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Biomedical subjects

L Zhang

Publications and source records attributed to L Zhang.

At least 235 records · Page 13Linked to original sources
PubMed2002

Activation of delta-isoform of protein kinase C is required for oxidant-induced disruption of both the microtubule cytoskeleton and permeability barrier of intestinal epithelia.

Using monolayers of intestinal (Caco-2) cells, we showed that oxidants disassemble the microtubule cytoskeleton and disrupt barrier integrity (permeability) (Banan et al., 2000a). Because exposure of our parental cells to oxidants causes protein kinase C (PKC)-delta to be translocated to particulate fractions, we hypothesized that PKC-delta activation is required for these oxidant effects. Monolayers of parental Caco-2 cells were incubated with oxidant (H(2)O(2)) +/- modulators. Other cells were transfected with an inducible plasmid to stably overexpress PKC-delta or with a dominant negative plasmid to stably inhibit the activity of native PKC-delta. In parental cells, oxidants caused translocation of PKC-delta to the particulate (membrane + cytoskeletal) fractions, activation of PKC-delta isoform, increases in monomeric (S1) tubulin and decreases in polymerized (S2) tubulin, disruption of the microtubule cytoarchitecture, and loss of barrier integrity (hyperpermeability). In transfected cells, induction of PKC-delta overexpression by itself (3.5-fold over its basal level) led to oxidant-like disruptive effects. Disruption induced by PKC-delta overexpression was potentiated by oxidants. Overexpressed PKC-delta resided in particulate fractions, indicating its activation. Stable inhibition of native PKC-delta activity (98%) by dominant negative transfection substantially protected against all measures of oxidative disruption. We conclude that 1) oxidants induce loss of intestinal epithelial barrier integrity by disassembling the microtubules in large part through the activation of the PKC-delta isoform; and 2) overexpression and activation of PKC-delta is by itself a sufficient condition for disruption of these cytoskeleton and permeation pathways. Thus, PKC-delta activation may play a key role in intestinal dysfunction in oxidant-induced diseases such as inflammatory bowel disease.

Aminoglycosides↗
PubMed2002

The beta 1 isoform of protein kinase C mediates the protective effects of epidermal growth factor on the dynamic assembly of F-actin cytoskeleton and normalization of calcium homeostasis in human colonic cells.

Using intestinal monolayers, we showed that F-actin cytoskeletal stabilization and Ca(2+) normalization contribute to epidermal growth factor (EGF)-mediated protection against oxidant injury. However, the intracellular mediator responsible for these protective effects remains unknown. Since the protein kinase C-beta1 (PKC-beta1) isoform is abundant in our naive (N) cells, we hypothesized that PKC-beta1 is essential to EGF protection. Monolayers of N Caco-2 cells were exposed to H(2)O(2) +/- EGF, PKC, or Ca(2+) modulators. Other cells were transfected to over-express PKC-beta1 or to inhibit its expression and then pretreated with low or high doses of EGF or a PKC activator, OAG (1-oleoyl-2-acetyl-sn-glycerol), before H(2)O(2). In N monolayers exposed to oxidant, pretreatment with EGF or PKC activators activated PKC-beta1, enhanced (45)Ca(2+) efflux, normalized Ca(2+), decreased monomeric G-actin, increased stable F-actin, and protected the cytoarchitecture of the actin. PKC inhibitors prevented these protective effects. Transfected cells stably over-expressing PKC-beta1 (+3.1-fold) but not N cell monolayers were protected from injury by even lower doses of EGF or OAG. EGF or OAG rapidly activated the over-expressed PKC-beta1. Antisense inhibition of PKC-beta1 expression (-90%) prevented all measures of EGF protection. Inhibitors of Ca(2+)-ATPase prevented EGF protection in N cells as well as protective synergism in transfected cells. EGF protects the assembly of the F-actin cytoskeleton in intestinal monolayers against oxidants in large part through the activation of PKC-beta1. EGF normalizes Ca(2+) by enhancing Ca(2+) efflux through PKC-beta1. We have identified novel biologic functions, protection of actin and Ca(2+) homeostasis, among the classical isoforms of PKC.

Actins↗
PubMed2002

Alterations in NF-kappaB and RBP-Jkappa by arenavirus infection of macrophages in vitro and in vivo.

Pichinde virus is an arenavirus that infects guinea pigs and serves as an animal model for human Lassa fever. An attenuated Pichinde virus variant (P2) and a virulent variant (P18) are being used to delineate pathogenic mechanisms that culminate in shock. In guinea pigs, the infection has been shown to begin in peritoneal macrophages following intraperitoneal inoculation and then spreads to the spleen and other reticuloendothelial organs. We show here that infection of the murine monocytic cell line P388D1 with either Pichinde virus variant resulted in the induction of inflammatory cytokines and effectors, including interleukin-6 and tumor necrosis factor alpha. Since these genes are regulated in part by the cellular transcription factors NF-kappaB and RBP-Jkappa, we compared the activities of NF-kappaB and RBP-Jkappa in P388D1 cells following infection with Pichinde virus. The attenuated P2 virus inhibited NF-kappaB activation and caused a shift in the size of the RBP-Jkappa complex. The virulent P18 virus showed less inhibition of NF-kappaB and failed to alter the size of the RBP-Jkappa complex. Peritoneal cells from P2-infected guinea pigs showed induction of NF-kappaB RelA/p50 heterodimer and p50/p50 homodimer and manifested an increase in the size of RBP-Jkappa. By contrast, P18 induced large amounts of the NF-kappaB p50/p50 dimer but failed to induce RelA/p50 or to cause an increase in the RBP-Jkappa size. Taken together, these changes suggest that the attenuated viral strain induces an "activation" of macrophages, while the virulent form of the virus does not.

Animals↗
PubMed2002

PKC-zeta prevents oxidant-induced iNOS upregulation and protects the microtubules and gut barrier integrity.

Using intestinal (Caco-2) monolayers, we reported that inducible nitric oxide synthase (iNOS) activation is key to oxidant-induced barrier disruption and that EGF protects against this injury. PKC-zeta was required for protection. We thus hypothesized that PKC-zeta activation and iNOS inactivation are key in EGF protection. Wild-type (WT) Caco-2 cells were exposed to H(2)O(2) (0.5 mM) +/- EGF or PKC modulators. Other cells were transfected to overexpress PKC-zeta or to inhibit it and then pretreated with EGF or a PKC activator (OAG) before oxidant. Relative to WT cells exposed to oxidant, pretreatment with EGF protected monolayers by 1) increasing PKC-zeta activity; 2) decreasing iNOS activity and protein, NO levels, oxidative stress, tubulin oxidation, and nitration); 3) increasing polymerized tubulin; 4) maintaining the cytoarchitecture of microtubules; and 5) enhancing barrier integrity. Relative to WT cells exposed to oxidant, transfected cells overexpressing PKC-zeta (+2.9-fold) were protected as indicated by decreases in all measures of iNOS-driven pathways and enhanced stability of microtubules and barrier function. Overexpression-induced inhibition of iNOS was OAG independent, but EGF potentiated this protection. Antisense inhibition of PKC-zeta (-95%) prevented all measures of EGF protection against iNOS upregulation. Thus EGF protects against oxidative disruption of the intestinal barrier by stabilizing the cytoskeleton in large part through the activation of PKC-zeta and downregulation of iNOS. Activation of PKC-zeta is by itself required for cellular protection against oxidative stress of iNOS. We have thus discovered novel biologic functions, suppression of the iNOS-driven reactions and cytoskeletal oxidation, among the atypical PKC isoforms.

Caco-2 Cells↗
PubMed2002

PKC-zeta is required in EGF protection of microtubules and intestinal barrier integrity against oxidant injury.

Using monolayers of human intestinal (Caco-2) cells, we showed that epidermal growth factor (EGF) protects intestinal barrier integrity against oxidant injury by protecting the microtubules and that protein kinase C (PKC) is required. Because atypical PKC-zeta isoform is abundant in wild-type (WT) Caco-2 cells, we hypothesized that PKC-zeta mediates, at least in part, EGF protection. Intestinal cells (Caco-2 or HT-29) were transfected to stably over- or underexpress PKC-zeta. These clones were preincubated with low or high doses of EGF or a PKC activator [1-oleoyl-2-acetyl-sn-glycerol (OAG)] before oxidant (0.5 mM H(2)O(2)). Relative to WT cells exposed to oxidant, only monolayers of transfected cells overexpressing PKC-zeta (2.9-fold) were protected against oxidant injury as indicated by increases in polymerized tubulin and decreases in monomeric tubulin, enhancement of architectural stability of the microtubule cytoskeleton, and increases in monolayer barrier integrity toward control levels (62% less leakiness). Overexpression-induced protection was OAG independent and even EGF independent, but EGF significantly potentiated PKC-zeta protection. Most overexpressed PKC-zeta (92%) resided in membrane and cytoskeletal fractions, indicating constitutive activation of PKC-zeta. Stably inhibiting PKC-zeta expression (95%) with antisense transfection substantially attenuated EGF protection as demonstrated by reduced tubulin assembly and increased microtubule disassembly, disruption of the microtubule cytoskeleton, and loss of monolayer barrier integrity. We conclude that 1) activation of PKC-zeta is necessary for EGF-induced protection, 2) PKC-zeta appears to be an endogenous stabilizer of the microtubule cytoskeleton and of intestinal barrier function against oxidative injury, and 3) we have identified a novel biological function (protection) among the atypical isoforms of PKC.

Caco-2 Cells↗
PubMed2002

Analysis of mixed chimerism in patients after allogeneic stem cell transplantation using a capillary electrophoresis system.

We analyzed mixed chimerism (MC) after allogeneic stem cell transplantation (SCT) using a capillary electrophoresis system with four kinds of fluorescence-labeled primers for microsatellites (D3S1359, D6S89, ACTBP2, HGH). The sensitivities of all microsatellites were at least 3%. The present method is sufficiently rapid: only 3-4 h are needed to perform all the procedures. For analysis of MC in 30 patients who had undergone allogeneic SCT, heterozygosity of all microsatellites was over 88% and informativeness of ACTBP2 and HGH was over 73%. We analyzed MC using this technique to determine whether it was useful for prediction of the prognosis of 22 patients who had undergone allogeneic SCT. MC was more frequently observed in patients who were treated without total body irradiation (TBI) than in patients who were treated with TBI (p = 0.009). MC was also seen in a larger percentage of patients without acute graft-versus-host disease (p = 0.027). Six patients developed graft failure or relapse among 12 patients with MC. The graft failure or relapse was higher in patients with MC than in patients with complete chimerism (CC) (p = 0.009) especially if they were over 30 years of age (p = 0.0005). In contrast, graft failure or relapse was not higher in patients with MC compared with patients with CC under 30 years of age (p = 0.78). These results show that MC is an important predictive factor, especially in patients over 30 years of age.

Adolescent↗
PubMed2002

Apolipoprotein E-deficient mice created by systemic administration of antisense oligodeoxynucleotides: a new model for lipoprotein metabolism studies.

Atherosclerotic cardiovascular disease results from complex interactions among multiple genetic and environmental factors. Thus, it is important to elucidate the influence of each factor on cholesterol metabolism. For this purpose, transgenic/gene-targeting technology is a powerful tool for studying gene functions. However, this technology has several disadvantages such as being time consuming and expensive. Accordingly, we established new animal models using in vivo gene transfer technology. In this study, we examined the feasibility of the creation of a new animal model for the study of atherosclerosis. We hypothesized that apolipoprotein (apo) E-deficient mice can be created by systemic administration of antisense apo E oligodeoxynucleotides (ODN) coupled to the HVJ-liposome complex. Initially, we examined the localization and cellular fate of FITC-labeled antisense ODN administered intravenously. FITC-labeled ODN transfection by the HVJ-liposome method resulted in fluorescence in the liver, spleen and kidney, but not in other organs such as brain. Moreover, fluorescence with the HVJ-liposome method was sustained for up to 2 weeks after transfection, which resulted in a striking difference from transfection of ODN alone or ODN in liposomes without HVJ, which showed rapid disappearance of fluorescence (within 1 day). Given these unique characteristics of the HVJ-liposome method, we next examined transfection of antisense apo E ODN by intravenous administration. Transfection of antisense apo E ODN resulted in a marked reduction of apo E mRNA levels in the liver, but no change in apo B and beta-actin mRNA levels. In mice fed a normal diet, a transient increase in cholesterol and triglyceride levels was observed in the antisense apo E-treated group, but they returned to normal levels by 6 days after transfection. Similar findings were also found in mice fed a high cholesterol diet. Neither scrambled nor mismatched ODN resulted in any increase in cholesterol. To make chronic hypercholesterolemic mice, we therefore performed repeated injections of apo E antisense ODN. Whenever antisense apo E ODN were injected, mice showed a transient increase in cholesterol and triglyceride. Cumulative administration of antisense apo E ODN resulted in a sustained increase in cholesterol for up to 3 weeks after the last transfection. Finally, mice treated with repeated injections of antisense apo E every week developed sustained hypercholesterolemia and hypertriglyceridemia until withdrawal of injections. Apolipoprotein-deficient mice created by intravenous administration of antisense ODN are a promising new animal model to help understand the role of apolipoprotein in vivo and develop a new drug therapy targeting apolipoprotein.

Animals↗
PubMed2002

Nonradioactive assay of FLAG-tagged MAPK using ANTI-FLAG antibody-coated multiwell plates.

We have developed a rapid, sensitive, and quantitative 96-well microplate-based nonradioactive immunoprecipitation/kinase assay to evaluate mitogen-activated protein kinase (MAPK) activity. Three quantitative nonradioactive imunoprecipitation/kinase assays of MAPK were demonstrated on a 96-well microplate coated with ANTI-FLAG M2 antibody (ANTI-FLAG M2 plate): (i) the capture of phosphorylated FLAG-tagged MAPK fusion protein (FLAG-MAPK) from phorbol esters-stimulated, FLAG-MAPK-transfected COS-7 cells, coupled with a very sensitive ELISA procedure to quantitate the level of phosphorylation of FLAG-MAPK; (ii) the in vitro kinase reaction of FLAG-MAPK activity with a substrate and ATP in the same well used to captured the phosphorylated FLAG-MAPK; and (iii) the in vitro kinase reaction of captured non-activated FLAG-MAPK by its upstream kinase from phorbol 12-myristate 13-acetate (PMA)-stimulated COS-7 cells. These results demonstrate that the ANTI-FLAG M2 plate allows for the rapid and quantitative determination of phosphorylation of FLAG-MAPK directly from stimulated, transfected cell lysate. Captured, phosphorylated FLAG-MAPK retains catalytic activity as demonstrated by the phosphorylation of Elk-1 in the same well. Furthermore, phosphorylation of captured FLAG-MAPK by the upstream kinases can be observed directly on the plate. These assays are sensitive, specific, and suitable for handling multiple samples. Thus, the ANTI-FLAG M2 plate forms the basis of a high-throughput screening platform in kinase analysis.

Animals↗
PubMed2002

Hyaluronan metabolism in rat tail skin following blockage of the lymphatic circulation.

This study was undertaken to explore the effects of lymphatic blockage on the metabolism of hyaluronan in the skin. In initial experiments, [3H] hyaluronan was injected subcutaneously into the tail skin of rats that either had no surgical intervention (control) or into those that had their lymphatic drainage blocked two hours earlier (acute lymphedema) or after the lymphatics had been blocked for three months (chronic lymphedema). The removal of tritiated hyaluronan from the injection sites was determined by the appearance of [3H] in the plasma. The results showed that the clearance of injected hyaluronan was delayed in rats with lymphatic blockage. The half- life of injected hyaluronan in the controls was approximately 70-75 hr, compared with approximately 105-110 hr in the lymph blocking rats. The levels of radioactivity in the plasma from rats with both acute and chronically blocked lymphatics were lower than that of control rats during the entire follow up period. In addition, biochemical analysis revealed that there was a significant increased amount of hyaluronan in the tail skin three months after lymphatic blocking. These results suggest that lymph absorption is an important factor in the transport of hyaluronan from the interstitium. Blockage of regional draining lymphatics likely impairs the catabolism of hyaluronan, which stagnates in skin tissue.

Animals↗
PubMed2002

[Cloning and expression of gynecophoral canal protein gene of Schistosoma japonicum (Chinese strain)].

A 1949 bp cDNA fragment was amplified by RT-PCR from adult Schistosoma japonicum (Chinese strain) mRNA with 3 pair of primers that were designed according to published SmGCP gene encoding gynecophoral canal protein of Schistosoma mansoni and SjGCP1 gene encoding the conservative region of gynecophoral canal protein of Schistosoma japonicum. Sequence analysis indicated that this fragment, named SjGCP, with 85% identity to SmGCP, contained a complete open reading frame (ORF) of gynecophoral canal protein gene of Schistosoma japonicum (Chinese strain). The amino acid sequence shared 83.7% identity with gynecophoral canal protein of Schistosoma mansoni. This fragment was cloned into the expression vector pET28c(+) and subsequently expressed in Escherichia coli. SDS-PAGE revealed that the molecular weight of this expressed product was 80 kD. Western blotting showed that the recombinant protein reacted well with the rabbit serum immunized with Sj worm antigen, indicating that this expressed product had good antigenicity.

Amino Acid Sequence↗
PubMed2001

Nuclear export of phosphorylated C/EBPbeta mediates the inhibition of albumin expression by TNF-alpha.

Decreased albumin expression is a frequent feature of cachexia patients afflicted with chronic diseases, including cancer, and a major contributor to their morbidity. Here we show that tumor necrosis-alpha (TNF-alpha) treatment of primary mouse hepatocytes or TNF-alpha overexpression in a mouse model of cachexia induces oxidative stress, nitric oxide synthase (NOS) expression and phosphorylation of C/EBPbeta on Ser239, within the nuclear localization signal, thus inducing its nuclear export, which inhibits transcription from the albumin gene. SIN-1, a NO donor, duplicated the TNF-alpha effects on hepatocytes. We found similar molecular abnormalities in the liver of patients with cancer-cachexia. The cytoplasmic localization and association of C/EBPbeta-PSer239 with CRM1 (exportin-1) in TNF-alpha-treated hepatocytes was inhibited by leptomycin B, a blocker of CRM1 activity. Hepatic cells expressing the non-phosphorylatable C/EBPbeta alanine mutant were refractory to the inhibitory effects of TNF-alpha on albumin transcription since the mutant remained localized to the nucleus. Treatment of TNF-alpha mice with antioxidants or NOS inhibitors prevented phosphorylation of C/EBPbeta on Ser239 and its nuclear export, and rescued the abnormal albumin gene expression.

Active Transport, Cell Nucleus↗
PubMed2001

Postischemic (6-Hour) treatment with recombinant human tissue plasminogen activator and proteasome inhibitor PS-519 reduces infarction in a rat model of embolic focal cerebral ischemia.

BACKGROUND AND PURPOSE: The proteasome inhibitor PS-519 blocks activation of nuclear factor-kappaB, a major mediator of inflammation. We tested the hypothesis that combination treatment of recombinant human tissue plasminogen activator (rhtPA) and PS-519 extends the therapeutic window for treatment of stroke with rhtPA without increasing incidence of hemorrhagic transformation. METHODS: The middle cerebral artery (MCA) of male Wistar rats (n=56) was occluded by an embolus. After embolization, animals were randomly divided into the following groups: PS-519 treatment groups: PS-519 was given at 2, 4, or 6 hours after MCA occlusion; rhtPA treatment groups: rhtPA was given at 2 or 4 hours after MCA occlusion; combination treatment groups: PS-519 and rhtPA were given at 2, 4, or 6 hours after MCA occlusion; control group: the same volume of saline was given at 2 hours after MCA occlusion. RESULTS: Administration of PS-519 alone at 2 or 4 hours, but not 6 hours, significantly (P<0.05) reduced infarct volume and improved neurological recovery compared with the control group. Administration of rhtPA alone at 2 hours, but not 4 hours, significantly (P<0.05) reduced infarct volume and improved neurological recovery compared with the control group. Furthermore, combination treatment with rhtPA and PS-519 even at 6 hours significantly (P<0.05) reduced infarct volume, improved neurological recovery, and did not increase the incidence of hemorrhagic transformation compared with the control group or the group treated with PS-519 alone. CONCLUSIONS: Our data suggest that combination treatment with PS-519 and rhtPA extends the neuroprotective effect to at least 6 hours after embolization.

Acetylcysteine↗
PubMed2001

Striatal neuronal loss or dysfunction and choline rise in children with attention-deficit hyperactivity disorder: a 1H-magnetic resonance spectroscopy study.

Twelve previously untreated boys suffering from attention-deficit hyperactivity disorder (ADHD) were investigated by using proton magnetic resonance spectroscopy (1H MRS) before and after one dose (10 mg) of methylphenidate. Pre- and post-methylphenidate spectra were acquired bilaterally in the globus pallidus. Peaks of N-acetylaspartate (NAA), choline (Cho), myo-inositol, glutamate and creatine (Cr) were measured and the ratios of the peaks were calculated and compared with data from ten matched controls. In children having ADHD, NAA/Cr ratio decreased significantly in the bilateral striatum while Cho/Cr ratio showed a mild unilateral increase. One oral dose of methylphenidate did not affect the ratios significantly. These findings suggest that the striatum was bilaterally involved in pediatric ADHD patients. Approximately 20-25% of neurons may have died or may be severely dysfunctional. There seems to be a mild hyperactivity of the cholinergic system.

Adolescent↗
PubMed2001

Numerical estimation of multicomponent adsorption isotherms in preparative chromatography: implications of experimental error.

Since experimental methods for measuring multicomponent adsorption isotherms are extremely tedious, numerical approaches are an attractive alternative. Here, the variance in isotherm parameters as a function of experimental error in measured effluent concentrations is quantified. The number of experimental replicates needed to obtain isotherm parameters to a desired level of accuracy is calculated explicitly. After the covariance matrix of the parameters has been determined, Monte Carlo methods are found to be rapid and effective. The use of different kinds of experiments, the effect of resolution and loading, and the impact of the number of measured data points are described.

Adsorption↗
PubMed2001

Reassortant analysis of guinea pig virulence of pichinde virus variants.

The new world arenavirus Pichinde (PIC) is the basis of an accepted small animal model for human Lassa fever. PIC (Munchique strain) variant P2 is attenuated in guinea pigs, whereas variant P18 is extremely virulent. Previous sequence analysis of the S segments of these two viruses indicated a small number of possible virulence markers in the glycoprotein precursor (GPC) and nucleoprotein (NP) genes. In order to determine the role of these S segment genes in guinea pig virulence in this system, we have generated reassortant viruses. When tested in outbred guinea pigs, the reassortant containing the S segment from the virulent parent P18 (S18L2) caused significantly higher morbidity than the reciprocal reassortant. This increased morbidity was associated with higher viral titers in serum and spleen. However, the S18L2 reassortant was not as fully virulent in this system as the P18 parent, indicating a role for L segment genes in virulence.

Animals↗