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Biomedical subjects

L J Zhang

Publications and source records attributed to L J Zhang.

At least 19 recordsLinked to original sources
PubMed2007

Regulation of oxidant-induced intestinal permeability by metalloprotease-dependent epidermal growth factor receptor signaling.

Inflammatory bowel disease (IBD) affects more than 1 million Americans with more than 30,000 new cases diagnosed each year. IBD increases patient morbidity and susceptibility to colorectal cancer, yet its etiology remains unknown. Current models identify two key determinants of IBD pathogenesis: hyperpermeability of the gut epithelial barrier to bacterial products and an abnormal immune response to these products. Two factors seem critical for hyperpermeability: oxidant-induced stress and proinflammatory cytokines (e.g., tumor necrosis factor-alpha). The aim of this study was to investigate the role of oxidant stress-mediated transactivation of the epidermal growth factor receptor (EGFR) in intestinal hyperpermeability. This study used the Caco-2 human colonic epithelial cell in vitro model of intestinal epithelium. Cells were grown on inserts for permeability and signaling studies and glass coverslips for microscopy studies. show that oxidant-induced intestinal hyperpermeability can be blocked by specific inhibitors of the EGFR, tumor necrosis factor convertase (TACE) metalloprotease, transforming growth factor (TGF)-alpha, and mitogen-activated protein kinases, especially extracellular signal-regulated kinase 1/2. We also show that oxidant initiates these signaling events, in part by causing translocation of TACE to cell-cell contact zones. In this study, our data identify a novel mechanism for oxidant-induced intestinal hyperpermeability relevant to IBD. We propose a new intestinal permeability model in which oxidant transactivates EGFR signaling by activation of TACE and cleavage of precursor TGF-alpha. These data could have a significant effect on our view of IBD pathogenesis and provide new therapeutic targets for IBD treatment.

ADAM Proteins↗
PubMed2006

Evolution and adaptation of H5N1 influenza virus in avian and human hosts in Indonesia and Vietnam.

Highly pathogenic avian influenza virus H5N1 is endemic in poultry in East and Southeast Asia with disease outbreaks recently spreading to parts of central Asia, Europe and Africa. Continued interspecies transmission to humans has been reported in Vietnam, Thailand, Cambodia, Indonesia and China, causing pandemic concern. Here, we genetically characterize 82 H5N1 viruses isolated from poultry throughout Indonesia and Vietnam and 11 human isolates from southern Vietnam together with sequence data available in public databases to address questions relevant to virus introduction, endemicity and evolution. Phylogenetic analysis shows that all viruses from Indonesia form a distinct sublineage of H5N1 genotype Z viruses suggesting this outbreak likely originated from a single introduction that spread throughout the country during the past two years. Continued virus activities in Indonesia were attributed to transmission via poultry movement within the country rather than through repeated introductions by bird migration. Within Indonesia and Vietnam, H5N1 viruses have evolved over time into geographically distinct groups within each country. Molecular analysis of the H5N1 genotype Z genome shows that only the M2 and PB1-F2 genes were under positive selection, suggesting that these genes might be involved in adaptation of this virus to new hosts following interspecies transmission. At the amino acid level 12 residues were under positive selection in those genotype Z viruses, in the HA and PB1-F2 proteins. Some of these residues were more frequently observed in human isolates than in avian isolates and are related to viral antigenicity and receptor binding. Our study provides insight into the ongoing evolution of H5N1 influenza viruses that are transmitting in diverse avian species and at the interface between avian and human hosts.

Animals↗
PubMed2006

Establishment of multiple sublineages of H5N1 influenza virus in Asia: implications for pandemic control.

Preparedness for a possible influenza pandemic caused by highly pathogenic avian influenza A subtype H5N1 has become a global priority. The spread of the virus to Europe and continued human infection in Southeast Asia have heightened pandemic concern. It remains unknown from where the pandemic strain may emerge; current attention is directed at Vietnam, Thailand, and, more recently, Indonesia and China. Here, we report that genetically and antigenically distinct sublineages of H5N1 virus have become established in poultry in different geographical regions of Southeast Asia, indicating the long-term endemicity of the virus, and the isolation of H5N1 virus from apparently healthy migratory birds in southern China. Our data show that H5N1 influenza virus, has continued to spread from its established source in southern China to other regions through transport of poultry and bird migration. The identification of regionally distinct sublineages contributes to the understanding of the mechanism for the perpetuation and spread of H5N1, providing information that is directly relevant to control of the source of infection in poultry. It points to the necessity of surveillance that is geographically broader than previously supposed and that includes H5N1 viruses of greater genetic and antigenic diversity.

Animals↗
PubMed2006

Imaging of primary adrenal lymphoma: case report and literature review.

Primary adrenal lymphoma is very rare, and correct preoperative diagnosis is difficult because of its nonspecific imaging findings. We report a case of bilateral primary adrenal lymphoma, with the largest dimension of 18 cm, which is the largest one reported so far, to our knowledge. Computed tomography (CT) and magnetic resonance (MR) findings were not specific enough to establish a correct diagnosis, but positron emission tomography/CT (PET/CT) showed high fluorodeoxyglucose (FDG) uptake, indicating its malignant nature. We describe the imaging findings and review the related literature to highlight this uncommon entity.

Adrenal Gland Neoplasms↗
PubMed2005

theta Isoform of protein kinase C alters barrier function in intestinal epithelium through modulation of distinct claudin isotypes: a novel mechanism for regulation of permeability.

Using monolayers of intestinal Caco-2 cells, we discovered that the isoform of protein kinase C (PKC), a member of the "novel" subfamily of PKC isoforms, is required for monolayer barrier function. However, the mechanisms underlying this novel effect remain largely unknown. Here, we sought to determine whether the mechanism by which PKC- disrupts monolayer permeability and dynamics in intestinal epithelium involves PKC--induced alterations in claudin isotypes. We used cell clones that we recently developed, clones that were transfected with varying levels of plasmid to either stably suppress endogenous PKC- activity (antisense, dominant-negative constructs) or to ectopically express PKC- activity (sense constructs). We then determined barrier function, claudin isotype integrity, PKC- subcellular activity, claudin isotype subcellular pools, and claudin phosphorylation. Antisense transfection to underexpress the PKC- led to monolayer instability as shown by reduced 1) endogenous PKC- activity, 2) claudin isotypes in the membrane and cytoskeletal pools ( downward arrowclaud-1, downward arrowclaud-4 assembly), 3) claudin isotype phosphorylation ( downward arrow phospho-serine, downward arrow phospho-threonine), 4) architectural stability of the claudin-1 and claudin-4 rings, and 5) monolayer barrier function. In these antisense clones, PKC- activity was also substantially reduced in the membrane and cytoskeletal cell fractions. In wild-type (WT) cells, PKC- (82 kDa) was both constitutively active and coassociated with claudin-1 (22 kDa) and claudin-4 (25 kDa), forming endogenous PKC-/claudin complexes. In a second series of studies, dominant-negative inhibition of the endogenous PKC- caused similar destabilizing effects on monolayer barrier dynamics, including claudin-1 and -4 hypophosphorylation, disassembly, and architectural instability as well as monolayer disruption. In a third series of studies, sense overexpression of the PKC- caused not only a mostly cytosolic distribution of this isoform (i.e., <12% in the membrane + cytoskeletal fractions, indicating PKC- inactivity) but also led to disruption of claudin assembly and barrier function of the monolayer. The conclusions of this study are that PKC- activity is required for normal claudin assembly and the integrity of the intestinal epithelial barrier. These effects of PKC- are mediated at the molecular level by changes in phosphorylation, membrane assembly, and/or organization of the subunit components of two barrier function proteins: claudin-1 and claudin-4 isotypes. The ability of PKC- to alter the dynamics of permeability protein claudins is a new function not previously ascribed to the novel subfamily of PKC isoforms.

Caco-2 Cells↗
PubMed2005

Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003.

Avian H9N2 influenza A virus has caused repeated human infections in Asia since 1998. Here we report that an H9N2 influenza virus infected a 5-year-old child in Hong Kong in 2003. To identify the possible source of the infection, the human isolate and other H9N2 influenza viruses isolated from Hong Kong poultry markets from January to October 2003 were genetically and antigenically characterized. The findings of this study show that the human H9N2 influenza virus, A/Hong Kong/2108/03, is of purely avian origin and is closely related to some viruses circulating in poultry in the markets of Hong Kong. The continued presence of H9N2 influenza viruses in poultry markets in southern China increases the likelihood of avian-to-human interspecies transmission.

Antigens, Viral↗
PubMed2004

Critical role of the atypical {lambda} isoform of protein kinase C (PKC-{lambda}) in oxidant-induced disruption of the microtubule cytoskeleton and barrier function of intestinal epithelium.

Oxidant injury to epithelial cells and gut barrier disruption are key factors in the pathogenesis of inflammatory bowel disease. Studying monolayers of intestinal (Caco-2) cells, we reported that oxidants disrupt the cytoskeleton and cause barrier dysfunction (hyperpermeability). Because the lambda isoform of protein kinase C (PKC-lambda), an atypical diacylglycerol-independent isozyme, is abundant in parental (wild type) Caco-2 cells and is translocated to the particulate fractions upon oxidant exposure, we hypothesized that PKC-lambda is critical to oxidative injury to the assembly and architecture of cytoskeleton and the intestinal barrier function. To this end, Caco-2 cells were transfected with an inducible plasmid, a tetracycline-responsive system, to create novel clones stably overexpressing native PKC-lambda. Other cells were transfected with a dominant-negative plasmid to stably inhibit the activity of native PKC-lambda. Cells were exposed to oxidant (H(2)O(2)) +/- modulators. Parental Caco-2 cells were treated similarly. We then monitored barrier function (fluorescein sulfonic acid clearance), microtubule cytoskeletal stability (confocal microscopy, immunoblotting), subcellular distribution of PKC-lambda (immunofluorescence, immunoblotting, immunoprecipitation), and PKC-lambda isoform activity (in vitro kinase assay). Monolayers were also processed to assess alterations in tubulin assembly, polymerized tubulin (S2, an index of cytoskeletal integrity), and monomeric tubulin (S1, an index of cytoskeletal disassembly) (polyacrylamide gel electrophoresis fractionation and immunoblotting. In parental cells, oxidant caused: 1) translocation of PKC-lambda from the cytosol to the particulate (membrane + cytoskeletal) fractions, 2) activation of native PKC-lambda, 3) tubulin pool instability (increased monomeric S1 and decreased polymerized S2), 4) disruption of cytoskeletal architecture, and 5) barrier dysfunction (hyperpermeability). In transfected clones, overexpression of the atypical (74 kDa) PKC-lambda isoform by itself ( approximately 3.2-fold increase) led to oxidant-like disruptive effects, including cytoskeletal and barrier hyperpermeability. Overexpressed PKC-lambda was mostly found in particulate cell fractions (with a smaller cytosolic distribution) indicating its activation. Disruption by PKC-lambda overexpression was also potentiated by oxidant challenge. Stable inactivation of endogenous PKC-lambda ( approximately 99.6%) by a dominant-negative protected against all measures of oxidant-induced disruption. We conclude that: 1) oxidant induces disruption of epithelial barrier integrity by disassembling the cytoskeleton, in large part, through the activation of PKC-lambda isoform; and 2) activation of PKC-lambda by itself appears to be sufficient for disruption of cellular cytoskeleton and monolayer barrier permeability. The unique ability to mediate an oxidant-like injury and cytoskeletal depolymerization and instability is a novel mechanism not previously attributed to the atypical subfamily of PKC isoforms.

Blotting, Western↗
PubMed2004

Novel effect of NF-kappaB activation: carbonylation and nitration injury to cytoskeleton and disruption of monolayer barrier in intestinal epithelium.

Using monolayers of intestinal cells, we reported that upregulation of inducible nitric oxide synthase (iNOS) is required for oxidative injury and that activation of NF-kappaB is key to cytoskeletal instability. In the present study, we hypothesized that NF-kappaB activation is crucial to oxidant-induced iNOS upregulation and its injurious consequences: cytoskeletal oxidation and nitration and monolayer dysfunction. Wild-type (WT) cells were pretreated with inhibitors of NF-kappaB, with or without exposure to oxidant (H(2)O(2)). Other cells were transfected with an IkappaBalpha mutant (an inhibitor of NF-kappaB). Relative to WT cells exposed to vehicle, oxidant exposure caused increases in IkappaBalpha instability, NF-kappaB subunit activation, iNOS-related activity (NO, oxidative stress, tubulin nitration), microtubule disassembly and instability (increased monomeric and decreased polymeric tubulin), and monolayer disruption. Monolayers pretreated with NF-kappaB inhibitors (MG-132, lactacystin) were protected against oxidation, showing decreases in all measures of the NF-kappaB --> iNOS --> NO pathway. Dominant mutant stabilization of IkappaBalpha to inactivate NF-kappaB suppressed all measures of the iNOS/NO upregulation while protecting monolayers against oxidant insult. In these mutants, we found prevention of tubulin nitration and oxidation and enhancement of cytoskeletal and monolayer stability. We concluded that 1) NF-kappaB is required for oxidant-induced iNOS upregulation and for the consequent nitration and oxidation of cytoskeleton; 2) NF-kappaB activation causes cytoskeletal injury following upregulation of NO-driven processes; and 3) the molecular event underlying the destabilizing effects of NF-kappaB appears to be increases in carbonylation and nitrotyrosination of the subunit components of cytoskeleton. The ability to promote NO overproduction and cytoskeletal nitration/oxidation is a novel mechanism not previously attributed to NF-kappaB in cells.

Blotting, Western↗
PubMed2004

Theta-isoform of PKC is required for alterations in cytoskeletal dynamics and barrier permeability in intestinal epithelium: a novel function for PKC-theta.

Using intestinal Caco-2 cells, we previously showed that assembly of cytoskeleton is required for monolayer barrier function, but the underlying mechanisms remain poorly understood. Because the theta-isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC-theta is crucial for changes in cytoskeletal and barrier dynamics. We have created the first multiple sets of gastrointestinal cell clones transfected with varying levels of cDNA to stably inhibit native PKC-theta (antisense, AS; dominant negative, DN) or to express its activity (sense). We studied transfected and WT Caco-2 cells. First, relative to WT cells, AS clones underexpressing PKC-theta showed monolayer injury as indicated by decreased native PKC-theta activity, reduced tubulin phosphorylation, increased tubulin disassembly (decreased polymerized and increased monomeric pools), reduced architectural integrity of microtubules, reduced stability of occludin, and increased barrier hyperpermeability. In these AS clones, PKC-theta was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC-theta was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-theta/tubulin complex. Second, DN transfection to inhibit the endogenous PKC-theta led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC-theta led to a mostly cytosolic distribution of theta-isoform (<10% in particulate fractions), indicating its inactivation. In these sense clones, we also found disruption of occludin and microtubule assembly and increased barrier dysfunction. In conclusion, 1). PKC-theta isoform is required for changes in the cytoskeletal assembly and barrier permeability in intestinal monolayers, and 2). the molecular event underlying this novel biological effect of PKC-theta involves changes in phosphorylation and/or assembly of the subunit components of the cytoskeleton. The ability to alter the cytoskeletal and barrier dynamics is a unique function not previously attributed to PKC-theta.

Caco-2 Cells↗
PubMed2004

Inhibition of oxidant-induced nuclear factor-kappaB activation and inhibitory-kappaBalpha degradation and instability of F-actin cytoskeletal dynamics and barrier function by epidermal growth factor: key role of phospholipase-gamma isoform.

Using monolayers of intestinal (Caco-2) cells as a model for studying inflammatory bowel disease (IBD), we previously showed that nuclear factor-kappaB (NF-kappaB) activation is required for oxidant-induced disruption of cytoskeletal and barrier integrity. Epidermal growth factor (EGF) stabilizes the F-actin cytoskeleton and protects against oxidant damage, but the mechanism remains unclear. We hypothesized that the mechanism involves activation of phospholipase C-gamma (PLC-gamma), which prevents NF-kappaB activation and the consequences of this activation, namely, cytoskeletal and barrier disruption. We studied wild-type and transfected cells. The latter were transfected with varying levels (1-5 microg) of cDNA to either stably overexpress PLC-gamma or to inhibit its activation. Cells were pretreated with EGF before exposure to oxidant (H(2)O(2)). Stably overexpressing PLC-gamma (+2.0-fold) or preincubating with EGF protected against oxidant injury as indicated by 1) decreases in several NF-kappaB-related variables [NF-kappaB (p50/p65 subunit) nuclear translocation, NF-kappaB subunit activity, inhibitory-kappaBalpha (I-kappaBalpha) phosphorylation and degradation]; 2) increases in F-actin and decreases in G-actin; 3) stabilization of the actin cytoskeletal architecture; and 4) enhancement of barrier function. Overexpression induced inactivation of NF-kappaB was potentiated by EGF. PLC-gamma was found mostly in membrane and cytoskeletal fractions (<9% in the cytosolic fractions), indicating its activation. Dominant negative inhibition of endogenous PLC-gamma (-99%) substantially prevented all measures of EGF protection against NF-kappaB activation. We concluded 1) EGF protects against oxidant-induced barrier disruption through PLC-gamma activation, which inactivates NF-kappaB; 2) Activation of PLC-gamma by itself is protective against NF-kappaB activation; 3) the ability to modulate the dynamics of NF-kappaB/I-kappa Balpha is a novel mechanism not previously attributed to the PLC family of isoforms in cells; and 4) development of PLC-gamma mimetics represents a possible new therapeutic strategy for IBD.

Actins↗
PubMed2003

PKC-beta1 isoform activation is required for EGF-induced NF-kappaB inactivation and IkappaBalpha stabilization and protection of F-actin assembly and barrier function in enterocyte monolayers.

Using monolayers of intestinal Caco-2 cells, we reported that activation of NF-kappaB is required for oxidative disruption and that EGF protects against this injury but the mechanism remains unclear. Activation of the PKC-beta1 isoform is key to monolayer barrier integrity. We hypothesized that EGF-induced activation of PKC-beta1 prevents oxidant-induced activation of NF-kappaB and the consequences of NF-kappaB activation, F-actin, and barrier dysfunction. We used wild-type (WT) and transfected cells. The latter were transfected with varying levels of cDNA to overexpress or underexpress PKC-beta1. Cells were pretreated with EGF or PKC modulators +/- oxidant. Pretreatment with EGF protected monolayers by increasing native PKC-beta1 activity, decreasing IkappaBalpha phosphorylation/degradation, suppressing NF-kappaB activation (p50/p65 subunit nuclear translocation/activity), enhancing stable actin (increased F-actin-to-G-actin ratio), increasing stability of actin cytoskeleton, and reducing barrier hyperpermeability. Cells stably overexpressing PKC-beta1 were protected by low, previously nonprotective doses of EGF or modulators. In these clones, we found enhanced IkappaBalpha stabilization, NF-kappaB inactivation, actin stability, and barrier function. Low doses of the modulators led to increases in PKC-beta1 in the particulate fractions, indicating activation. Stably inhibiting endogenous PKC-beta1 substantially prevented all measures of EGF's protection against NF-kappaB activation. We conclude that EGF-mediated protection against oxidant disruption of the intestinal barrier function requires PKC-beta1 activation and NF-kappaB suppression. The molecular event underlying this unique effect of PKC-beta1 involves inhibition of phosphorylation and increases in stabilization of IkappaBalpha. The ability to inhibit the dynamics of NF-kappaB/IkappaBalpha and F-actin disassembly is a novel mechanism not previously attributed to the classic subfamily of PKC isoforms.

Actins↗
PubMed2003

Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China.

A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome (SARS). SCoV-like viruses were isolated from Himalayan palm civets found in a live-animal market in Guangdong, China. Evidence of virus infection was also detected in other animals (including a raccoon dog, Nyctereutes procyonoides) and in humans working at the same market. All the animal isolates retain a 29-nucleotide sequence that is not found in most human isolates. The detection of SCoV-like viruses in small, live wild mammals in a retail market indicates a route of interspecies transmission, although the natural reservoir is not known.

Amino Acid Sequence↗
PubMed2003

Zeta isoform of protein kinase C prevents oxidant-induced nuclear factor-kappaB activation and I-kappaBalpha degradation: a fundamental mechanism for epidermal growth factor protection of the microtubule cytoskeleton and intestinal barrier integrity.

Oxidant damage and gut barrier disruption contribute to the pathogenesis of a variety of inflammatory gastrointestinal disorders, including inflammatory bowel disease (IBD). In our studies using a model of the gastrointestinal (GI) epithelial barrier, monolayers of intestinal (Caco-2) cells, we investigated damage to and protection of the monolayer barrier. We reported that activation of nuclear factor-kappaB (NF-kappaB) via degradation of its endogenous inhibitor I-kappaBalpha is key to oxidant-induced disruption of barrier integrity and that growth factor (epidermal growth factor, EGF) protects against this injury by stabilizing the cytoskeletal filaments. Protein kinase C (PKC) activation seems to be required for monolayer maintenance, especially activation of the atypical zeta isoform of PKC. In an attempt to investigate, at the molecular level, the fundamental events underlying EGF protection against oxidant disruption, we tested the intriguing hypothesis that EGF-induced activation of PKC-zeta prevents oxidant-induced activation of NF-kappaB and the consequences of NF-kappaB activation, namely, cytoskeletal and barrier disruption. Monolayers of wild-type (WT) Caco-2 cells were incubated with oxidant (H2O2) with or without EGF or modulators. In other studies, we used the first gastrointestinal cell clones created by stable transfection of varying levels (1-5 microg) of cDNA to either overexpress PKC-zeta or to inhibit its expression. Transfected cell clones were then pretreated with EGF or a PKC activator (diacylglycerol analog 1-oleoyl-2-acetyl-glycerol, OAG) before oxidant. We monitored the following endpoints: monolayer barrier integrity, stability of the microtubule cytoskeleton, subcellular distribution and activity of the PKC-zeta isoform, intracellular levels and phosphorylation of the NF-kappaB inhibitor I-kappaBalpha, and nuclear translocation and activity of NF-kappaB subunits p65 and p50. Monolayers were also fractionated and processed to assess alterations in the structural protein of the microtubules, polymerized tubulin (S2), and monomeric tubulin (S1). Our data indicated that relative to WT monolayers exposed only to oxidant, pretreatment with EGF protected cell monolayers by 1) increasing native PKC-zeta activity; 2) decreasing several variables related to NF-kappaB activation [NF-kappaB (both p50 and p65 subunits) nuclear translocation, NF-kappaB subunits activity, I-kappaBalpha degradation, and phosphorylation]; 3) increasing stable tubulin (increased polymerized S2 tubulin and decreased monomeric S1 tubulin); 4) maintaining the cytoarchitectural integrity of microtubules; and 5) preventing hyperpermeability (barrier disruption). In addition, relative to WT cells exposed to oxidant, monolayers of transfected cells stably overexpressing PKC-zeta (approximately 3.0-fold increase) were protected as indicated by decreases in all measures of NF-kappaB activation as well as enhanced stability of microtubule cytoarchitecture and barrier function. Overexpression induced stabilization of I-kappaBalpha and inactivation of NF-kappaB was OAG-independent, although EGF potentiated this protection. Approximately 90% of the overexpressed PKC-zeta resided in particulate (membrane + cytoskeletal) fractions (with less than 10% in cytosolic fractions), indicating constitutive activation of the zeta isoform of PKC. Furthermore, antisense transfection to stably inhibit native PKC-zeta expression (-95%) and activation (-99%) prevented all measures of EGF-induced protection against NF-kappaB activation and monolayer disruption. We conclude the following: 1) EGF protects against oxidant disruption of the intestinal barrier integrity, in large part, through the activation of PKC-zeta and inactivation of NF-kappaB (an inflammatory mediator); 2) activation of PKC-zeta is by itself required for monolayer protection against oxidant stress of NF-kappaB activation; 3) the mechanism underlying this novel biological effect of the atypical PKC isoform zeta seems to involve suppression of phosphorylation and enhancement of stabilization of I-kappaBalpha; and 4) development of agents that can mimic or enhance PKC-zeta-induced suppression of NF-kappaB activation may be a useful therapeutic strategy for preventing oxidant damage to GI mucosal epithelium in disorders such as IBD. To our knowledge, this is the first report that PKC-zeta can inhibit the dynamics of NF-kappaB and cytoskeletal disassembly in cells.

Active Transport, Cell Nucleus↗
PubMed2003

Key role of PLC-gamma in EGF protection of epithelial barrier against iNOS upregulation and F-actin nitration and disassembly.

Upregulation of inducible nitric oxide synthase (iNOS) is key to oxidant-induced disruption of intestinal (Caco-2) monolayer barrier, and EGF protects against this disruption by stabilizing the cytoskeleton. PLC-gamma appears to be essential for monolayer integrity. We thus hypothesized that PLC-gamma activation is essential in EGF protection against iNOS upregulation and the consequent cytoskeletal oxidation and disarray and monolayer disruption. Intestinal cells were transfected to stably overexpress PLC-gamma or to inhibit its activation and were then pretreated with EGF +/- oxidant (H2O2). Wild-type (WT) intestinal cells were treated similarly. Relative to WT monolayers exposed to oxidant, pretreatment with EGF protected monolayers by: increasing native PLC-gamma activity; decreasing six iNOS-related variables (iNOS activity/protein, NO levels, oxidative stress, actin oxidation/nitration); increasing stable F-actin; maintaining actin stability; and enhancing barrier integrity. Relative to WT cells exposed to oxidant, transfected monolayers overexpressing PLC-gamma (+2.3-fold) were protected, as indicated by decreases in all measures of iNOS-driven pathway and enhanced actin and barrier integrity. Overexpression-induced inhibition of iNOS was potentiated by low doses of EGF. Stable inhibition of PLC-gamma prevented all measures of EGF protection against iNOS upregulation. We conclude that 1) EGF protects against oxidative stress disruption of intestinal barrier by stabilizing F-Actin, largely through the activation of PLC-gamma and downregulation of iNOS pathway; 2) activation of PLC-gamma is by itself essential for cellular protection against oxidative stress of iNOS; and 3) the ability to suppress iNOS-driven reactions and cytoskeletal oxidation and disassembly is a novel mechanism not previously attributed to the PLC family of isoforms.

Actin Cytoskeleton↗
PubMed2003

The delta-isoform of protein kinase C causes inducible nitric-oxide synthase and nitric oxide up-regulation: key mechanism for oxidant-induced carbonylation, nitration, and disassembly of the microtubule cytoskeleton and hyperpermeability of barrier of intestinal epithelia.

Using intestinal (Caco-2) cells, we found that oxidant-induced disruption of barrier integrity requires microtubule disassembly. Protein kinase C (PKC)-delta isoform seems to be essential for disruption, but the mechanism is unknown. Because inducible nitric-oxide synthase (iNOS) is key to oxidant stress, we hypothesized that PKC-delta activation is essential in oxidant-induced iNOS up-regulation and the consequent cytoskeletal oxidation and disarray and monolayer barrier dysfunction. Cells were transfected with an inducible plasmid to overexpress native PKC-delta or with a dominant-negative to inhibit the activity of native PKC-delta. Clones were then incubated with oxidant (H(2)O(2)) +/- modulators. Parental cells were treated similarly. Exposure to oxidant-disrupted monolayers by increasing native PKC-delta activity, increasing six iNOS-related variables (iNOS activity and protein, nitric oxide, oxidative stress, tubulin oxidation and nitration), decreasing polymerized tubulin, disrupting the cytoarchitecture of microtubules, and causing monolayer dysfunction. Induction of PKC-delta overexpression by itself (3.5-fold) led to oxidant-like disruptive effects, including activation of the iNOS-driven pathway. Overexpression-induced up-regulation of iNOS was potentiated by oxidants. iNOS inhibitors or oxidant scavengers were protective. Dominant inhibition of native PKC-delta activity (99.5%) prevented all measures of oxidant-induced iNOS up-regulation and protected the monolayer barrier. The conclusions are as follows. 1) Oxidants induce loss of epithelial barrier integrity by oxidizing and disassembling the cytoskeleton, in part, through the activation of PKC-delta and up-regulation of iNOS. 2) Overexpression and activation of PKC-delta are by themselves key for cellular injury by oxidative stress of iNOS. 3) We thus report a pathophysiological mechanism, activation of iNOS pathway and its injurious consequences to the cytoskeleton, including oxidation and nitration, among the "novel" subfamily of PKC isoforms.

Blotting, Western↗
PubMed2003

Migration and differentiation of adult rat subventricular zone progenitor cells transplanted into the adult rat striatum.

Adult brain subventricular zone progenitor cells undergo neurogenesis in the olfactory bulb. We tested the hypothesis that cultured adult subventricular zone progenitor cells migrate and differentiate into neurons when transplanted into the adult striatum. Cells in the adult rat subventricular zone were isolated and cultured for 8 days in medium containing basic fibroblast growth factor. These cells proliferated as assayed by bromodeoxyuridine immunostaining, and the majority of them were neuron-specific class III beta-tubulin (TuJ1) immunoreactive at 8 days of culture. These cultured cells were labeled in vitro with bromodeoxyuridine or with lipophilic dye-coated particles and were transplanted into the adult rat striatum. Twenty-eight days after transplantation, the cells migrated 0.5-1.5 mm from the midline of the graft to the surrounding host striatum. Migration of grafted cells in the host striatum was also detected on magnetic resonance imaging in living rats. Morphological analysis revealed that many of these migrated cells exhibited multibranched processes from the cell soma resembling host medium-size striatal projection neurons. Only a few astrocyte-like cells were detected. Double immunostaining showed that many bromodeoxyuridine immunoreactive cells were microtubule-associated protein 2 or immunoreactive with a mouse monoclonal antibody against neuronal nuclear protein, whereas only a few bromodeoxyuridine immunoreactive cells had glial fibrillary acidic protein immunoreactivity. Morphology of bromodeoxyuridine and microtubule-associated protein 2 immunoreactive cells was similar to those of host microtubule-associated protein 2 immunoreactive cells. These results suggest that transplanted cultured adult subventricular zone progenitor cells can migrate and differentiate in response to guidance cues within the adult striatum.

Age Factors↗
PubMed2002

Human marrow stromal cell therapy for stroke in rat: neurotrophins and functional recovery.

OBJECTIVE: To test the effect of i.v.-injected human bone marrow stromal cells (hMSC) on neurologic functional deficits after stroke in rats. METHODS: Rats were subjected to transient middle cerebral artery occlusion and IV injected with 3 x 10(6) hMSC 1 day after stroke. Functional outcome was measured before and 1, 7, and 14 days after stroke. Mixed lymphocyte reaction and the development of cytotoxic T lymphocytes measured the immune rejection of hMSC. A monoclonal antibody specific to human cellular nuclei (mAb1281) was used to identify hMSC and to measure neural phenotype. ELISA analyzed neurotrophin levels in cerebral tissue from hMSC-treated or nontreated rats. Bromodeoxyuridine injections were used to identify newly formed cells. RESULTS: Significant recovery of function was found in rats treated with hMSC at 14 days compared with control rats with ischemia. Few (1 to 5%) hMSC expressed proteins phenotypic of brain parenchymal cells. Brain-derived neurotrophic factor and nerve growth factor significantly increased, and apoptotic cells significantly decreased in the ischemic boundary zone; significantly more bromodeoxyuridine-reactive cells were detected in the subventricular zone of the ischemic hemisphere of rats treated with hMSC. hMSC induced proliferation of lymphocytes without the induction of cytotoxic T lymphocytes. CONCLUSION: Neurologic benefit resulting from hMSC treatment of stroke in rats may derive from the increase of growth factors in the ischemic tissue, the reduction of apoptosis in the penumbral zone of the lesion, and the proliferation of endogenous cells in the subventricular zone.

Animals↗