Biomedical subjects

H Wang

Publications and source records attributed to H Wang.

At least 1,297 records · Page 72Linked to original sources

PubMed1998

[Effect of allicin on ethanol-induced hepatotoxicity in mice].

To study the effect of allicin, an effective component of garlic, on ethanol-induced hepatotoxicity in mice. The results showed that allicin (10 mg/kg ig, qd x 10) could reverse the higher activities of serum ALT and glutathione s-transferase (GST) in ethanol-treated mice. Furthermore, allicin could significantly enhance the content of hepatic reduced glutathione (GSH), and the activities of hepatic glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Re) and GST in ethanol-induced hepatotoxicity mice. There were no remarkable changes in the hepatic catalase (Cat) and superoxide dismutase(SOD) activities. These results suggested that allicin have the effective hepato-protection on ethanol-induced hepatotoxicity, which is related to its selective effect on the glutathione-related enzyme system.

Animals↗

PubMed1998

Fetuin (alpha2-HS-glycoprotein) opsonizes cationic macrophagedeactivating molecules.

Macrophages become activated by bacterial endotoxin (lipopolysaccharide) and other stimuli to release proinflammatory cytokines and NO. To prevent release of toxic or potentially lethal quantities of these factors, the state of macrophage activation is counter-regulated by anti-inflammatory mediators (e.g., glucocorticoid hormones, interleukin 10, and transforming growth factor type beta). Fetuin, a negative acute-phase protein, recently was implicated as an anti-inflammatory mediator, because it is required for macrophage deactivation by spermine. In the present studies, we found that fetuin is necessary for macrophages to respond to CNI-1493, a tetravalent guanylhydrazone inhibitor of p38 mitogen-activated protein kinase phosphorylation. Fetuin dose-dependently increases macrophage uptake of CNI-1493, which can be specifically inhibited by anti-human fetuin antibodies. Anti-human fetuin antibodies render primary human peripheral blood mononuclear cells insensitive to deactivation by CNI-1493. Thus, macrophages use fetuin as an opsonin for cationic-deactivating molecules, both endogenous (e.g., spermine) and pharmacologic (e.g., CNI-1493). This role of fetuin as an opsonic participant in macrophage-deactivating mechanisms has implications for understanding and manipulating the innate immune response.

Amino Acid Sequence↗

PubMed1998

Energy transduction in the F1 motor of ATP synthase.

ATP synthase is the universal enzyme that manufactures ATP from ADP and phosphate by using the energy derived from a transmembrane protonmotive gradient. It can also reverse itself and hydrolyse ATP to pump protons against an electrochemical gradient. ATP synthase carries out both its synthetic and hydrolytic cycles by a rotary mechanism. This has been confirmed in the direction of hydrolysis after isolation of the soluble F1 portion of the protein and visualization of the actual rotation of the central 'shaft' of the enzyme with respect to the rest of the molecule, making ATP synthase the world's smallest rotary engine. Here we present a model for this engine that accounts for its mechanochemical behaviour in both the hydrolysing and synthesizing directions. We conclude that the F1 motor achieves its high mechanical torque and almost 100% efficiency because it converts the free energy of ATP binding into elastic strain, which is then released by a coordinated kinetic and tightly coupled conformational mechanism to create a rotary torque.

Adenosine Triphosphate↗

PubMed1998

Dominant-negative suppression of HNF-1alpha function results in defective insulin gene transcription and impaired metabolism-secretion coupling in a pancreatic beta-cell line.

Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) have been linked to subtype 3 of maturity-onset diabetes of the young (MODY3), which is characterized by a primary defect in insulin secretion. The role of HNF-1alpha in the regulation of pancreatic beta-cell function was investigated. Gene manipulation allowed graded overexpression of HNF-1alpha and controlled dominant-negative suppression of HNF-1alpha function in insulinoma INS-1 cells. We show that HNF-1alpha is essential for insulin gene transcription, as demonstrated by a pronounced decrease in insulin mRNA expression and in insulin promoter activity under dominant-negative conditions. The expression of genes involved in glucose transport and metabolism including glucose transporter-2 and L-type pyruvate kinase is also regulated by HNF-1alpha. Loss of HNF-1alpha function leads to severe defects in insulin secretory responses to glucose and leucine, resulting from impaired glucose utilization and mitochondrial oxidation. The nutrient-evoked ATP production and subsequent changes in plasma membrane potential and intracellular Ca2+ were diminished by suppression of HNF-1alpha function. These results suggest that HNF-1alpha function is essential for maintaining insulin storage and nutrient-evoked release. The defective mitochondrial oxidation of metabolic substrates causes impaired insulin secretion, indicating a molecular basis for the diabetic phenotype of MODY3 patients.

Adenosine Triphosphate↗

PubMed1998

Selective suppression of stress-activated protein kinase pathway by protein phosphatase 2C in mammalian cells.

Protein phosphatase 2Calpha (PP2Calpha) or PP2Cbeta-1 expressed in COS7 cells suppressed anisomycin- and NaCl-enhanced phosphorylations of p38 co-expressed in the cells. PP2Calpha or PP2Cbeta-1 expression also suppressed both basal and stress-enhanced phosphorylations of MKK3b and MKK6b, which are upstream protein kinases of p38, and of MKK4, which is one of the major upstream protein kinases of JNK. Basal activity of MKK7, another upstream protein kinase of JNK, was also suppressed by PP2Calpha or PP2Cbeta-1 expression. However, basal as well as serum-activated phosphorylation of MKK1alpha, an upstream protein kinase of ERKs, was not affected by PP2Cbeta or PP2Cbeta-1. A catalytically inactive mutant of PP2Cbeta-1 further enhanced the NaCl-stimulated phosphorylations of MMK3b, MKK4 and MKK6b, suggesting that this mutant PP2Cbeta-1 works as a dominant negative form. These results suggest that PP2C selectively inhibits the SAPK pathways through suppression of MKK3b, MKK4, MKK6b and MKK7 activities in mammalian cells.

Animals↗

PubMed1998

Morphology and function of human benign tumors and normal thyroid tissues maintained in severe combined immunodeficient mice.

In the improved SCID (severe combined immunodeficient) mice, various human benign tumors of the head and neck region were well maintained morphologically and functionally for 3 years until the experiments were terminated, e.g. transplanted parathyroid adenoma secreted parathyroid hormone (PTH) in the SCID mice for more than 1 year. Normal human thyroid tissue was also well maintained in the SCID mice for 3 years. Rapid and high uptake of radioiodine into the transplanted human thyroid tissue was observed. Furthermore, transplanted human thyroid tissue secreted thyroid hormone (T3) and T3 secretion was stimulated by the injection of human thyroid stimulating hormone (TSH). These findings suggest that the improved SCID mice will provide an invaluable experimental system for investigating the function of normal human tissues and the influence of endogenous and exogenous factors on human tissues.

Adenoma↗

PubMed1998

Vascular endothelial growth factor upregulates the expression of matrix metalloproteinases in vascular smooth muscle cells: role of flt-1.

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis that stimulates proliferation, migration, and proteolytic activity of endothelial cells. Although the mitogenic activity of VEGF is endothelial cell specific, recent reports indicate VEGF is able to stimulate chemotaxis and tissue factor production in monocytes. VEGF-stimulated activity in monocytes is mediated by the VEGF receptor flt-1. The purpose of the present study was to investigate the effects of VEGF on another major cell type in the vascular wall, namely, the vascular smooth muscle cell (SMC). Using cultured cells, we showed that VEGF has a minimal mitogenic effect on SMCs, which is in accordance with published data. However, VEGF treatment significantly enhanced production of matrix metalloproteinase (MMP)-1, -3, and -9 by human SMCs. The upregulation of MMP-1 and MMP-9 was pronounced, and the stimulation for MMP-3 was less prominent. Stimulation could be demonstrated at both protein and mRNA levels, as reflected by ELISA, zymography, and Northern blot analysis. To explore the signal transduction pathway for the effect of VEGF on SMCs, we studied the expression of 2 high-affinity VEGF receptors, the kinase insert domain-containing receptor (KDR) and flt-1, in human SMCs. Both reverse transcriptase-polymerase chain reaction and immunoblotting revealed the expression of flt-1. Immunoprecipitation followed by immunoblotting illustrated phosphorylation of the flt-1 receptor after VEGF treatment. Similar methodology failed to detect expression of KDR in human SMCs. These data suggest the role of flt-1 in mediating VEGF-stimulated MMP expression of SMCs. The physiological relevance of MMP upregulation was studied by examining VEGF-stimulated SMC migration through 2 synthetic extracellular matrix barriers, Matrigel and Vitrogen. Our results indicate that VEGF treatment accelerated SMC migration through both barriers, and that this response was blocked by MMP inhibition in Matrigel, which supports a permissive role of MMP in SMC migration. These data are the first to show a direct effect of VEGF on SMCs. SMC-derived MMPs may be an additional source of proteases to digest vascular basement membrane, which is a crucial step in the initial stage of angiogenesis. The MMPs may also contribute to SMC migration in angiogenesis and atherogenesis.

Aorta↗

PubMed1998

NMR structure and mutagenesis of the N-terminal Dbl homology domain of the nucleotide exchange factor Trio.

Guanine nucleotide exchange factors for the Rho family of GTPases contain a Dbl homology (DH) domain responsible for catalysis and a pleckstrin homology (PH) domain whose function is unknown. Here we describe the solution structure of the N-terminal DH domain of Trio that catalyzes nucleotide exchange for Rac1. The all-alpha-helical protein has a very different structure compared to other exchange factors. Based on site-directed mutagenesis, functionally important residues of the DH domain were identified. They are all highly conserved and reside in close proximity on two a helices. In addition, we have discovered a unique capability of the PH domain to enhance nucleotide exchange in DH domain-containing proteins.

Blood Proteins↗

PubMed1998

An early phase of embryonic Dlx5 expression defines the rostral boundary of the neural plate.

Relatively little is known about the molecular events that specify the rostrocaudal axis of the neural plate. Here we show that a member of the Distal-less (Dlx) homeobox gene family, Dlx5, is one of the earliest known markers for the most rostral ectoderm, before the formation of an overt neural plate. During late gastrulation Dlx5 expression becomes localized to the anterior neural ridge, which defines the rostral boundary of the neural plate, and also extends caudolaterally, marking the region of the presumptive neural crest. Subsequently, Dlx5 is expressed in tissues (olfactory epithelium, ventral cephalic epithelium) that are believed to derive from the anterior neural ridge, based on the avian fate map. The early phase of Dlx5 expression in the anterior neural ridge and its derivatives is distinct from a later phase of expression in the ventral telencephalon and diencephalon and also appears to be unique for Dlx5 among members of the Dlx family. Another distinctive feature of Dlx5 expression is the occurrence of an alternative transcript (deltaDlx5), which encodes a truncated protein lacking the homeodomain, and represents a significant fraction of total Dlx5 transcripts at all embryonic stages that were examined. In contrast with full-length DLX5, the deltaDLX5 truncated protein is deficient in DNA-binding activity and does not interact with the homeoprotein partner MSX1. Taken together, our findings suggest that Dlx5 activity may be regulated via the expression of an alternative transcript and demonstrate that Dlx5 marks the anterior boundary of the neural plate.

Alternative Splicing↗

PubMed1998

Isolation and characterization of mammalian homologs of the Drosophila gene glial cells missing.

The glial cells missing (gcm) gene in Drosophila encodes a transcription factor that determines the choice between glial and neuronal fates. We report here the isolation of two mammalian gcm homologs, Gcm1 and Gcm2, and the characterization of their expression patterns during embryonic development. Although Gcm2 is expressed in neural tissues at a low level, the major sites of expression for both of the mammalian genes are nonneural, suggesting that the functions of the mammalian homologs have diverged and diversified. However, when expressed ectopically, Gcm1 can substitute functionally for Drosophila gcm by transforming presumptive neurons into glia. Thus, certain biochemical properties, although not the specificity of the tissue in which the gene is expressed, have been conserved through the evolution of the Gcm gene family.

Amino Acid Sequence↗

PubMed1998

Isoform-specific phosphorylation of fission yeast type 2C protein phosphatase.

Protein phosphatase 2C (PP2C) is one of the four major protein serine/threonine phosphatases of eukaryotes and is implicated in the regulation of various cellular functions. With the goal of elucidating the mechanism responsible for regulating PP2C functions, we investigated the significance of phosphorylation of fission yeast Ptc1, Ptc2, and Ptc3, the yeast orthologs of mammalian PP2C. Both Ptc2 and Ptc3 but not Ptc1 were phosphorylated stoichiometrically by casein kinase II on serine residues at their carboxy-terminal regions. Mutational analysis of Ptc2 and Ptc3 revealed that serine residues of the conserved sequence (Ser-X-Ser-X-X-Glu/Asp) of these proteins were the phosphorylation sites. Interestingly, the activities of Ptc2 and Ptc3 were decreased 25 +/- 7.5% and increased 55 +/- 3.7%, respectively, by phosphorylation. In addition, the same site(s) of Ptc2 was phosphorylated when the protein was expressed in fission yeast cells. These results suggest that phosphorylation of PP2C plays important physiological roles in fission yeast cells.

Amino Acid Sequence↗

PubMed1998

Functional role of cysteine residues in the Na+/H+ exchanger effects of mutation of cysteine residues on targeting and activity of the Na+/H+ exchanger.

We investigated the role of cysteine residues in activity and localization of the NHE1 isoform of the Na+/H+ exchanger. Each of the nine cysteine residues was mutated to serine or arginine. Mutation of the first serine (amino acid number 9) and serine number six (amino acid number 477) resulted in dramatic decreases in detectable activity of the Na+/H+ exchanger when transfected into AP-1 cells. Some other mutations resulted in minor decreases in activity of the protein. Confocal and light microscopy of mutant cells with decreased activity showed that the antiporter protein was mostly retained in an intracellular compartment which colocalized with the medial-Golgi cisternae. Smaller amounts of active protein still remained targeted to the plasma membrane in these mutants. Treatment of wild-type cells with DTT also caused the retention of the Na+/H+ exchanger to the same intracellular compartment. The results suggest that cysteines play an important role in intracellular folding and trafficking of the Na+/H+ exchanger.

Amino Acid Substitution↗

PubMed1998

Structure-function relationships of human liver cytochromes P450 3A: aflatoxin B1 metabolism as a probe.

Cytochromes P450 3A4 and 3A5, the dominant drug-metabolizing enzymes in the human liver, share >85% primary amino acid sequence identity yet exhibit different regioselectivity toward aflatoxin B1 (AFB1) biotransformation [Gillam et al., (1995) Arch. Biochem. Biophys. 317, 374-384]. P450 3A4 apparently prefers AFB1 3alpha-hydroxylation, which results in detoxification and subsequent elimination of the hepatotoxin, over AFB1 exo-8,9-oxidation. In contrast, P450 3A5 is incapable of appreciable AFB1 3alpha-hydroxylation and converts it predominantly to the exo-8,9-oxide which is genotoxic. To elucidate the structural features that govern the regioselectivity of the human liver 3A enzymes in AFB1 metabolism and bioactivation, a combination of approaches including sequence alignment, homology modeling, and site-directed mutagenesis was employed. Specifically, the switch in AFB1 regioselectivity was examined after individual substitution of the divergent amino acids in each of the six putative substrate recognition sites (SRSs) of P450 3A4 with the corresponding amino acid of P450 3A5. Of the P450 3A4 mutants examined, P107S, F108L, N206S, L210F, V376T, S478D, and L479T mutations resulted in a significant switch of P450 3A4 regioselectivity toward that of P450 3A5. The results confirmed the importance of some of these residues in substrate contact in the active site, with residue N206 (SRS-2) being critical for AFB1 detoxification via 3alpha-hydroxylation. Moreover, the P450 3A4 mutant N206S most closely mimicked P450 3A5, not only in its regioselectivity of AFB1 metabolism but also in its overall functional capacity. Furthermore, the other SRS-2 mutant, L210F, also resembled P450 3A5 in its overall AFB1 metabolism and regioselectivity. These findings reveal that a single P450 3A5 SRS domain (SRS-2) is capable of conferring the P450 3A5 phenotype on P450 3A4. In addition, some of these P450 3A4 mutations that affected AFB1 regioselectivity had little influence on testosterone 6beta-hydroxylation, thereby confirming that each substrate-P450 active site fit is indeed unique.

Aflatoxin B1↗

PubMed1998

Treatment of cardiac allografts with established leukocyte infiltration by modulation of alpha4 (CD49d) and leukocyte function-associated antigen-1 (CD11a/CD18) integrin function.

BACKGROUND: Leukocyte infiltration is a landmark feature of organ rejection. The present study was undertaken to determine whether monoclonal antibodies (mAb) against alpha4 (CD49d) and/or leukocyte function-associated antigen-1 (LFA-1; CD11a/CD18) would reverse ongoing rejection in a mouse C57BL/6-to-BALB/c heart transplant model. METHODS: Control animals had rejection on postoperative day (POD) 8. Treatment with mAb started on POD 4 when leukocyte infiltration was well established. The recipients were treated with (1) mAb LFA-1, (2) mAb alpha4, and (3) mAbs LFA-1 + alpha4 at a dose of 6 mg/kg/day i.v. on PODs 4, 5, and 7. Untreated and rat IgG-treated animals were used as controls. RESULTS: Control animals experienced rejection on POD 8. Treatment with mAb against LFA-1 or alpha4 alone prolonged allograft survival to 17.0+/-3.2 and 24.3+/-4.6 days, respectively (P < 0.01 vs. controls). Combination therapy with both mAb increased allograft survival to 28.2+/-3.7 days (P < 0.01 vs. controls). Sequential pathological studies showed the mAb to alpha4, but not LFA-1, markedly reduced the degree of lymphocytic infiltration in cardiac allografts. In contrast, a different pattern was observed using in vitro studies: mAb to LFA-1, not alpha4, significantly reduced proliferative responses in mixed lymphocyte culture and interleukin-2 production from recipient splenocytes on POD 8. CONCLUSION: These data indicate that integrins play an important role in rejection. Although the effect of mAb against alpha4 and LFA-1 may involve different mechanisms, treatment with mAbs to integrins may be valuable in future clinical transplantation by averting ongoing rejection and prolonging graft survival.

Animals↗

PubMed1998

Desensitization of mitochondrial Ca2+ and insulin secretion responses in the beta cell.

The role of mitochondria in the desensitization of insulin secretion was investigated. In rat pancreatic beta cells, both insulin secretion and mitochondrial [Ca2+] increases were desensitized following two challenges with the mitochondrial substrate methyl succinate. In the beta cell line INS-1, similar results were observed when a 5-min interval separated two 5-min pulses. In contrast, ATP generation monitored in luciferase-expressing INS-1 cells was stimulated to the same extent during both exposures to methyl succinate. Succinate, like alpha-glycerophosphate, activates the electron transport chain at complex II. As a consequence, the mitochondrial membrane hyperpolarizes, promoting ATP synthesis and Ca2+ influx into the mitochondria through the uniporter. The mitochondrial desensitization was further studied in permeabilized INS-1 cells. Increasing extramitochondrial [Ca2+] from 100 to 500 nM enhanced succinate oxidation 4-fold. At 500 nM Ca2+, 1 mM succinate caused a blunted mitochondrial [Ca2+] increase upon the second, compared with the first, stimulation. These effects were mimicked by alpha-glycerophosphate, and there was cross-desensitization between the two compounds. Succinate hyperpolarized the mitochondrial membrane during both the first and second applications. This suggests that the uniporter itself, rather than the respiratory chain, is desensitized. These results emphasize the key role of the mitochondria not only in the stimulation of insulin secretion, but also in its desensitization.

Adenosine Triphosphate↗

PubMed1998

Altered c-fos expression in autonomic regulatory centers of genetically obese (ob/ob) mouse brain.

This study examined c-fos-like immunoreactivity (FLI) in the brain of obese (ob/ob) and lean (+/+) mice during states of satiety and hunger. The results demonstrated that there is an altered neural activity primarily in the hypothalamus of the obese mouse brain. Most interestingly, only obese mice had an increased FLI in the dorsomedial and supramammillary nuclei. Conversely, only lean mice showed increase in FLI in the medial part of perifornical nucleus following food deprivation for 24 h.

Animals↗

PubMed1998

Cerebral ischemia enhances polyamine oxidation: identification of enzymatically formed 3-aminopropanal as an endogenous mediator of neuronal and glial cell death.

To elucidate endogenous mechanisms underlying cerebral damage during ischemia, brain polyamine oxidase activity was measured in rats subjected to permanent occlusion of the middle cerebral artery. Brain polyamine oxidase activity was increased significantly within 2 h after the onset of ischemia in brain homogenates (15.8 +/- 0.9 nmol/h/mg protein) as compared with homogenates prepared from the normally perfused contralateral side (7.4 +/- 0.5 nmol/h/mg protein) (P <0.05). The major catabolic products of polyamine oxidase are putrescine and 3-aminopropanal. Although 3-aminopropanal is a potent cytotoxin, essential information was previously lacking on whether 3-aminopropanal is produced during cerebral ischemia. We now report that 3-aminopropanal accumulates in the ischemic brain within 2 h after permanent forebrain ischemia in rats. Cytotoxic levels of 3-aminopropanal are achieved before the onset of significant cerebral cell damage, and increase in a time-dependent manner with spreading neuronal and glial cell death. Glial cell cultures exposed to 3-aminopropanal undergo apoptosis (LD50 = 160 microM), whereas neurons are killed by necrotic mechanisms (LD50 = 90 microM). The tetrapeptide caspase 1 inhibitor (Ac-YVAD-CMK) prevents 3-aminopropanal-mediated apoptosis in glial cells. Finally, treatment of rats with two structurally distinct inhibitors of polyamine oxidase (aminoguanidine and chloroquine) attenuates brain polyamine oxidase activity, prevents the production of 3-aminopropanal, and significantly protects against the development of ischemic brain damage in vivo. Considered together, these results indicate that polyamine oxidase-derived 3-aminopropanal is a mediator of the brain damaging sequelae of cerebral ischemia, which can be therapeutically modulated.

Aldehydes↗

PubMed1998

Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1.

Cytohesin-1 (B2-1) is a guanine nucleotide exchange factor for human ADP ribosylation factor (Arf) GTPases, which are important for vesicular protein trafficking and coatamer assembly in the cell. Cytohesin-1 also has been reported to promote cellular adhesion via binding to the beta2 integrin cytoplasmic domain. The solution structure of the Sec7 domain of cytohesin-1, which is responsible for both the protein's guanine nucleotide exchange factor function and beta2 integrin binding, was determined by NMR spectroscopy. The structure consists of 10 alpha-helices that form a unique tertiary fold. The binding between the Sec7 domain and a soluble, truncated version of human Arf-1 was investigated by examining 1H-15N and 1H-13C chemical shift changes between the native protein and the Sec7/Arf-1 complex. We show that the binding to Arf-1 occurs through a large surface on the C-terminal subdomain that is composed of both hydrophobic and polar residues. Structure-based mutational analysis of the cytohesin-1 Sec7 domain has been used to identify residues important for binding to Arf and for mediating nucleotide exchange. Investigations into the interaction between the Sec7 domain and the beta2 integrin cytoplasmic domain suggest that the two proteins do not interact in the solution phase.

ADP-Ribosylation Factors↗