressed by prostate cancer cells. Since PTHrP enhances the growth and osteolytic potential of prostate cancer cells, it is important to control PTHrP expression in these cells. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) downregulates PTHrP expression in a variety of human cell types, including prostate cancer cells. Therefore, downregulation of PTHrP gene expression by 1,25(OH)2D3 may enhance the therapeutic benefits of 1,25(OH)2D3 by neutralizing PTHrP’s contribution to the pathogenesis and progression of prostate carcinoma and its tendency to metastasize to bone. In this study, we show that 1,25(OH)2D3 and its non-hypercalcemic analog EB1089 decrease cell proliferation and suppress PTHrP mRNA and protein levels in the human prostate cancer cell lines LNCaP, C4-2, and PC-3. These cell lines represent early to advanced stages of prostate cancer, since LNCaP cells are weakly metastatic, androgen receptor (AR)-positive and androgen-responsive cells, C4-2 cells are AR-positive and androgen-independent LNCaP variants, and PC-3 cells are highly metastatic AR-negative cells. We identified a negative vitamin D response element (nVDREhPTHrP) within the human PTHrP gene; interaction of the vitamin D receptor (VDR) with this nVDRE is decreased by 1,25(OH)2D3. However, transient transfection of nVDREhPTHrP cloned upstream of the SV40 promoter downregulated promoter activity in response to 1,25(OH)2D3 or EB1089 treatment in LNCaP and C4-2, but not in PC-3, cells. The retinoid X receptor (RXR) is a frequent heterodimeric partner of the VDR. We show that RXRα forms part of the nuclear protein complex that interacts with nVDREhPTHrP with the VDR in LNCaP, C4-2, and PC-3 cells. The RXR ligand 9-cis-retinoic acid (9-cis-RA) downregulates PTHrP mRNA levels in both LNCaP and C4-2 cells; this decrease is more pronounced in LNCaP than in C4-2 cells. 9-cis-RA also enhances the 1,25(OH)2D3-mediated downregulation of PTHrP expression in both cell lines; this effect is more pronounced in LNCaP cells. Co-treatment with 1,25(OH)2D3 or EB1089 and 9-cis-RA further decreased promoter activity driven via nVDREhPTHrP in LNCaP, but not C4-2, cells. The proliferation of LNCaP, but not C4-2, cells is decreased by 9-cis-RA. These results indicate that PTHrP gene expression is regulated by 1,25(OH)2D3 in a cell line-specific manner in prostate cancer cells.
Cytokine patterns in a comparative model of arenavirus infection: Implications for virulence and control of viral replication
(2005-07-12) Erin P. Scott; Judith Aronson; Thomas K. Hughes; Lynn Soong; David N. McMurray; Clarence J. Peters
Guinea pig infection with the arenavirus Pichinde provides an animal model for human Lassa fever, a disease that affects 300,000 to 500,000 people a year in western Africa. Low passage Pichinde virus (P2) induces a mild disease with low viremia, while high passage Pichinde (P18) induces a severe disease with high viremia, ending in terminal shock. We hypothesized that severe disease would be associated with a suppression of potentially antiviral cytokines early in infection, and high levels of potentially pathogenic pro-inflammatory cytokines late in infection. Cytokine responses to P2 and P18 infection were measured from primary guinea pig peritoneal macrophages (PM) in vitro when measured by real time RT-PCR. In general, neither P2 nor P18 infection altered cytokine production from unstimulated PM. P18 infected PM did have lower mRNA levels of IL-1beta, IL-12p40, and MCP-1 after LPS addition when compared to P2 infected PM. During experimental guinea pig infection, P18 infection was associated with markedly increased IFN-gamma and MCP-1 mRNA levels from the initial peritoneal target cells relative to P2. P18 infected peritoneal cells had slightly decreased TNF-alpha, IL-8, and IL-12p40 transcripts relative to mock infected peritoneal cells. Late in infection, P18 infected spleens and livers had similar cytokine patterns relative to P2, but P18 infected PBL had decreased TNF-alpha, IFN-gamma, and RANTES transcripts. We also examined the ability of a decoy AP-1 thioaptamer, XBY-S2, to alter morbidity, mortality, and cytokine expression during P18 infection of guinea pigs. After two doses of XBY-S2, 50% (p=.024) of treated guinea pigs survived infection and had undetectable viremia. XBY-S2-treated P18 infected guinea pigs over time had overall increased cytokine mRNA expression of TNF-alpha, IL-8, IL-1beta, and IL-10 compared to PBS-treated P18 infected guinea pigs. A suppression of PBL IL-1beta and RANTES mRNA at day 12 of P18 infection was repeatedly observed. Conclusions from these experiments are 1) macrophage-derived cytokines do not explain the differential replication of P2 and P18 viruses, 2) high levels of IFN-gamma and MCP-1 may contribute to virulence of P18 virus, 3) over-expression of pro-inflammatory cytokines in PBL, liver, or spleen is not associated with terminal shock, 4) boosting of pro-inflammatory cytokines by an AP-1 aptamer correlates with reduced viremia and survival of P18 infection.

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