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H Wang

Publications and source records attributed to H Wang.

At least 1,531 records · Page 85Linked to original sources
PubMed1997

Cloning and characterization of cDNAs encoding chicken mitogen-activated protein kinase kinase type 2, MEK2: downregulation of MEK2 in response to inhibition of mitochondrial DNA expression.

The present work was initiated with the aim of identifying nuclear genes whose expression is sensitive to the mitochondrial DNA (mtDNA) status of transformed chicken DU24 cells. We cloned and sequenced cDNAs for the mitogen-activated protein kinase kinase type 2, MEK2, a protein involved in the mitogenic growth factor signal transduction pathway in vertebrates. Sequence comparisons between the chicken protein and its mammalian counterparts indicated that MEK2 proteins are highly conserved among vertebrates. Southern blot analysis of endonuclease-digested genomic DNA from primary chick embryo fibroblasts (CEF) suggested that MEK2 is a single-copy gene in this vertebrate species. The steady-state level of MEK2 transcripts is decreased in DUS3 mtDNA-less (rho0) cells developed by long-term exposure of DU24 rho+ cells to ethidium bromide (EtdBr). Run-on in vitro transcription assays and mRNA stability studies indicated that the decrease in MEK2 mRNA content is associated with post-transcriptional regulation. In parental DU24 cells, MEK2 mRNA content decreased after inhibition of mtDNA transcription by EtdBr and inhibition of translation on mitoribosomes by chloramphenicol (CAM). Cytoplasmic hybrids (cybrids) constructed by fusion of chicken rho0 cells with enucleated parental cells and CEF recovered a basal level of MEK2 expression. The MEK2 protein content is decreased in DUS3 rho0 cells and in parental DU24 rho+ cells treated with EtdBr and CAM for 6 days, while that of MEK1, a closely related kinase, remained unchanged. On the basis of these observations, we propose that mitochondria participate in the mitogenic signal transduction pathway in chicken cells through regulation of MEK2 expression.

Amino Acid Sequence↗
PubMed1997

Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry.

The larger of two diuretic hormones of the tobacco hornworm, Manduca sexta, (Mas-DH) is a peptide of 41 residues. It is one of a family of seven currently known insect diuretic hormones that are similar to the corticotropin-releasing factor-urotensin-sauvagine family of peptides. We investigated the possible inactivation of Mas-DH by incubating it in vitro with larval Malpighian tubules (Mt), the target organ of the hormone. The medium was analyzed, and degradation products were identified, using on-line microbore reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry (RPLC-ESI-MS). This sensitive technique allows identification of metabolites of Mas-DH (present at an initial level of approximately 1 microM). An accurate Mr value for a metabolite is usually sufficient for unambiguous identification. Mas-DH is cleaved by Mt proteases initially at L29-R30 and R30-A31 under our assay conditions; some Mas-DH is also oxidized, apparently at M2 and M11. The proteolysis can be inhibited by 5 mM EDTA, suggesting that divalent metals are needed for peptide cleavage. The oxidation of the hormone can be inhibited by catalase or 1 mM methionine, indicating that H2O2 or related reactive oxygen species are responsible for the oxidative degradation observed. RPLC-ESI-MS is shown here to be an elegant and efficient method for studying peptide hormone metabolism resulting from unknown proteases and pathways.

Amino Acid Sequence↗
PubMed1997

Experimental antitumor activity of the Ce(IV)-mitoxantrone complex and its interaction with deoxyribonucleic acid.

The Ce(IV)-mitoxantrone complex exhibits a higher lethality to Ehrlich ascites tumor cells than that of the free drug and shows stronger inhibition ability on the DNA synthesis of the tumor cells. Thus the Ce(IV)-mitoxantrone complex may become a more potent antitumor drug than mitoxantrone. The different interaction model of mitoxantrone and its Ce(IV) complex with DNA were studied by the methods of spectroscopy, electrochemistry, and electrophoresis. Ce(IV) ions chelate with oxygens of the hydroxyl groups at the 1,4 position and the carbonyl function on C-9 and C-10, then intercalate into the base pairs of DNA together. The complexation of Ce(IV) gives rise to more compact binding of mitoxantrone with DNA, and leads to an additional change on the normal conformation and the double-helical structure of DNA; this may be related to the more stronger action of the complex on DNA synthesis and survival of cultured tumor cells.

Animals↗
PubMed1997

Expression levels of functional folate receptors alpha and beta are related to the number of N-glycosylated sites.

In a previous study with inhibitors of N-glycosylation, it was proposed that core glycosylation of the folate receptor (FR) is required for the proper folding of the protein [Luhrs (1991) Blood 77, 1171-1180]. The human FR isoforms type alpha and type beta have three and two candidate sites for N-glycosylation respectively, only one of which is conserved. The significance of N-glycosylation at each of these loci in the expression and function of FR was examined by eliminating the sites both individually and in combination by introducing Asn-->Gln substitutions. Translation experiments in vitro showed that the mutations did not alter the synthetic rates of the polypeptides. The recombinant proteins were expressed in human 293 fibroblasts. Treatment with N-glycanase and analysis by Western blotting of the wild-type and mutant proteins revealed that all of the candidate sites in both FR-alpha and FR-beta are glycosylated. When all of the N-glycosylation sites were abolished, 2% and 8% of FR-alpha and FR-beta respectively were expressed on the cell surface compared with the corresponding wild-type proteins; the residual FR polypeptides in the cell lysates were unable to bind [3H]folic acid. In both the proteins, the inclusion of each additional N-glycosylation site partly contributed to restoration of cell surface [3H]folic acid binding and receptor-mediated folate transport. Further, in FR-beta the introduction of an additional unnatural site of N-glycosylation resulted in the enhancement of the expression of the cell surface receptor compared with the wild-type protein. The results indicate that the total mass of N-glycosylation, not a specific locus of the modification, is critical for the efficient folding and optimal expression of functional FR-alpha and FR-beta.

Amino Acid Sequence↗
PubMed1997

Structure of the carboxyl-terminal dimerization domain of the HIV-1 capsid protein.

The carboxyl-terminal domain, residues 146 to 231, of the human immunodeficiency virus-1 (HIV-1) capsid protein [CA(146-231)] is required for capsid dimerization and viral assembly. This domain contains a stretch of 20 residues, called the major homology region (MHR), which is conserved across retroviruses and is essential for viral assembly, maturation, and infectivity. The crystal structures of CA(146-231) and CA(151-231) reveal that the globular domain is composed of four helices and an extended amino-terminal strand. CA(146-231) dimerizes through parallel packing of helix 2 across a dyad. The MHR is distinct from the dimer interface and instead forms an intricate hydrogen-bonding network that interconnects strand 1 and helices 1 and 2. Alignment of the CA(146-231) dimer with the crystal structure of the capsid amino-terminal domain provides a model for the intact protein and extends models for assembly of the central conical core of HIV-1.

Amino Acid Sequence↗
PubMed1997

The Na+/H+ antiporter potentiates growth and retinoic acid-induced differentiation of P19 embryonal carcinoma cells.

The Na+/H+ exchanger is a ubiquitous plasma membrane protein that is responsible for pH regulation and is activated by growth factors. We examined the role of the Na+/H+ exchanger in cell growth and differentiation. Treatment of P19 cells with the Na+/H+ exchanger inhibitor Hoe 694 eliminated retinoic acid-induced differentiation in this cell line. We developed a P19 embryonal carcinoma cell line that was deficient in the Na+/H+ antiporter. Na+/H+ exchanger-deficient cells were reduced in the rate of cell growth and this effect was enhanced by the removal of added HCO3- and by reducing extracellular pH. The antiporter-deficient cells were also markedly deficient in their ability to differentiate to neuronal-like cells and recovered this ability when the Na+/H+ antiporter was reintroduced. The results show that the absence of Na+/H+ antiport as a pH regulatory mechanism can result in deficiencies in both cell growth and differentiation in embryonal carcinoma cells.

Animals↗
PubMed1997

Interactions of a high mobility group-like protein with human mitochondrial DNA.

A number of nuclear-encoded proteins are known to bind to the mitochondrial genome and may be involved in mitochondrial replication and in mitochondrial diseases. Mitochondrial diseases such as Kearns-Sayre syndrome (KSS) are flanked by common direct repeats. To study protein binding to these mitochondria DNA regions we used gel mobility shift binding assays. Proteins present in nuclear or in mitochondrial extracts from normal or KSS-derived fibroblasts bound to a 280-bp mitochondrial DNA fragment encompassing a deletion breakpoint in the mitochondrial genome. In addition, nuclear and mitochondrial protein bound to a nearby surrounding fragment and to a synthetic oligonucleotide with a related consensus DNA sequence. Southwestern blot analysis showed that the DNA binding ability resided in a 35-kDa protein. The amino terminal sequence of the 35-kDa protein was very similar to human high mobility group proteins. These results suggest that this protein may be involved in mitochondrial DNA deletions.

Amino Acid Sequence↗
PubMed1997

Human syntaxin 7: a Pep12p/Vps6p homologue implicated in vesicle trafficking to lysosomes.

The movement of hydrolases and other proteins to lysosomes is accomplished by vesicle trafficking. Specific vesicles are targeted from the trans-Golgi network via a prelysosomal compartment to lysosomes. The specificity of vesicle transport is thought to occur through the interaction of vesicle proteins with receptors on a particular target membrane. The syntaxins are a family of transmembrane proteins that have been implicated as vesicle receptors involved in vesicle docking and fusion. Syntaxins 1-4 are localized to the plasma membrane, and in particular, syntaxin 1a mediates synaptic vesicle docking in the nerve terminal. Syntaxins 5 and 6 have been localized to cis-Golgi and trans-Golgi network compartments, respectively. We now report the identification of syntaxin 7 from a human brain cDNA library. The syntaxin 7 gene is localized to human chromosome 6. By Northern analysis, the syntaxin RNA was found to be broadly distributed. Based on its homology to yeast and plant vacuolar syntaxins, we propose that syntaxin 7 has a role in vesicle trafficking between the Golgi complex and lysosomes. In vitro binding studies reveal that syntaxin 7 binds alphaSNAP, a key regulator of transport vesicle fusion at multiple stages of the secretory pathway.

Amino Acid Sequence↗
PubMed1997

Acute glucose intolerance in insulinoma cells with unbalanced overexpression of glucokinase.

The INS-r3-GK27 insulinoma cells are endowed with artificially inducible glucokinase under control of the reverse tetracycline-dependent transcriptional activator. Moderate induction of glucokinase has been shown to result in proportionate increases in glycolytic flux and in potentiation of glucose effects on insulin secretion and pyruvate kinase gene expression. In cells with 20-fold overexpression of glucokinase, however, glucose activation of secretion and gene expression was severely impaired. Measurements of the glycolytic flux in cells with 7- and 21-fold increases in glucokinase activity and determination of the flux control coefficient of this enzyme showed that control of glycolysis at the glucokinase step was lost in the cells at the higher level of overexpression. Challenging the cells with glucose above 6 mM resulted in massive accumulation of glucose 6-phosphate and caused a rapid and sustained depletion of cellular ATP, in contrast with the glucose-induced rise in ATP in cells with wild-type glucokinase levels. Loss of cell viability ensued upon prolonged culture in high glucose. In summary, in insulinoma beta cells strongly overexpressing glucokinase, an imbalance between glucose phosphorylation and turnover of glucose 6-phosphate resulted in acute glucose intolerance due to trapping of cellular orthophosphate in dead-end product and severe paralysis of energy metabolism.

Animals↗
PubMed1997

Activation of CPP32-like proteases is not sufficient to trigger apoptosis: inhibition of apoptosis by agents that suppress activation of AP24, but not CPP32-like activity.

The 24-kD apoptotic protease (AP24) is a serine protease that is activated during apoptosis and has the capacity to activate internucleosomal DNA fragmentation in isolated nuclei. This study examined the following: (a) the functional relationship between AP24 and the CPP32-like proteases of the caspase family; and (b) whether activation of CPP32-like proteases is sufficient to commit irreversibly a cell to apoptotic death. In three different leukemia cell lines, we showed that agents that directly (carbobenzoxy-Ala-Ala-borophe (DK120) or indirectly inhibit activation of AP24 (protein kinase inhibitors, basic fibroblast growth factor, tosylphenylalaninechloromethylketone, and caspase inhibitors) protected cells from apoptosis induced by TNF or UV light. Only the caspase inhibitors, however, prevented activation of CPP32-like activity as revealed by cleavage of the synthetic substrate, DEVD-pNa, by cell cytosols, and also by in vivo cleavage of poly (ADP-ribosyl) polymerase, a known substrate of CPP32. Activation of DEVD-pNa cleaving activity without apoptosis was also demonstrated in two variants derived from the U937 monocytic leukemia in the absence of exogenous inhibitors. Cell-permeable peptide inhibitors selective for CPP32-like proteases suppressed AP24 activation and apoptotic death. These findings indicate that CPP32-like activity is one of several upstream signals required for AP24 activation. Furthermore, activation of CPP32-like proteases alone is not sufficient to commit irreversibly a cell to apoptotic death under conditions where activation of AP24 is inhibited.

Apoptosis↗
PubMed1997

A floating microwire technique for multichannel chronic neural recording and stimulation in the awake freely moving rat.

We describe a cheap, and relatively simple, method for the construction and implantation of bundles of six fully-floating 25 microm microwire electrodes in the rat central nervous system (CNS). Wires are stiffened for implantation by temporarily attaching them to a micropipette guide with sucrose which subsequently dissolves in the brain. The associated headpiece connector mates with a plug-in FET which gives high quality recordings, free of movement artefacts, even when used with 3 m of unscreened cable. Different electrode configurations are easily selected and sufficient space is available on the headpiece to accommodate injection guide cannulae. The electrode performance was evaluated in 42 implanted rats where the system was used successfully for long term recording of superior colliculus (SC) deep layer neurons and behavioural responses evoked by electrical stimulation of the same wires. We obtained neural recordings on 81% of the 252 implanted wires, with 79% of these providing good, reliably discriminable, single unit responses following post-operative recovery. During a five-week testing period on a subsample of the 42 'best' electrodes (one per animal), we found the same sensory or motor response 1 week after initial testing in 91% wires, 67% after 2 weeks and 24% after 5 weeks. Using waveform templating techniques we were able to show that 62% of the electrodes still working at 5 weeks were reliably recording the same cell.

Animals↗
PubMed1997

Inhibition of growth and p21ras methylation in vascular endothelial cells by homocysteine but not cysteine.

Although hyperhomocysteinemia has been recognized recently as a prevalent risk factor for myocardial infarction and stroke, the mechanisms by which it accelerates arteriosclerosis have not been elucidated, mostly because the biological effects of homocysteine can only be demonstrated at very high concentrations and can be mimicked by cysteine, which indicates a lack of specificity. We found that 10-50 microM of homocysteine (a range that overlaps levels observed clinically) but not cysteine inhibited DNA synthesis in vascular endothelial cells (VEC) and arrested their growth at the G1 phase of the cell cycle. Homocysteine in this same range had no effect on the growth of vascular smooth muscle cells (VSMC) or fibroblasts. Homocysteine decreased carboxyl methylation of p21(ras) (a G1 regulator whose activity is regulated by prenylation and methylation in addition to GTP-GDP exchange) by 50% in VEC but not VSMC, a difference that may be explained by the ability of homocysteine to dramatically increase levels of S-adenosylhomocysteine, a potent inhibitor of methyltransferase, in VEC but not VSMC. Moreover, homocysteine-induced hypomethylation in VEC was associated with a 66% reduction in membrane-associated p21(ras) and a 67% reduction in extracellular signal-regulated kinase 1/2, which is a member of the mitogen-activated protein (MAP) kinase family. Because the MAP kinases have been implicated in cell growth, the p21(ras)-MAP kinase pathway may represent one of the mechanisms that mediates homocysteine's effect on VEC growth. VEC damage is a hallmark of arteriosclerosis. Homocysteine-induced inhibition of VEC growth may play an important role in this disease process.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

Down-regulation of the cyclin A promoter by transforming growth factor-beta1 is associated with a reduction in phosphorylated activating transcription factor-1 and cyclic AMP-responsive element-binding protein.

Transforming growth factor (TGF)-beta1 prevents cell cycle progression by inhibiting several regulators, including cyclin A. To study the mechanisms by which TGF-beta1 down-regulates cyclin A gene expression, we transfected reporter plasmids driven by the cyclin A promoter into mink lung epithelial cells in the absence and presence of TGF-beta1. The TGF-beta1-induced down-regulation of cyclin A promoter activity appeared to be mediated via the activating transcription factor (ATF) site, because mutation of this site abolished down-regulation. Surprisingly, although TGF-beta1 treatment for 24 h markedly decreased cyclin A promoter activity, it did not decrease the abundance of the ATF-binding proteins ATF-1 and cyclic AMP-responsive binding protein (CREB). However, we detected 90 and 78% reductions (by Western analysis) in phosphorylated CREB and ATF-1, respectively, in mink lung epithelial cells treated with TGF-beta1. TGF-beta1-induced down-regulation of cyclin A promoter activity was reversed by okadaic acid (a phosphatase inhibitor) and by cotransfection with plasmids expressing the cAMP-dependent protein kinase catalytic subunit or the simian virus small tumor antigen (Sm-t, an inhibitor of PP2A). These data indicate that TGF-beta1 may down-regulate cyclin A promoter activity by decreasing phosphorylation of CREB and ATF-1.

Activating Transcription Factor 1↗
PubMed1997

Decreased CP-1 (NF-Y) activity results in transcriptional down-regulation of topoisomerase IIalpha in a doxorubicin-resistant variant of human multiple myeloma RPMI 8226.

Decreased topoisomerase II (Topo II) activity results in resistance to antineoplastic agents targeting this enzyme. Dox1V derived from human multiple myeloma RPMI 8226 demonstrated a 4-fold resistance to doxorubicin in the absence of MDR1 overexpression or topo II mutations (Futscher B.W., Foley N., Gleason-Guzman M., Meltzer P.S., Sullivan D.M., and Dalton W.S., Int'l. J. Cancer, 66: 520-5, 1996.). Consistent with its drug resistant phenotype, a 2- to 3-fold decrease in topo II expression was identified. To investigate the molecular basis for decreased topo II expression in Dox1V, a semi-quantitative analysis of Topo II activity, protein level and mRNA transcript were performed. The results demonstrated that reduced Topo II activity is due to a decreased mRNA level. Southern blot and sequencing experiments revealed wild-type sequence of the topo II promoter in the drug resistant cells. Transient gene expression assays demonstrated that topo II is transcriptionally down-regulated in Dox1V independent of the promoter sequence of the endogenous alleles. Instead, the activity of a ubiquitous transcription factor CP-1 (NF-Y) interacting with the topo II promoter is decreased. The decrease in CP-1/NF-Y activity in Dox1V is correlated well with the decrease in topo II transcriptional activity, transcript level, Topo II protein and enzyme activity. Therefore, transcriptional down-regulation resulted from a reduced CP-1/NF-Y activity is responsible for decreased topo II expression in Dox1V cells.

Antigens, Neoplasm↗
PubMed1997

Regulation of CYP3A9 gene expression by estrogen and catalytic studies using cytochrome P450 3A9 expressed in Escherichia coli.

Sexual dimorphism in the expression of CYP3A9, a novel form of CYP3A from rat brain, is shown for the first time in rat brain as well as in rat liver. CYP3A9 expression is female specific in rat liver as judged by its 10-fold higher expression in females than in males. CYP3A9 gene expression was inducible by estrogen treatment both in male and in female rats. Ovariectomy of adult female rats elicited a drastic reduction on the mRNA level of CYP3A9 which could be fully restored by estrogen replacement. These results suggest that estrogen may play an important role in the female-specific expression of the CYP3A9 gene. P450 3A9 recombinant protein was expressed in Escherichia coli by means of the pCWOri+ expression vector and the MALLLAVF amino terminal sequence modification. This construct gave a high level of expression (130 nmol P450 3A9/liter culture) and the recombinant protein of the modified P450 3A9 was purified to electrophoretic homogeneity with a specific content of 10.1 nmol P450/mg protein from solubilized fractions through two chromatographic steps. The purified P450 3A9 protein was active in the metabolism of imipramine, erythromycin, benzphetamine, and ethylmorphine as well as 17beta-estradiol in a reconstituted system containing lipid and rat NADPH-P450 reductase. Of special interest is the finding that P450 3A9 can catalyze the formation of desipramine with a turnover number of 4.9 nmol/min/nmol P450, suggesting the possible involvement of this isoform in the metabolism of imipramine in brain. Optimal reconstitution conditions for P450 3A9 activities required a lipid mixture (1:1:1 mixture of L-alpha-dilauroyl phosphatidylcholine, L-alpha-dioleoyl phosphatidylcholine, and phosphatidylserine) and GSH.

Animals↗
PubMed1997

The degradation of apolipoprotein B100 is mediated by the ubiquitin-proteasome pathway and involves heat shock protein 70.

Apolipoprotein B (apoB) is the major protein component of atherogenic lipoproteins of hepatic origin. In HepG2 cells, the standard cell culture model of human hepatic lipoprotein metabolism, there is a limited availability of core lipids in the endoplasmic reticulum for association with nascent apoB. Under these conditions, apoB is partially translocated, interacts with cytosolic Hsp70, and undergoes rapid degradation. We show that increasing the expression of Hsp70 in HepG2 cells promotes apoB degradation. In addition, apoB is polyubiquitinated and its degradation both normally and after Hsp70 induction is blocked by inhibitors of the proteasome. The apoB that accumulates after proteasome inhibition is endoplasmic reticulum-associated and can be assembled into lipoproteins and secreted if new lipid synthesis is stimulated. Thus, apoB is the first example of a wild-type mammalian protein whose secretion is regulated by degradation in the cytosol via the ubiquitin-proteasome pathway. Furthermore, targeting of this secretory protein to the proteasome is regulated by the molecular chaperone Hsp70 and the availability of apoB's lipid-ligands.

Apolipoprotein B-100↗
PubMed1997

Stability enhancement for peptide analysis by electrospray using the triple quadrupole mass spectrometer.

Electrospray ionization sources, used with triple quadrupole mass spectrometers from PE/Sciex (API III+), Micromass (Quattro II), and Finnigan (TSQ 7000), were modified with a 35-gauge stainless steel needle. The dimensions of the needle were 63 microm i.d. by 145 microm o.d. with variable length, depending on the specific instrument. This modification led to enhanced signal stability, improved signal/noise ratios, and lowered sample consumption for a wide range of peptides. Stable baselines were observed with flow rates in the range of 50 nL/min to 5 microL/min. An alternative design, based on a metal wire housed within a fused silica capillary, led to the most stable signals of all during infusion, but caused excessive peak broadening with capillary chromatography. The Finnigan interface was further modified with an external postcolumn addition tee, used in conjunction with capillary liquid chromatography columns of 30 and 50 microm internal diameter. The best results with the modified Finnigan interface were acquired using the 50-microm column at a flow rate of 150 to 200 nL/min.

Factor VII↗