Search PubMed⌕ Search

Biomedical subjects

X Meng

Publications and source records attributed to X Meng.

At least 19 recordsLinked to original sources
PubMed1999

p38 MAPK inhibition decreases TNF-alpha production and enhances postischemic human myocardial function.

INTRODUCTION: TNF-alpha is a proinflammatory cytokine implicated in myocardial dysfunction following ischemia/reperfusion (I/R). I/R results in myocardial production of TNF-alpha and TNF-alpha suppresses myocardial contractility. p38 mitogen-activated protein kinase (MAPK) is a redox-sensitive protein kinase involved in intracellular signaling leading to TNF-alpha production. It remains unknown if the human heart produces TNF-alpha after I/R and, if so, whether p38 MAPK is involved. HYPOTHESIS: p38 MAPK inhibition enhances human myocardial post-I/R contractile function by inhibition of myocardial TNF-alpha production. METHODS: Human atrial trabeculae were suspended in organ baths, field simulated at 1 Hz, and force development was recorded. Following a 90-min equilibration, trabeculae were exposed to a p38 MAPK inhibitor (SB 203580, 1 microM) or vehicle (each n = 6) prior to simulated ischemia (45 min hypoxia, substrate-free, rapid pacing at 3 Hz) followed by 120 min reoxygenation. Myocardial TNF-alpha levels were measured by ELISA at end reoxygenation. RESULTS: I/R increased human myocardial TNF-alpha levels from 26.9 +/- 9.3 to 83.9 +/- 19.2 pg/g wet tissue (P < 0.05 perfusion vs I/R; ANOVA Bonferroni/Dunn), while p38 MAPK inhibition decreased post-I/R myocardial TNF-alpha levels to 32.3 +/- 8.0 pg/g wet tissue (P > 0.05 p38 MAPK inhibition vs I/R). p38 MAPK inhibition improved postischemic force development from 18.5 +/- 2.1 to 37.0 +/- 2.0% baseline developed force (%BDF; P < 0.05 I/R vs p38 MAPK inhibition). CONCLUSIONS: (1) The human heart produces TNF-alpha after I/R, (2) p38 MAPK mediates myocardial I/R-induced TNF-alpha production, (3) p38 MAPK inhibition limits functional impairment after I/R, and (4) inhibition of ischemia-induced TNF-alpha production may represent a potent therapeutic strategy for improving myocardial function after angioplasty, coronary bypass, or heart transplantation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

LPS-Induced NF-kappaB activation and TNF-alpha release in human monocytes are protein tyrosine kinase dependent and protein kinase C independent.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) is an important mediator of septic shock. Endotoxin (LPS) signal transduction in human monocytes leads to activation of nuclear factor-kappa B (NF-kappaB) and TNF-alpha release. Previous studies have implicated activation of both protein kinase C (PKC) and protein tyrosine kinases (PTK) in LPS-induced NF-kappaB activation and TNF-alpha production. We hypothesized that inhibition of either PKC or PTK would decrease LPS-induced NF-kappaB DNA binding and TNF-alpha release in human monocytes. MATERIALS AND METHODS: Human monocytes were stimulated with PMA (50 ng/ml) alone or LPS (100 ng/ml) with and without a nonspecific serine/threonine protein kinase inhibitor staurosporine (Stauro), a specific pan-PKC inhibitor bisindolylmaleimide (Bis), or an inhibitor of PTK genistein (Gen). TNF-alpha release in culture supernatants was measured by an ELISA. NF-kappaB DNA binding was evaluated by electrophoretic mobility shift assay. RESULTS: LPS increased NF-kappaB DNA binding and TNF-alpha release in human monocytes. Nonspecific protein kinase inhibition inhibited NF-kappaB activation and TNF-alpha release, while specific PKC inhibition with Bis had no effect on LPS-induced NF-kappaB DNA binding or TNF-alpha release. PTK inhibition with Gen attenuated both LPS-induced NF-kappaB DNA binding and TNF-alpha production in human monocytes. Direct activation of PKC with PMA induced both NF-kappaB activation and TNF-alpha production by human monocytes. CONCLUSIONS: These results suggest that LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes are independent of PKC activity. Furthermore, our results provide evidence that PTK plays a role in LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes and thus could be a potential therapeutic target in inflammatory states.

Cells, Cultured↗
PubMed1999

Liposomal delivery of purified inhibitory-kappaBalpha inhibits tumor necrosis factor-alpha-induced human vascular smooth muscle proliferation.

Vessel injury results in the elaboration of various cytokines, including tumor necrosis factor-alpha (TNF-alpha), which may influence vascular smooth muscle cell (VSMC) function and contribute to atherogenesis. We tested the hypothesis that TNF-alpha-induced VSMC proliferation requires activation of the transcription factor nuclear factor-kappaB (NF-kappaB), which could be prevented by delivery of the NF-kappaB inhibitory peptide, IkappaBalpha. TNF-alpha induced concentration-dependent human VSMC proliferation, and neutralizing antibody to interleukin-6 reduced TNF-alpha-induced VSMC proliferation by 65%. In TNF-alpha-stimulated VSMCs, there was a 3-fold increase in NF-kappaB-dependent luciferase reporter activity that was associated with degradation of IkappaBalpha. To determine an essential role for NF-kappaB in TNF-alpha-induced VSMC proliferation, recombinant IkappaBalpha was introduced into VSMCs via liposomal delivery. Under these conditions, TNF-alpha-induced NF-kappaB nuclear translocation and DNA binding were inhibited, NF-kappaB-dependent luciferase activity was reduced by 50%, there was no degradation of native IkappaBalpha detected, interleukin-6 production was reduced by 54%, and VSMC proliferation was decreased by 60%. In conclusion, the mitogenic effect of TNF-alpha on human arterial VSMCs is dependent on NF-kappaB activation and may be prevented by exogenously delivered IkappaBalpha. Furthermore, liposomal delivery of endogenous inhibitory proteins may represent a novel, therapeutically accessible method for selective transcriptional suppression in the response to vascular injury.

Cell Division↗
PubMed1999

Toll receptor-mediated Drosophila immune response requires Dif, an NF-kappaB factor.

The induction of immunity genes in Drosophila has been proposed to be dependent on Dorsal, Dif, and Relish, the NF-kappaB-related factors. Here we provide genetic evidence that Dif is required for the induction of only a subset of antimicrobial peptide genes. The results show that the presence of Dif without Dorsal is sufficient to mediate the induction of drosomycin and defensin. We also demonstrate that Dif is a downstream component of the Toll signaling pathway in activating the drosomycin expression. These results reveal that individual members of the NF-kappaB family in Drosophila have distinct roles in immunity and development.

Androgen-Binding Protein↗
PubMed1999

Delimitation of two regions in the 90-kDa heat shock protein (Hsp90) able to interact with the glucocorticosteroid receptor (GR).

The role of the 90-kDa heat shock protein (Hsp90) as a chaperone and its regulatory functions for cellular proteins such as the glucocorticosteroid receptor (GR) depends on the direct interaction of the Hsp90 with the corresponding protein as part of a multiprotein complex. The search for the amino acid sequence(s) in Hsp90 involved in interaction with the human GR has been carried out by mutational deletion analysis in whole cells, studying the effects of interaction on the nucleocytoplasmic distributions of transiently expressed Hsp90 and GR derivatives in COS-7 cells. Using a recently developed confocal microscopic immunofluorescence method that allows quantification of the nucleocytoplasmic ratios of the proteins in individual cells and statistical comparison of cell populations, two subregions of the Hsp90 molecule have been defined that allow interaction with GR (residues 206-291 and 446-581). The latter region may play a fundamental role in the interaction, while the former may merely stabilize the binding to GR of the intact Hsp90 molecule. Moreover, the dissection of the Hsp90 molecule allowed us to define two regions displaying nuclear localization activity (residues 1-206 and 381-581), followed by two regions having a predominantly cytoplasmic localization activity (residues 287-381 and 581-728) and counteracting the nuclear localization activities.

Animals↗
PubMed1999

The molecular chaperone Hsp90 can negatively regulate the activity of a glucocorticosteroid-dependent promoter.

Hsp90, a molecular chaperone required for the functioning of glucocorticosteroid receptor (GR), ensures, by direct interaction, the conformational competence of the steroid-binding pocket. In addition to having this positive function, Hsp90 maintains steroid receptors in an inactive form in the absence of hormone. However, neither the participation of Hsp90 once the pathway has been activated by the ligand nor the importance of increased Hsp90 levels in determining the amplitude of the response has ever been assessed directly. Here, by increasing the Hsp90/GR ratio in the nuclear compartment, we found an attenuation of the response to glucocorticosteroids that was not due to a nonspecific or toxic effect of the Hsp90 modified by nuclear targeting. Since this negative effect was more pronounced at high levels of hormone, when receptor and Hsp90 are maximally dissociated, the possibility of an interaction between Hsp90 and GR, already activated to a DNA-binding form, was directly investigated. Indeed GR, after in vivo activation by ligand, was still able to reassociate with Hsp90, suggesting that this interaction plays a role in vivo, possibly in receptor recycling. Moreover, the GR binding to its DNA response element was inhibited by an excess of Hsp90, pointing to a function of Hsp90 in the nuclear compartment. It is thus proposed that an increased Hsp90/GR ratio influences the responsiveness to ligand at a step that is after receptor activation. This increased ratio may be of pathophysiological relevance in the different circumstances that lead to an elevated level of nuclear Hsp90.

Amino Acid Sequence↗
PubMed1999

Isolation and characterisation of neurotoxigenic Clostridium butyricum from soil in China.

Soil specimens collected from a site around the home of patients with food-borne type E. botulism probably caused by neurotoxigenic Clostridium butyricum in Guanyun, Jiangsu province, China, were examined for the presence of neurotoxigenic C. butyricum. Five lakeside sites of Weishan lake, in an area near to the sites where the type E. botulism outbreaks caused by neurotoxigenic C. butyricum occurred were also surveyed. Type E toxin-producing C. butyricum was isolated from soil from four sites including the site in Guanyun. Polymerase chain reaction assay demonstrated the presence of the type E toxin gene in all the toxigenic isolates. The biochemical properties of the isolates from the Guanyun soil and the lakeside soil were identical except for inulin fermentation and starch hydrolysis properties. These results indicate that neurotoxigenic C. butyricum has its principal habitat in soil.

Bacterial Toxins↗
PubMed1999

Adrenergic induction of bimodal myocardial protection: signal transduction and cardiac gene reprogramming.

This study tested the hypothesis that in vivo norepinephrine (NE) treatment induces bimodal cardiac functional protection against ischemia and examined the roles of alpha1-adrenoceptors, protein kinase C (PKC), and cardiac gene expression in cardiac protection. Rats were treated with NE (25 micrograms/kg iv). Cardiac functional resistance to ischemia-reperfusion (25/40 min) injury was examined 30 min and 1, 4, and 24 h after NE treatment with the Langendorff technique, and effects of alpha1-adrenoceptor antagonism and PKC inhibition on the protection were determined. Northern analysis was performed to examine cardiac expression of mRNAs encoding alpha-actin and myosin heavy chain (MHC) isoforms. Immunofluorescent staining was performed to localize PKC-betaI in the ventricular myocardium. NE treatment improved postischemic functional recovery at 30 min, 4 h, and 24 h but not at 1 h. Pretreatment with prazosin or chelerythrine abolished both the early adaptive response at 30 min and the delayed adaptive response at 24 h. NE treatment induced intranuclear translocation of PKC-betaI in cardiac myocytes at 10 min and increased skeletal alpha-actin and beta-MHC mRNAs in the myocardium at 4-24 h. These results demonstrate that in vivo NE treatment induces bimodal myocardial functional adaptation to ischemia in a rat model. alpha1-Adrenoceptors and PKC appear to be involved in signal transduction for inducing both the early and delayed adaptive responses. The delayed adaptive response is associated with the expression of cardiac genes encoding fetal contractile proteins, and PKC-betaI may transduce the signal for reprogramming of cardiac gene expression.

Actins↗
PubMed1998

A novel human gene, WSTF, is deleted in Williams syndrome.

Williams syndrome (WS) is a developmental disorder caused by deletion of multiple genes at chromosome 7q11.23. Here, we report the identification and characterization of a novel gene, WSTF, that maps to the common WS deletion region. WSTF encodes a novel protein of 1425 amino acids with unknown function. It contains one PHD-type zinc finger motif followed by a bromodomain. Both motifs are found in many transcription regulators, suggesting that WSTF may function as a transcription factor. WSTF is ubiquitously expressed in both adult and fetal tissues. The WSTF gene consists of 20 exons spanning about 80 kb. Fluorescence in situ hybridization analysis shows that WSTF is deleted in 50/50 WS individuals. Hemizygous deletion of WSTF may contribute to WS.

Amino Acid Sequence↗
PubMed1998

Osteoblast recruitment and bone formation enhanced by cell matrix-associated heparin-binding growth-associated molecule (HB-GAM).

Bone has an enormous capacity for growth, regeneration, and remodeling. This capacity is largely due to induction of osteoblasts that are recruited to the site of bone formation. The recruitment of osteoblasts has not been fully elucidated, though the immediate environment of the cells is likely to play a role via cell- matrix interactions. We show here that heparin-binding growth-associated molecule (HB-GAM), an extracellular matrix-associated protein that enhances migratory responses in neurons, is prominently expressed in the cell matrices that act as target substrates for bone formation. Intriguingly, N-syndecan, which acts as a receptor for HB-GAM, is expressed by osteoblasts/osteoblast precursors, whose ultrastructural phenotypes suggest active cell motility. The hypothesis that HB-GAM/N-syndecan interaction mediates osteoblast recruitment, as inferred from developmental studies, was tested using osteoblast-type cells that express N-syndecan abundantly. These cells migrate rapidly to HB-GAM in a haptotactic transfilter assay and in a migration assay where HB-GAM patterns were created on culture wells. The mechanism of migration is similar to that previously described for the HB-GAM-induced migratory response of neurons. Our hypothesis that HB-GAM/N-syndecan interaction participates in regulation of osteoblast recruitment was tested using two different in vivo models: an adjuvant-induced arthritic model and a transgenic model. In the adjuvant-induced injury model, the expression of HB-GAM and of N-syndecan is strongly upregulated in the periosteum accompanying the regenerative response of bone. In the transgenic model, the HB-GAM expression is maintained in mesenchymal tissues with the highest expression in the periosteum. The HB-GAM transgenic mice develop a phenotype characterized by an increased bone thickness. HB-GAM may thus play an important role in bone formation, probably by mediating recruitment and attachment of osteoblasts/osteoblast precursors to the appropriate substrates for deposition of new bone.

Animals↗
PubMed1998

Induction of keratinocyte proliferation and lymphocytic infiltration by in vivo introduction of the IL-6 gene into keratinocytes and possibility of keratinocyte gene therapy for inflammatory skin diseases using IL-6 mutant genes.

To understand biological function of IL-6 in the skin in vivo, we constructed a vector that strongly expressed human IL-6 in keratinocytes and introduced it into rat keratinocytes in vivo by the naked DNA method. The overexpression of IL-6 induced macroscopic erythema and histologically evident keratinocyte proliferation and lymphocytic infiltration in the treated area of rat skin. Since previous studies using IL-6 transgenic mice have not shown skin inflammation of these mice, our result provides the first evidence that IL-6 is related to the pathogenesis of inflammatory skin diseases. ELISA suggested that a certain degree of transgenic IL-6 expression in keratinocytes was required for inducing skin inflammation. Cytokine profile in rat keratinocytes after the gene introduction was examined by reverse transcriptase-PCR assay and revealed that gene expression of rat IL-1alpha and TNF-alpha showed no marked change until 24 h, whereas that of rat IL-6 and TGF-alpha increased with time. We then introduced and expressed the IL-6 mutant genes, which were designed to behave as IL-6Ralpha antagonists, and found that their ability to induce erythema was lower than that of the wild-type gene. Furthermore, preintroduction of some mutant genes delayed the erythema induced by postintroduction of the wild-type IL-6 gene, suggesting that the mutant forms of IL-6 prevent wild-type IL-6 from binding to IL-6Ralpha. This result indicates that keratinocyte gene therapy may be possible for inflammatory skin diseases using IL-6 mutant genes.

Animals↗
PubMed1998

Ischemic preconditioning decreases postischemic myocardial tumor necrosis factor-alpha production. Potential ultimate effector mechanism of preconditioning.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is an autocrine contributor to myocardial dysfunction and cardiomyocyte death in ischemia-reperfusion (I/R) injury, sepsis, chronic heart failure, and cardiac allograft rejection. Cardiac resident macrophages, infiltrating leukocytes, and cardiomyocytes themselves produce TNF-alpha. Although adenosine reduces macrophage TNF-alpha production and protects myocardium against I/R, it remains unknown whether ischemic preconditioning, which is mediated by adenosine, decreases postischemic myocardial TNF-alpha production. METHODS AND RESULTS: Isolated rat hearts were crystalloid perfused with the Langendorff method and subjected to global, normothermic I/R (20/40 minutes), with or without prior transient ischemic preconditioning (5 minutes) or adenosine pretreatment. Postischemic cardiac TNF-alpha (ELISA) and function were determined (Langendorff). I/R increased cardiac TNF-alpha and impaired myocardial function. Ischemic preconditioning or adenosine decreased myocardial TNF-alpha and improved postischemic functional recovery. Sequestration of myocardial TNF-alpha (TNF binding protein) during the I/R experiments similarly improved postischemic myocardial function. CONCLUSIONS: This study constitutes the initial demonstration that in addition to its other beneficial effects, preconditioning decreases postischemic myocardial TNF-alpha, an autocrine contributor to postischemic myocardial dysfunction. Reduced myocardial TNF-alpha production may represent the distal effector mechanism of preconditioning.

Animals↗
PubMed1998

A novel human gene FKBP6 is deleted in Williams syndrome.

Williams syndrome (WS) is a developmental disorder caused by haploinsufficiency of genes at 7q11.23. We have shown that hemizygosity of elastin is responsible for one feature of WS, supravalvular aortic stenosis. We have also implicated LIM-kinase 1 hemizygosity as a contributing factor to impaired visual-spatial constructive cognition in WS. Here we identify and characterize a novel gene, FKBP6, within the common WS deletion region. FKBP6 shows homology to the FK-506 binding protein (FKBP) class of immunophilins. FKBP6 has a putative N-terminal FK-506 binding and peptidylproyl isomerase (rotamase) domain and, like known high-molecular-weight FKBPs, an imperfect C-terminal tetratricopeptide repeat domain. FKBP6 is expressed in testis, heart, skeletal muscle, liver, and kidney. FKBP6 consists of nine exons and is completely contained within a 35-kb cosmid clone. Fluorescence in situ hybridization experiments show that FKBP6 gene is deleted in 40/40 WS individuals. Hemizygous deletion of FKBP6 may contribute to certain defects such as hypercalcemia and growth delay in WS.

Amino Acid Sequence↗
PubMed1998

Quantification of the nucleocytoplasmic distribution of wild type and modified proteins using confocal microscopy: interaction between 90-kDa heat shock protein (Hsp90 alpha) and glucocorticosteroid receptor (GR).

The investigation of molecular interactions in whole cells by immunofluorescence was developed recently, based on the targeting of the protein partners to different cellular compartments and analysis of the modifications in their subcellular distribution resulting from their interaction. This paper describes the adaptation of the confocal microscopy to the quantification of the partitioning of transiently coexpressed proteins between nucleus and cytoplasm. We defined a nucleocytoplasmic ratio R, corresponding to the difference between nuclear and cytoplasmic fluorescence intensities divided by their sum (N - C/N + C), which does not refer to absolute fluorescence intensities. Interaction was detected by statistically comparing the distribution of R value frequencies in cell populations expressing one or both proteins. The convenience of this whole cell method was demonstrated by detecting and analyzing interaction between the human glucocorticosteroid receptor (GR) and the chick 90-kDa heat shock protein (Hsp90), using various combinations of wild-type and nuclear- or cytoplasmic-targeted GR and Hsp90. In addition, three Hsp90 deletion/ truncation mutants were tested: the C-terminal truncated mutant NC4 interacted slightly, indicating the contribution of this part of the molecule to the interaction with GR, while the shorter truncated mutant NC6 did not interact with GR, likely resulting from an incorrect folding of the molecule. No role for the first charged region (delta A') was found as shown by the strong interaction detected for the delta A'Hsp90. This method can fruitfully be applied to the delimitation of the amino-acid sequences involved in protein-protein interaction by mutational analysis, especially to seek confirmation of other methods or when other approaches have failed.

Animals↗
PubMed1998

N-acetyl-cysteine decreases the matrix-degrading capacity of macrophage-derived foam cells: new target for antioxidant therapy?

BACKGROUND: Atherosclerotic plaque destabilization triggers clinical cardiovascular disease and thus represents an attractive therapeutic target. Weakening of tissue through the action of matrix-degrading enzymes, called matrix metalloproteinases (MMPs), released by resident macrophages was previously implicated in unstable vascular syndromes. METHODS AND RESULTS: We used a hypercholesterolemic rabbit model of atherosclerosis to investigate the gelatinolytic activity associated with macrophage-derived foam cells (FCs). Gelatinolytic activity and expression of MMP-9 but not of MMP-2 cosegregated with macrophage FCs in aortic lesions. Macrophage-derived gelatinases were further investigated in vitro. MMP-9 was identified as the main macrophage-derived gelatinase in cells isolated from aortic lesions and from granuloma induced in the same rabbits to increase cell yield. Importantly, detection of activated MMP-9 in the FC culture medium supports the notion that these cells can independently initiate processing of secreted MMP zymogens to active enzymes. We further examined whether FC gelatinolytic activity is dependent on the presence of reactive oxygen species (ROS). We found that treatment (1 to 5 days) with 1 to 10 mmol/L N-acetyl-L-cysteine (NAC), an ROS scavenger, decreased not only gelatinolytic activity but also gelatinase expression by FCs. Similarly, NAC treatment of explanted lesions abolished in situ gelatinolytic activity and MMP-9 expression. CONCLUSIONS: Macrophage FCs are an abundant source of gelatinolytic activity that can be inhibited in vitro and in situ by NAC. This newly described action of antioxidant therapy might prove useful to inhibit matrix degradation and to improve vascular stability.

Acetylcysteine↗
PubMed1998

Keratinocyte gene therapy for systemic diseases. Circulating interleukin 10 released from gene-transferred keratinocytes inhibits contact hypersensitivity at distant areas of the skin.

This study has examined the systemic effects of a circulating gene product, human interleukin 10 (IL-10), released from transduced keratinocytes. IL-10 is an anti-inflammatory cytokine which has an inhibitory effect on contact hypersensitivity (CHS). An expression vector (phIL-10) was constructed for human IL-10 and was injected into the dorsal skin of hairless rats. Local expression of IL-10 mRNA and protein was detected by reverse-transcriptase polymerase chain reaction and immunohistochemical staining, respectively. Enzyme-linked immunosorbent assay showed that the amount of IL-10 in the local keratinocytes and in the circulation increased with the dose of phIL-10 transferred. To determine whether circulating IL-10 could inhibit the effector phase of CHS at a distant area of the skin, various doses of phIL-10 were injected into the dorsal skin of sensitized rats before challenge on the ears. Our results showed that the degree of swelling of the ears of phIL-10- treated rats was significantly lower than that in the negative control animals. These results suggest that IL-10 released from transduced keratinocytes can enter the bloodstream and cause biological effects at distant areas of the skin. This study demonstrates that it may be possible to treat systemic disease using keratinocyte gene therapy.

Animals↗
PubMed1998

Genes don't count.

It is the regulation of gene expression that determines phenotype and cellular response. Several families of proteins control gene expression in cells and influence the pathogenesis of multiple organ failure, the acute phase response, atherosclerosis, and graft-vs-host disease. Understanding the basics of the regulation of gene transcription will allow the knowledgeable surgeon to target gene expression as a therapeutic modality in multiple diseases. We examine nuclear factor kappa B as an example of a transcription factor that is involved in multiple surgical diseases and has pharmacological inhibitors available to knowledgeable surgeons.

Animals↗