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Biomedical subjects

W Tang

Publications and source records attributed to W Tang.

At least 217 records · Page 12Linked to original sources
PubMed1996

In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins.

It has been shown recently in China that arsenic trioxide (As2O3) is a very effective treatment for acute promyelocytic leukemia (APL). APL patients resistant to all-trans retinoic acid (ATRA) and conventional chemotherapy can still respond to AS2O3. In this study, we addressed the possible cellular and molecular mechanisms of this treatment by using NB4 cells as a model. The results show that: (1) As2O3 triggers relatively specific NB4 cell apoptosis at micromolar concentration, as proved by morphology, histogramic related nuclear DNA contents, and DNA gel eletrophoresis. (2) As2O3 does not influence bax, bcl-x, c-myc, and p53 gene expression, but downregulates bcl-2 gene expression at both mRNA and protein levels. (3) As2O3 induces a significant modulation of the PML staining pattern in NB4 cells and HL-60 cells. The micropunctates characteristic of PML-RAR alpha in NB4 cells dissappear after treatment with As2O3, whereas a diffuse PML staining occurs in the perinuclear cytoplasmic region. In addition, a low percentage of untreated NB4 cells exhibits an accumulation of PML positive particles in a compartment of cytoplasm. The percentage of these cells can be significantly increased after As2O3 treatment. A similar PML staining pattern is observed in apoptotic cells. (4) ATRA pretreatment does not influence As2O3-induced apoptosis. These results suggest that induction of cell apoptosis can be one of the mechanisms of the therapeutic effect of As2O3. Moreover, this apoptosis induction occurs independently of the retinoid pathway and may be mediated, at least partly, through the modulation of bcl-2, as well as PML-RAR alpha and/ or PML proteins.

Apoptosis↗
PubMed1996

2D-NMR studies of the effects of axial substitution on two helices in horse cytochrome c.

The sequential amino acids from Gly-1 to Cys-14 in the N-terminal segment and from Lys-88 to Glu-104 in the C-terminal segment of cytochrome c-imidazole complex (Im-cyt c) have been studied by two-dimensional nuclear magnetic resonance techniques. Resonance assignments for the main-chain and side-chain protons are reported. Qualitative interpretation of nuclear Overhauser enhancement data allows the secondary structure of the two segments to be described, which indicate the patterns of NOEs found are consistent with an alpha-helix between Val-3 and Cys-14 in the N-terminus, and between Lys-88 and Asn-103 in the C-terminus. Two alpha-helices are found to be maintained. Comparison of the long-range NOEs of Im-cyt c relative to the crystal structure of native cytochrome c reveals apparent conformational changes of some side-chains especially those close to heme pocket within the N- and C-terminal helices resulting from the binding of imidazole to iron by displacing native Met-80 side-chain. The explanation for ligand-induced changes within the N- and C-helices is therefore suggested.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and characterization of a novel human diacylglycerol kinase zeta.

Diacylglycerol (DAG) occupies a central position in the synthesis of complex lipids and also has important signaling roles. For example, DAG is an allosteric regulator of protein kinase C, and the cellular levels of DAG may influence a variety of processes including growth and differentiation. We previously demonstrated that human endothelial cells derived from umbilical vein express growth-dependent changes in their basal levels of diacylglycerol and diacylglycerol kinase activity (Whatley, R. E., Stroud, E. D., Bunting, M., Zimmerman, G. A., McIntyre, T. M., and Prescott, S. M. (1993) J. Biol. Chem. 268, 16130-16138). To further explore the role of diacylglycerol metabolism in endothelial responses, we used a degenerate reverse transcription-polymerase chain reaction method to identify diacylglycerol kinase isozymes expressed by human endothelial cells. We report the isolation of a 3.5-kilobase cDNA encoding a novel diacylglycerol kinase (hDGKzeta) with a predicted molecular mass of 103.9 kDa. Human DGK zeta contains two zinc fingers, an ATP binding site, and four ankyrin repeats near the carboxyl terminus. A unique feature, as compared with other diacylglycerol kinases, is the presence of a sequence homologous to the MARCKS phosphorylation site domain. From Northern blot analysis of multiple tissues, we observed that hDGKzeta mRNA is expressed at highest levels in brain. COS-7 cells transfected with the hDGKzeta cDNA express 117-kDa and 114-kDa proteins that react specifically with an antibody to a peptide derived from a unique sequence in hDGK zeta. The transfected cells also express increased diacylglycerol kinase activity, which is not altered in the presence of R59949, an inhibitor of human platelet DGK activity. The hDGKzeta displays stereoselectivity for 1,2-diacylglycerol species in comparison to 1,3-diacylglycerol, but does not exhibit any specificity for molecular species of long chain diacylglycerols.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of a novel human diacylglycerol kinase highly selective for arachidonate-containing substrates.

Diacylglycerol (DAG) is a second messenger that activates protein kinase C and also occupies a central role in phospholipid biosynthesis. Conversion of DAG to phosphatidic acid by DAG kinase regulates the amount of DAG and the route it takes. We used degenerate primers to amplify polymerase chain reaction products from cDNA derived from human endothelial cells. A product with a novel sequence was identified and used to clone a 2.6-kilobase cDNA from an endothelial cell library. When transfected with a truncated version of this cDNA, COS-7 cells had a marked increase in DAG kinase activity, which demonstrated clear selectivity for arachidonoyl-containing species of diacylglycerol. The open reading frame of this clone has 567 residues with a predicted protein of 64 kDa. This enzyme, which we designated DGK epsilon, has two distinctive zinc finger-like structures in its N-terminal region, but does not contain the E-F hand motifs found in several other mammalian DGKs. The catalytic domain of DGK epsilon, which is related to other DGKs, contains two ATP-binding motifs. Northern blotting demonstrated that DGK epsilon is expressed predominantly in testis. This unique diacylglycerol kinase may terminate signals transmitted through arachidonoyl-DAG or may contribute to the synthesis of phospholipids with defined fatty acid composition.

Amino Acid Sequence↗
PubMed1996

High frequency and error-prone DNA recombination in ataxia telangiectasia cell lines.

The only specific DNA repair defect found in ataxia telangiectasia (A-T) cells is mis-repair of cleaved DNA. In this report we measured DNA recombination, given its role in DNA repair and genetic instability. Using plasmids containing selectable reporter genes, we found a higher frequency of both chromosomal recombination (>100 times) and extra-chromosomal recombination (27 times) in SV40-transformed A-T cell lines compared with in an SV40-transformed normal fibroblast cell line. Southern analysis of single A-T colonies exhibiting post-integration recombination revealed that 24/27 had undergone aberrant rearrangements; recombination in normal fibroblast colonies was achieved by gene conversion in 8/11 clones and 10/11 clones showed unchanged copies of the plasmid. Using co-transfection of two integrating plasmids, each containing a separate deletion in the xgprt reporter gene, the 27 times difference in extra-chromosomal recombination was found when the plasmids were cleaved at a distance from the reporter gene. When the plasmids were cleaved within the reporter gene, the co-transfection frequency was reduced in A-T, but was increased in normal cells. We conclude that A-T cell lines have not only a high frequency chromosomal and extra-chromosomal recombination, but also exhibit error-prone recombination of cleaved DNA.

Ataxia Telangiectasia↗
PubMed1996

Rhinovirus stimulation of interleukin-6 in vivo and in vitro. Evidence for nuclear factor kappa B-dependent transcriptional activation.

To further understand the biology of rhinovirus (RV), we determined whether IL-6 was produced during RV infections and characterized the mechanism by which RV stimulates lung cell IL-6 production. In contrast to normals and minimally symptomatic volunteers, IL-6 was detected in the nasal washings from patients who developed colds after RV challenge. RV14 and RV1A, major and minor receptor group RVs, respectively, were potent stimulators of IL-6 protein production in vitro. These effects were associated with significant increases in IL-6 mRNA accumulation and gene transcription. RV was also a potent stimulator of IL-6 promoter-driven luciferase activity. This stimulation was modestly decreased by mutation of the nuclear factor (NF)-IL-6 site and abrogated by mutation of the NF-kappa B site in this promoter. An NF-kappa B-DNA binding activity, mediated by p65, p50, and p52 NF-kappa B moieties, was rapidly induced in RV-infected cells. Activator protein 1-DNA binding was not similarly altered. These studies demonstrate that IL-6 is produced during symptomatic RV infections, that RVs are potent stimulators of IL-6 elaboration, and that RV stimulation IL-6 production is mediated by an NF-kappa B-dependent transcriptional stimulation pathway. IL-6 may play an important role in the pathogenesis of RV infection, and NF-kappa B activation is likely to be an important event in RV-induced pathologies.

Adult↗
PubMed1996

Characterization of thiol-conjugated metabolites of 2-propylpent-4-enoic acid (4-ene VPA), a toxic metabolite of valproic acid, by electrospray tandem mass spectrometry.

The hepatotoxicity of the anticonvulsant drug valproic acid (VPA) is most likely associated with the bioactivation of its metabolite 2-propylpent-4-enoic acid (4-ene VPA), which is known to induce hepatic microvesicular steatosis in rats. This paper presents an on-line liquid chromatographic/tandem mass spectrometric (LC/MS/MS) identification of new glutathione (GSH)-related conjugates of the reactive metabolites of 4-ene VPA. Bile samples collected from male Sprague-Dawley rats dosed intraperitoneally with 4-ene VPA or its [2H7]-analogue (100 mg kg-1) were injected on to an ODS column interfaced to a LC/MS/MS instrument using electrospray ionization. LC was developed such that no overlapping of peaks occurred among those metabolites which may potentially produce common fragment ions of interest. Subsequent comparison of LC retention times and MS/MS full fragment ion spectra generated for putative metabolites with that of authentic reference compounds made available by chemical synthesis confirmed the presence of the GSH, cysteinylglycine, cysteine and N-acetylcysteine (NAC) conjugates of 2-(2'-carboxypentanyl)oxirane (4,5-epoxy VPA) and (E)-2-propylpenta-2,4-dienoic acid ((E)-2,4-diene VPA), respectively. Quantitatively, the biliary thiol conjugates accounted for 5% of the dose. This observation is novel for 4-ene VPA metabolism in terms of the degradation of GSH conjugates to the corresponding mercapturic acids possibly occurring within the liver as opposed to an inter-organ process which involves the kidney. In addition, the GSH- and NAC-glucuronide di-conjugates of (E)-2,4-diene VPA were also identified as the biliary metabolites with the GSH-glucuronide di-conjugate being 10% of the corresponding mono-GSH conjugate. Taken together, these data clearly indicate that reactive metabolites of VPA can react with hepatic GSH via several different metabolic pathways and may subsequently produce depletion of GSH that leads to toxic consequences.

Animals↗
PubMed1996

The intratracheal administration of endotoxin: X. Dexamethasone downregulates neutrophil emigration and cytokine expression in vivo.

Intratracheal instillation of endotoxin (LPS) causes acute pulmonary inflammation characterized by the accumulation of plasma proteins and leukocytes within the pulmonary airways. The synthetic glucocorticoid dexamethasone 1) inhibits the LPS-initiated vascular leak of plasma proteins into the airspace, 2) inhibits the LPS-initiated emigration of neutrophils and lymphocytes into the airspace in a dose-dependent fashion, and 3) inhibits LPS-initiated mRNA and/or bronchoalveolar lavage protein expression of cytokines (TNF, IL-1 and IL-6) and chemokines (MIP-1 alpha, MIP-2 and MCP-1). In conclusion, dexamethasone inhibits both the vascular and cellular aspects of acute inflammation by downregulation of a broad spectrum of inflammatory cytokines and chemokines.

Animals↗
PubMed1996

Reaction of metallothionein with ethylenediamine-N,N,N',N'-tetraacetic acid.

The reactions of various rabbit MTs (Cd7Th, Zn7Th, Hg7Th, Bi7Th, Pb7Th) with EDTA were studied at different pH and EDTA concentrations, and were determined to be pseudo-first-order reactions. The reactions were all divided into two steps: one faster and EDTA-dependent, and the other EDTA-independent. The mechanism is also described in detail. Based on obtained results, the varied induction behaviors of MT formation for those metals are discussed.

Animals↗
PubMed1996

In vitro atherosclerotic plaque and calcium quantitation by intravascular ultrasound and electron-beam computed tomography.

The purpose of this investigation was to compare the accuracy of intravascular ultrasound (IVUS) and electron-beam computed tomography (EBCT) in quantitating human atherosclerotic plaque and calcium. In experiment 1, 12 human atherosclerotic arterial segments were obtained at autopsy and imaged by using IVUS and EBCT. The plaque from each arterial segment was dissected and a volume measurement of the dissected plaque was obtained by water displacement. The plaque from each arterial segment was ashed at 700 degrees F, and the weight of the remaining ashes was used as an estimate of the calcium mass. In experiment II, 11 calcified arterial segments were obtained at autopsy and imaged by using IVUS at one site along the artery. A corresponding histologic cross section stained with Masson's trichrome was prepared. In experiment I, the mean plaque volume measured by water displacement was 165.3 +/- 118.4 microliters. The mean plaque volume calculated by IVUS was 166.1 +/- 114.4 microliters and correlated closely with that by water displacement (r = 0.98, p < 0.0001). The mean calcium mass measured by ashing was 19.4 +/- 15.8 mg. The mean calculated calcium mass by EBCT was 19.9 mg and correlated closely with that by ashing (r=0.98, p<0.001). The mean calculated calcium volume by IVUS was 18.6 +/- 11.2 microliters and correlated linearly with the calcium mass by ashing (r = 0.87, p < 0.0003). In experiment II, the mean cross-sectional area of the calcified matrix was 1.71 +/- 0.66 mm2 by histologic examination compared with 1.44 +/- 0.66 mm2 by IVUS. There was a good correlation between the calcified cross-sectional area by histologic examination and IVUS (r = 0.76, p < 0.007); however, IVUS may underestimate the amount of calcium present depending on the intralesional calcium morphologic characteristics. In conclusion, IVUS accurately quantitates atherosclerotic plaque volume as well as the cross-sectional area and volume of intralesional calcium, especially if the calcium is localized at the base of the plaque. IVUS underestimates the amount of calcium present because of signal drop-off when the calcium is too thick for the ultrasound to completely penetrate. In comparison, EBCT accurately quantitates calcium mass regardless of the intralesional calcium morphologic characteristics; however, EBCT does not accurately quantitate plaque volume and will miss noncalcified atherosclerotic lesions.

Adolescent↗
PubMed1996

Detection of coronary calcification with electron-beam computed tomography: evaluation of interexamination reproducibility and comparison of three image-acquisition protocols.

The purpose of this investigation was to determine the variability of electron-beam computed tomography (EBCT) measurement of coronary calcification by using two commonly employed image-acquisition protocols and to compare this variability with that of a new image-acquisition protocol. We performed three EBCT scans within 15 minutes on each of 324 consecutive high-risk, asymptomatic subjects participating in an ongoing epidemiologic research investigation. Subjects were divided into three groups: group 1 (n = 175) received two scans with a standard 20-slice, 3 mm slice thickness image-acquisition protocol and a third scan with the new 20-slice, 6 mm slice thickness protocol; group 2 (n = 77) received two scans with a new 6 mm slice thickness image-acquisition protocol and a third scan with a standard 20-slice, 3 mm slice thickness protocol; group 3 (n = 72) received two scans with a 30-slice, 3 mm slice thickness image-acquisition protocol and a third scan with a 20-slice, 6 mm slice thickness protocol. Calcium score, calcium mass estimate, and calcium volume estimate were determined for each scan. We compared retest variability of calcium measurements for each of the three image-acquisition protocols. The variability of the new 6 mm slice thickness protocol was significantly lower than that of either the 20-slice 3 mm slice thickness protocol (p = 0.009) or the 30-slice 3 mm slice thickness protocol (p = 0.02) for measurement of calcium score, mass, or volume. Retest reproducibility for all three image-acquisition protocols was low; however, the 20-slice 6 mm slice thickness protocol gave reproducibility superior to either of the 3 mm slice thickness protocols. Variability of absolute indices increased, and variability of relative indices decreased as the value of these indices increased. EBCT is not sufficiently reproducible to allow serial quantitation of coronary calcium in individual patients over relatively short periods (< 2 years).

Aged↗
PubMed1996

Bioactivation of a toxic metabolite of valproic acid, (E)-2-propyl-2,4-pentadienoic acid, via glucuronidation. LC/MS/MS characterization of the GSH-glucuronide diconjugates.

The hepatotoxicity of the anticonvulsant drug valproic acid may be associated with the formation of potentially reactive metabolites, one of which is (E)-2-propyl-2,4-pentadienoic acid ((E)-2,4-diene VPA). This report describes the characterization of new GSH-related conjugates of this diene. Bile samples collected from male Sprague-Dawley rats dosed ip with (E)-2,4-diene VPA (100 mg/kg) were analyzed by LC/MS/MS. Initial Q1 parent in scanning indicated that the daughter ions m/z 162 and 123 could be derived from the ions at m/z 624 and 480, respectively. Subsequent collision-induced dissociation (CID) of these parent ions revealed a common neutral loss of 176 Da which is diagnostic for glucuronides. A similar neutral loss of 176 Da was observed in daughter ion spectra of the biliary metabolites arising from [2H7]-4-ene VPA dosed ip to rats, where the ion fragments containing the VPA portion were 7 amu higher than those derived from the unlabeled drug. CID of the ion at m/z 624 also gave fragments characteristics for GSH conjugates such as the loss of glycine and glutamate moieties. Based on the MS data, the metabolites were assigned the diconjugate structures 1-O-(2-propyl-5-(glutathion-S-yl)-3-pentenoyl)-beta-D-glucur onide (5-GS-3-ene VPA-glucuronide I, MH+, 624) and the corresponding 5-NAC-3-ene VPA-glucuronide (MH+, 480). Further proof of structural identity was obtained from 1H NMR of HPLC-purified metabolites. The amount of biliary 5-GS-3-ene VPA-glucuronide I was 7-fold greater than the corresponding 5-GS-3-ene VPA, the sum of the two metabolites accounting for 6.6% of the dose. Incubation of 1-O-(2-propyl-2,4-pentadienoyl)-beta-D-glucuronide (2,4-diene VPA-glucuronide) with GSH in the presence or absence of GST enzyme led to the formation of 5-GS-3-ene VPA-glucuronide I which was readily detected by LC/MS/MS, suggesting that in vivo the diconjugate may arise from the reaction of GSH with 2,4-diene VPA-glucuronide. To our knowledge, this is the first recorded instance in which glucuronide formation activates a drug to further conjugate with GSH via a Michael addition reaction.

Animals↗
PubMed1996

Use of microphysiometry for analysis of heterologous ion channels expressed in yeast.

Measurement of extracellular acidification rates by microphysiometry provides a means to analyze the function of ion channels expressed in yeast cells. These measurements depend on the proton pumping action of the H(+)-ATPase, a central component of the yeast plasma membrane. We used microphysiometry to analyze the activity of two ion channels expressed in yeast. In one example, an inwardly rectifying K+ channel, gpIRK1, provides a potassium uptake function when expressed in a potassium transporter-defective yeast strain. Rates of acidification in gpIRK1-expressing cells directly reflect channel function. Addition of cesium, an inhibitor of gpIRK1 activity, results in an immediate reduction in acidification rates. In a second example, expression of a nonselective cation channel, the influenza virus M2 protein, is believed to interfere with the maintenance of the electrochemical proton gradient by the H(+)-ATPase. In cells expressing the M2 channel, addition of inhibitors increases the rate of proton extrusion. Moreover, functional differences between two M2 inhibitors, amantadine and BL-1743, are distinguished by the microphysiometer. This application demonstrates the utility of the microphysiometer for functional studies of ion channels; it is adaptable to a screening process for compounds that modulate ion channel activity.

Biosensing Techniques↗
PubMed1996

Adrenergic vasopressor agents and mechanical ventilation for the treatment of experimental septic shock.

OBJECTIVE: Vasopressor agents and mechanical ventilation are routine interventions for the treatment of sepsis complicated by hypotension. It was our hypothesis that such treatment singly or in combination increases the duration of survival. DESIGN: Prospective, randomized, controlled study. SETTING: University research laboratory. SUBJECTS: Thirty male Sprague-Dawley rats anesthetized with intraperitoneal injection of pentobarbital. INTERVENTIONS: Peritonitis was induced by cecal ligation and spillage of cecal contents into the abdominal cavity. The first phase of this study was performed on 15 spontaneously breathing Sprague-Dawley rats that were randomized to three groups of five animals each. One group received treatment with dopamine. The second group received norepinephrine. The third group received only the diluent as a placebo. Concentrations of the vasopressor agents were increased such that mean arterial pressure was maintained at approximately 80% of baseline values; the volumes infused were kept constant. For the second phase of this study, the grouping of animals and the techniques of study were identical, except that rats were mechanically ventilated. MEASUREMENTS AND MAIN RESULTS: Mean arterial pressure was best maintained with norepinephrine. However, no statistically significant differences in duration of survival, cardiac index, arterial blood lactate concentration, or arterial and venous PCO2 and PO2 values were identified between groups. With mechanical ventilation, survival was prolonged (p < .01). Survival was increased from an average of 291 mins to 342 mins with dopamine, from 257 mins to 352 mins in placebo controls, and from 280 mins to 329 mins with norepinephrine. Again, no significant differences in hemodynamic and blood gas measurements, or in the duration of survival between vasopressor-treated and control animals were documented. CONCLUSIONS: No benefit or detriment was demonstrated when vasopressor agents were administered to sustain arterial pressure in the course of experimental peritonitis in this murine model of septic shock. This finding contrasted with highly significant prolongation of survival when animals were mechanically ventilated. There was no evidence that routine vasopressor therapy, under these controlled experimental conditions in rats, improved duration of survival.

Adrenergic alpha-Agonists↗
PubMed1996

Myocardial dysfunction after successful resuscitation from cardiac arrest.

OBJECTIVE: To investigate the functional and metabolic changes in the myocardium after successful resuscitation from cardiac arrest. DESIGN: Prospective, randomized, sham-controlled study. SETTING: Animal laboratory at a university center. SUBJECTS: Domestic pigs. INTERVENTIONS: Electric induction of ventricular fibrillation by alternating current delivered to the right ventricular endocardium through a pacing electrode. Electric defibrillation was attempted after an interval of 12 mins of ventricular fibrillation, which included 4 mins of untreated ventricular fibrillation and 8 mins of precordial compression in 13 animals, seven of which were successfully resuscitated. Seven additional animals were randomized to serve as "sham" controls, in which cardiac arrest was not induced. MEASUREMENTS AND MAIN RESULTS: Left ventricular pressure-volume relationships utilizing the conductance method were obtained in conjunction with conventional hemodynamic and metabolic measurements at baseline and during a 6-hr interval after successful cardiac resuscitation. Progressive and striking increases in left ventricular volumes were observed after successful cardiac resuscitation. The end-diastolic volume increased from a prearrest level of 89 +/- 21 mL to a maximum of 154 +/- 53 mL (p<.05) at 360 mins after successful resuscitation. The time-coincident end-systolic volume increased from 54 +/- 21 to 126 +/- 54 mL (p<.05), such that the ejection fraction was reduced from 0.41 +/- 0.10 to 0.20 +/- 0.07 ( p<.05). Ventricular dilation was associated with marked reductions in stroke volume and ventricular work. However, compensatory increases in heart rate maintained cardiac output at levels that sustained adequate systemic oxygen delivery. The slope of the end-systolic pressure-volume relationships progressively decreased from 5.04 +/- 1.88 to 2.00 +/- 0.57 mm Hg/mL (p<.05) at 360 mins after successful resuscitation. The volume intercept at left ventricular pressure of 100 mm Hg increased from 43 +/- 19 to 94 +/- 51 mL (p=.03). Both the decrease in the slope and the increase in the volume intercept were characteristic of progressive impairment in contractile function. The rate of left ventricular pressure decrease was unchanged. Accordingly, no substantial changes in lusitropic properties were identified. Despite large increases in end-diastolic volume, the end-diastolic pressure remained unchanged. CONCLUSION: Postresuscitation myocardial dysfunction in this animal model was characterized by impaired contractile function, decreased work capability, and ventricular dilation.

Animals↗