Radhakrishna Rao - US grants

DESCRIPTION (provided by applicant): Increased epithelial permeability and oxidative stress-induced epithelial injury have been implicated in the pathogenesis of a wide spectrum of intestinal inflammatory diseases including necrotizing enterocolitis and inflammatory bowel disease (Crohn's disease and Ulcerative colitis). Our studies so far have shown that oxidative stress increases paracellular permeability in intestinal epithelial monolayers. The mechanism of this oxidative stress-induced disruption of epithelial junctions involves activation of a proto-oncogene, c-Src, tyrosine phosphorylation of junctional proteins, disruption of protein-protein interactions among the junctional proteins, and loss of integrity of the junctional complexes that prevent epithelial permeability. On the basis of our results it is further hypothesized that: a) oxidative stress-induced phosphorylation of occludin on specific tyrosine residues reduces its ability to bind ZO-1, ZO-2 and ZO-3, b) translocation of PP2A to the tight junction (TJ)plays a crucial role in oxidative stress-induced dephosphorylation of occludin on threonine residues and disruption of the tight junction, and c) EGF prevents oxidative stress-induced translocation of PP2A to the tight junction by PKCbeta1 and MAPK dependent mechanism. Using the above mentioned model we propose to determine that: 1) tyrosine phosphorylation of C-terminal tail of occludin reduces its ability to bind ZO-1, ZO-2 and ZO-3, 2) phosphorylation of occludin on specific tyrosine residue(s) is responsible for the prevention of its binding to ZO-1, ZO-2 and ZO-3, 3) mutation of specific tyrosine residue(s) on the C-terminal tail of occludin prevents the oxidative stress-induced disruption of tight junction, 4) oxidative stress induces translocation of PP2A to the tight junction by c-Src dependent mechanism, 5) expression of dominant negative PP2A-Cot and reduced expression by antisense oligos prevent oxidative stress-induced disruption of the tight junction, 6) dephosphorylation of occludin on serine/threonine attenuates its interaction with F-actin, 7) EGF prevents oxidative stress-induced translocation of PP2A to the T J, 8) PKCbeta1 mediates EGF-mediated protection of TJ, and 9) MEK and ERK are involved in the mechanism of EGF-mediated protection of TJ. Information derived from this study has the potential to expand our understanding of oxidative stress-mediated injury in intestinal epithelium, by identifying some of the mechanisms of oxidant-induced disruption of paracellular junctional complexes and protection by EGF.

DESCRIPTION (provided by applicant): Evidence from clinical and experimental studies indicates that elevated intestinal permeability to endotoxins and the resulting endotoxemia play a crucial role in the pathogenesis of alcoholic liver disease. Our studies conducted so far have shown that acetaldehyde, the metabolic product of ethanol, is accumulated in the intestine of alcoholics and disrupts the intestinal epithelial barrier function and increases the permeability to endotoxins. The mechanism of this acetaldehyde-induced disruption of epithelial barrier function involves inhibition of a protein tyrosine phosphatase, PTP1B, tyrosine phosphorylation of junctional proteins, disruption of the interactions among the junctional proteins (that determine the barrier function), and loss of integrity of the junctional complexes. Our preliminary studies also indicated that epidermal growth factor (EGF) and L-glutamine prevent acetaldehyde-mediated increase in permeability to endotoxins. On the basis of our results it is further hypothesized that: a) acetaldehyde-induced phosphorylation of PTP1B and beta-catenin disrupts E-Cadherin-beta-Catenin-PTP1B complex, b) EGF and L-glutamine prevent acetaldehyde induced disruption of junctions by PLCgamma and MAP kinase mediated signals, and c) L-glutamine prevents acetaldehyde-induced disruption of junctions by EGF receptor-dependent mechanism. Using a cell culture model of the intestinal epithelium and human colonic biopsies we will to determine that: 1) interaction between E-cadherin and PTP1B is reduced by Thr-phosphorylation of PTP1B, 2) phosphorylation of beta-catenin on tyrosine residues prevents its binding to E-cadherin, 3) mutation of specific threonine residues on PTP1B and tyrosine residues in beta-catenin prevents the acetaldehyde-induced disruption of junctions, 4) EGF and glutamine prevent acetaldehyde-induced phosphorylation of PTP1B and beta-catenin, and disruption of E-cadherin/beta-catenin/PTP1B complex, 5) PLCgamma and MAP kinase signaling pathways mediate EGF- and glutamine-induced prevention of phosphorylation of PTP1B and beta-catenin, and disruption of E-cadherin/beta-catenin/PTP1B complex, 6) L-glutamine prevents acetaldehyde-induced phosphorylation of PTP1B and beta-catenin, and disruption of E-cadherin/beta-catenin/PTP1B complex by EGF receptor-dependent mechanism, 7) EGF receptor activation is involved in glutamine-mediated prevention of acetaldehyde-induced disruption of AJ and TJ, 9) glutamine prevents acetaldehyde-induced disruption of AJ and TJ by Src kinase dependent mechanism, and 10) EGF receptor activation by glutamine is mediated by extra cellular release of metalloproteinase and TGFalpha. Information derived from this study will expand our understanding of acetaldehyde-mediated injury in intestinal epithelium and endotoxin absorption in alcoholics. The studies on the protection of barrier function by epidermal growth factor and L-glutamine have the potential to provide the basis for the development of preventive and therapeutic strategies for alcoholic liver disease.

Neonatal necrotizing enterocolitis (NEC), the acute necrosis of the small intestine, is the most common non-respiratory, life-threatening disease among infants admitted to neonatal intensive care units. Although etiologic and epidemiologic characteristics of NEC are poorly understood, two major factors closely associated with NEC are prematurity and enteral feeding. A striking observation made by several groups of scientists is that feeding breast milk reduces the risk of NEC in premature infants. Such a beneficial effect was not observed by feeding infant formula. We recently demonstrated that human milk contains protective factors that protect the intestinal epithelial barrier function from oxidative-stress induced disruption. Our preliminary studies indicate that there are at least two protective factors in human milk, one heat stable and the other heat-sensitive. On the basis of preliminary results it is hypothesized that milk-borne factors protect the intestinal mucosal barrier function. Our long-range goal is to determine the mechanism involved in breast milk-mediated protection of the gastrointestinal mucosa, and to determine the role of milk-borne mucosal protective factors in preventing neonatal gastrointestinal diseases, such as NEC. Our next step towards this goal will be to isolate two distinct milk-borne protective factors that prevent hydrogen peroxide-induced barrier disruption, and to characterize their physical and chemical properties to reveal their identity. This goal will be achieved by: 1) isolation of protective factors to homogeneity by liquid chromatography, and 2) characterization of physical and chemical properties of isolated protective factors. These studies will reveal the identity of at least two distinct mucosal protective factors in breast milk, and provide the basis for further characterization of the mechanisms involved in mucosal protection by breast milk, and their role in preventing neonatal gastrointestinal diseases

Analysis, Data; Animal Model; Animal Models and Related Studies; Biochemical; Biochemistry; Brain; Brain Hypoxia-Ischemia; CRISP; Cell Communication and Signaling; Cell Signaling; Chemistry, Biological; Citellus; Computer Retrieval of Information on Scientific Projects Database; Creatine; Data; Data Analyses; Development; Disease Progression; Encephalon; Encephalons; Fe element; Funding; Glycine, N-(aminoiminomethyl)-N-methyl-; Grant; Hibernation; Huntington Chorea; Huntington Disease; Huntington's; Huntington's Disease; Huntington's Disease Pathway; Huntingtons Disease; Hydrogen Oxide; Hypoxia-Ischemia, Brain; Institution; Intracellular Communication and Signaling; Investigators; Iron; Mammals, Rodents; Measures; Method LOINC Axis 6; Methodology; Modeling; Monitor; NIH; National Institutes of Health; National Institutes of Health (U.S.); Neonatal; Nerve Degeneration; Nervous System, Brain; Neurochemistry; Neuron Degeneration; Process; Progressive Chorea, Hereditary, Chronic (Huntington); Range; Research; Research Personnel; Research Resources; Researchers; Resources; Rodent; Rodentia; Rodentias; Science of neurochemistry; Signal Transduction; Signal Transduction Systems; Signaling; Source; Spectroscopy; Spectrum Analyses; Spectrum Analysis; Spermophilus; Steam; Suslik; Time; Transgenic Organisms; United States National Institutes of Health; Water; biological signal transduction; cold temperature; ground squirrel; hypoxia ischemia; low temperature; model organism; mouse model; neural degeneration; neurochemistry; neurodegeneration; neuronal degeneration; transgenic

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Hypoglycemia is a common metabolic condition in newborn infants. Prolonged and recurrent hypoglycemia is associated with long-term hippocampal dysfunction. Despite extensive studies in humans and animal models, detailed mechanism is not well understood, especially the sequel of different processes providing fuel for brain cells, when glucose plasma is too low. The purpose of this project is to quantify neurochemical changes during acute and recurrent hypoglycemia on the hippocampus of rat pups using 1H NMR spectroscopy at 9.4 T.