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W Tang

Publications and source records attributed to W Tang.

At least 73 records · Page 4Linked to original sources
PubMed2001

Networking senescence-regulating pathways by using Arabidopsis enhancer trap lines.

The last phase of leaf development, generally referred to as leaf senescence, is an integral part of plant development that involves massive programmed cell death. Due to a sharp decline of photosynthetic capacity in a leaf, senescence limits crop yield and forest plant biomass production. However, the biochemical components and regulatory mechanisms underlying leaf senescence are poorly characterized. Although several approaches such as differential cDNA screening, differential display, and cDNA subtraction have been employed to isolate senescence-associated genes (SAGs), only a limited number of SAGs have been identified, and information regarding the regulation of these genes is fragmentary. Here we report on the utilization of enhancer trap approach toward the identification and analysis of SAGs. We have developed a sensitive large-scale screening method and have screened 1,300 Arabidopsis enhancer trap lines and have identified 147 lines in which the reporter gene GUS (beta-glucuronidase) is expressed in senescing leaves but not in non-senescing ones. We have systematically analyzed the regulation of beta-glucuronidase expression in 125 lines (genetically, each contains single T-DNA insertion) by six senescence-promoting factors, namely abscisic acid, ethylene, jasmonic acid, brassinosteroid, darkness, and dehydration. This analysis not only reveals the complexity of the regulatory circuitry but also allows us to postulate the existence of a network of senescence-promoting pathways. We have also cloned three SAGs from randomly selected enhancer trap lines, demonstrating that reporter expression pattern reflects the expression pattern of the endogenous gene.

Arabidopsis↗
PubMed2001

Effects of charged amino-acid mutation on the solution structure of cytochrome b(5) and binding between cytochrome b(5) and cytochrome c.

The solution structure of oxidized bovine microsomal cytochrome b(5) mutant (E48, E56/A, D60/A) has been determined through 1524 meaningful nuclear Overhauser effect constraints together with 190 pseudocontact shift constraints. The final family of 35 conformers has rmsd values with respect to the mean structure of 0.045+/-0.009 nm and 0.088+/-0.011 nm for backbone and heavy atoms, respectively. A characteristic of this mutant is that of having no significant changes in the whole folding and secondary structure compared with the X-ray and solution structures of wild-type cytochrome b(5). The binding of different surface mutants of cytochrome b(5) with cytochrome c shows that electrostatic interactions play an important role in maintaining the stability and specificity of the protein complex formed. The differences in association constants demonstrate the electrostatic contributions of cytochrome b(5) surface negatively charged residues, which were suggested to be involved in complex formation in the Northrup and Salemme models, have cumulative effect on the stability of cyt c-cyt b(5) complex, and the contribution of Glu48 is a little higher than that of Glu44. Moreover, our result suggests that the docking geometry proposed by Northrup, which is involved in the participation of Glu48, Glu56, Asp60, and heme propionate of cytochrome b(5), do occur in the association between cytochrome b(5) and cytochrome c.

Amino Acid Sequence↗
PubMed2001

Development and evaluation of high throughput functional assay methods for HERG potassium channel.

Three functional hERG channel assay methods have been developed and evaluated. The methods were tested against five known hERG channel inhibitors: dofetilide, terfenadine (Seldane), sertindole (Serdolect), astemizole (Hismanal), and cisapride (Propulsid). The DiBAC4(3)-based assays were found to be the most economical but had high false-hit rates as a result of the interaction of dye with the test compounds. The membrane potential dye assay had fewer color-quenching problems but was expensive and still gave false hits. The nonradioactive Rb+ efflux assay was the most sensitive of all the assays evaluated and had the lowest false-hit rate.

Animals↗
PubMed2001

A comparison of biphasic and monophasic waveform defibrillation after prolonged ventricular fibrillation.

STUDY OBJECTIVE: To compare the effects of biphasic defibrillation waveforms and conventional monophasic defibrillation waveforms on the success of initial defibrillation, postresuscitation myocardial function, and duration of survival after prolonged duration of untreated ventricular fibrillation (VF), including the effects of epinephrine. DESIGN: Prospective, randomized, animal study. SETTING: Animal laboratory and university-affiliated research and educational institute. PARTICIPANTS: Domestic pigs. INTERVENTIONS: VF was induced in 20 anesthetized domestic pigs receiving mechanical ventilation. After 10 min of untreated VF, the animals were randomized. Defibrillation was attempted with up to three 150-J biphasic waveform shocks or a conventional sequence of 200-J, 300-J, and 360-J monophasic waveform shocks. When reversal of VF was unsuccessful, precordial compression was performed for 1 min, with or without administration of epinephrine. The protocol was repeated until spontaneous circulation was restored or for a maximum of 15 min. MEASUREMENTS AND RESULTS: No significant differences in the success of initial resuscitation or in the duration of survival were observed. However, significantly less impairment of myocardial function followed biphasic shocks. Administration of epinephrine reduced the total electrical energy required for successful resuscitation with both biphasic and monophasic waveform shocks. CONCLUSIONS: Lower-energy biphasic waveform shocks were as effective as conventional higher-energy monophasic waveform shocks for restoration of spontaneous circulation after 10 min of untreated VF. Significantly better postresuscitation myocardial function was observed after biphasic waveform defibrillation. Administration of epinephrine after prolonged cardiac arrest decreased the total energy required for successful resuscitation.

Animals↗
PubMed2001

Heterotropic cooperativity of cytochrome P450 3A4 and potential drug-drug interactions.

Cytochromes P450 (CYP) 3A4 is the most abundant human hepatic CYP isoform catalyzing the metabolism of approximately 50% of therapeutic agents. In addition to inhibition or induction, CYP3A4 is subject to stimulation, termed homotropic (substrate stimulation) and heterotropic (stimulation by effectors) cooperativity. The heterotropic cooperativity of CYP3A4 may result from an increase in Vmax, a decrease in Km or a combination of the two and sometimes exhibits regio-selectivity when the enzyme is involved in two or more metabolic pathways for a single substrate. An effector of CYP3A4 can also be a substrate; its metabolism may or may not be inhibited by another substrate. These characteristics of heterotropic cooperativity of CYP3A4 have been interpreted in the context of two binding domains in the active site of the enzyme, two substrate binding plus a distinct allosteric binding site, multiple enzyme conformations or multiple binding sites accompanied by conformational changes. Examples of in vivo CYP cooperativity are rare; representative cases include flavone-dependent stimulation of zoxazolamine metabolism in rats and enhancement of CYP3A-mediated hepatic clearance of diclofenac by quinidine in monkeys. Effector-induced increases in CYP3A4 activity were observed during the 1'-hydroxylation of midazolam and 4'- and 10-hydroxylation of warfarin in human hepatocyte systems. These data imply that CYP cooperativity has the potential to cause in vivo drug-drug interactions. Because cooperative and inhibitory responses from CYP3A4 are known to be substrate-dependent, projection of the pharmacokinetics of an investigational drug and CYP-associated risks of drug-drug interactions in humans can be very complex. Further investigation of CYP cooperativity is warranted.

Animals↗
PubMed2001

Enzyme kinetics of cytochrome P450-mediated reactions.

The most common drug-drug interactions may be understood in terms of alterations of metabolism, associated primarily with changes in the activity of cytochrome P450 (CYP) enzymes. Kinetic parameters such as Km, Vmax, Ki and Ka, which describe metabolism-based drug interactions, are usually determined by appropriate kinetic models and may be used to predict the pharmacokinetic consequences of exposure to one or multiple drugs. According to classic Michaelis-Menten (M-M) kinetics, one binding site models can be employed to simply interpret inhibition (pure competitive, non-competitive and uncompetitive) or activation of the enzyme. However, some cytochromes P450, in particular CYP3A4, exhibit unusual kinetic characteristics. In this instance, the changes in apparent kinetic constants in the presence of inhibitor or activator or second substrate do not obey the rules of M-M kinetics, and the resulting kinetics are not straightforward and hamper mechanistic interpretation of the interaction in question. These unusual kinetics include substrate activation (autoactivation), substrate inhibition, partial inhibition, activation, differential kinetics and others. To address this problem, several kinetic models can be proposed, based upon the assumption that multiple substrate binding sites exist at the active site of a particular P450, and the resulting kinetic constants are, therefore, solved to adequately describe the observed interaction between multiple drugs. The following is an overview of some cytochrome P450-mediated classic and atypical enzyme kinetics, and the associated kinetic models. Applications of these kinetic models can provide some new insights into the mechanism of P450-mediated drug-drug interactions.

Algorithms↗
PubMed2001

Solution structure of cytochrome b(5) mutant (E44/48/56A/D60A) and its interaction with cytochrome c.

Using 1617 meaningful NOEs with 188 pseudocontact shifts, a family of 35 conformers of oxidized bovine microsomal cytochrome b5 mutant (E44/48/56A/D60A) has been obtained and is characterized by good resolution (rmsd to the mean structure are 0.047 +/- 0.007 nm and 0.095 +/- 0.008 nm for backbone and heavy atoms, respectively). The solution structure of the mutant, when compared with the X-ray structure of wild-type cytochrome b(5), has no significant changes in the whole folding and secondary structure. The binding between cytochrome b(5) and cytochrome c shows that the association constant of the mutant-cytochrome c complex is much lower than the one for wild-type complex (2.2 x 10(4) M(-1) vs. 5.1 x 10(3) M(-1)). The result suggests the four acidic residues have substantial effects on the formation of the complex between cytochrome b(5) and cytochrome c, and therefore it is concluded reasonably that the electrostatic interaction plays an important role in maintaining the stability and specificity of the complex formed. The competition between the ferricytochrome b(5) mutant and [Cr(oxalate)(3)](3-) for ferricytochrome c shows that site III of cytochrome c, which is a strong binding site to wild-type cytochrome b(5), still binds to the mutant with relatively weaker strength. Our results indicate that certain bonding geometries do occur in the interaction between the present mutant and cytochrome c and these geometries, which should be quite different from the ones of the Salemme and Northrup models.

Amino Acid Sequence↗
PubMed2001

In vitro stimulation of warfarin metabolism by quinidine: increases in the formation of 4'- and 10-hydroxywarfarin.

It has been demonstrated that the activity of cytochrome P450 (CYP)3A4 in certain cases is stimulated by quinidine (positive heterotropic cooperativity). We report herein that the 4'- and 10-hydroxylation of S- and R-warfarin are enhanced in human liver microsomal incubations containing quinidine. These reactions were catalyzed by CYP3A4, based on data derived from immunoinhibitory studies, with 4'-hydroxylation being preferentially associated with S-warfarin and 10-hydroxylation with R-warfarin. The 4'-hydroxylation of S-warfarin and 10-hydroxylation of R-warfarin increased with increasing quinidine concentrations and maximized at ~3- and 5-fold the values of controls, respectively. Stimulatory effects of quinidine also were observed with recombinant CYP3A4, suggesting that increases in warfarin metabolism were due to quinidine-mediated enhancement of CYP3A4 activity. This positive cooperativity of CYP3A4 was characterized by a 2.5-fold increase in V(max) for the 4'-hydroxylation of S-warfarin and a 5-fold increase in V(max) for the 10-hydroxylation of R-warfarin, with little change in K(m) values. Conversely, V(max) for the 3-hydroxylation of quinidine was not influenced by the presence of warfarin. These results are consistent with previous findings suggesting the existence of more than one binding site in CYP3A4 through which interactions may occur between substrate and effector at the active site of the enzyme. Such interactions were subsequently illustrated by a kinetic model containing two binding domains, and a good regression fit was obtained for the experimental data. Finally, stimulation of warfarin metabolism by quinidine was investigated in suspensions of human hepatocytes, and increases in the formation of 4'- and 10-hydroxywarfarin again were observed in the presence of quinidine, indicating that this type of drug-drug interaction occurs in intact cells.

Cytochrome P-450 CYP3A↗
PubMed2001

Bioactivation of diclofenac via benzoquinone imine intermediates-identification of urinary mercapturic acid derivatives in rats and humans.

The metabolism of diclofenac has been reported to produce reactive benzoquinone imine intermediates. We describe the identification of mercapturic acid derivatives of diclofenac in rats and humans. Three male Sprague-Dawley rats were administered diclofenac in aqueous solution (pH 7) at 50 mg/kg by intraperitoneal injection, and urine was collected for 24 h. Human urine specimens were obtained, and samples were pooled from 50 individuals. Urine samples were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Two metabolites with MH(+) ions at m/z 473 were detected in rat urine and identified tentatively as N-acetylcysteine conjugates of monohydroxydiclofenac. Based upon collision-induced fragmentation of the MH(+) ions, accurate mass measurements of product ions, and comparison of LC/MS/MS properties of the metabolites with those of synthetic reference compounds, one metabolite was assigned as 5-hydroxy-4-(N-acetylcystein-S-yl)diclofenac and the other as 4'-hydroxy-3'-(N-acetylcystein-S-yl)diclofenac. The former conjugate also was detected in the pooled human urine sample by multiple reaction-monitoring LC/MS/MS analysis. It is likely that these mercapturic acid derivatives represent degradation products of the corresponding glutathione adducts derived from diclofenac-2,5-quinone imine and 1',4'-quinone imine, respectively. Our data are consistent with previous findings, which suggest that oxidative bioactivation of diclofenac in humans proceeds via benzoquinone imine intermediates.

Acetylcysteine↗
PubMed2001

[Differences of nitric oxide synthase expression and activity between splanchnic arterial and venous vessels of cirrhotic rats].

OBJECTIVE: To understand the implication of differences of nitric oxide synthase (NOS) expression and activity between splanchnic arterial and venous vessels in the pathogenesis of portal hypertension. METHODS: Cirrhosis was induced in 100 Wistar rats by subcutaneously administration of carbon tetrachloride. NOS localization, activity and gene expression in the mesenteric artery and the portal vein vessels of both cirrhotic and normal rats were investigated by immunohistochemistry, chemoluminescence and reverse transcription-polymerase chain reaction, respectively. RESULTS: There was inducible NOS enzyme isoform in al1 layers of splanchnic vessels of cirrhotic rats, whereas endothelial NOS isoform largely in vascular endothelia. NOS activity and its mRNA expression all were significantly increased in cirrhotic rats when compared with normal rats (P<0.05 or 0.01).Moreover, the activities of general and constitutive NOS and the expression of endothelial NOS mRNA in cirrhotic rats were significantly higher in the mesenteric artery than in the portal vein (P<0.01). CONCLUSIONS: Enhanced expression and activity of endothelial NOS enzyme isoform may be mainly responsible for increased NO production of splanchnic vessels in cirrhotic rats, and the differences of NOS expression and activity between the mesenteric artery and the portal vein vessels may be one of the pathogeneses of portal hypertension in which NO might be involved.

Animals↗
PubMed2001

Changes of evoked potentials and evaluation of mild hypothermia for treatment of severe brain injury.

OBJECTIVE: To observe the changes of evoked potentials after severe brain injury and the effect of mild hypothermia on acute severe brain injury. METHODS: A total of 44 patients with severe closed head injury (GCS 3-8, admitted within 10 hours from injury) admitted from May 1998 to March 1999 were selected for this study. All patients were admitted into the intensive care unit and divided into 2 groups, Group A (GCS 3-5) and Group B (GCS 6-8). Patients were also randomly assigned to either normothermia or hypothermia subgroups. Patients in the hypothermia group were cooled to 32-34 degrees C. Median nerve short-latency somatosensory evoked potentials (SLSEP) and brain stem auditory evoked potentials (BAEP) were recorded before cooling and 4, 24, 48, 72, 96 and 120 hours, respectively after cooling and temperature resuming. SLSEP and BAEP were measured at the same time in the normothermia group (control group). The changes of evoked potentials (EP) were analyzed by statistical methods. RESULTS: In the Group B, N(20) amplitudes in SLSEP and I/V amplitudes in BAEP after mild hypothermia treatment in the hypothermia group differed significantly from those in the control group (P<0.05). However, in the Group A, no significant difference in all parameters was found. CONCLUSIONS: These results demonstrate that mild hypothermia treatment (32-34 degrees C) in the Group B has a significant neuroelectrophysiological effect on severe brain injury. Nevertheless, the effect of mild hypothermia in the Group A is not apparent and needs further studying.

Acute Disease↗
PubMed2001

[Expression of bone morphogenetic protein (BMP)-2, 4 in mouse embryonic tooth during the bud stage].

OBJECTIVE: The purpose of this paper was to investigate the distributions of bone morphogenic protein (BMP)-2, 4 in the developing molars of mice during the bud stage and, the functional relationship between BMP-2 and BMP-4. METHODS: The distribution of BMP-2, 4 were examined in developing mouse molars on the 14th (the 14th embryonic day), using monoclonal antibodies and immunohistochemistry methods. RESULTS: BMP-2, 4 were detected both in the epithelial cells and dental mesenchyme cells. CONCLUSION: There is a similar distribution pattern between BMP-2 and BMP-4. Therefore, there may be a functional relationship between BMP-2 and BMP-4 during the development of mouse embryonic teeth in the bud stage.

Animals↗
PubMed2001

The effects of leupeptin on cochlear blood flow, auditory sensitivity, and histology.

The purpose of this study was to evaluate the long-term safety of administering leupeptin (1 mg/ml in Hank's Balanced Salt Solution) to the round window membrane by investigating its effects on cochlear blood flow, auditory sensitivity (i.e., auditory brainstem response), and cochlear histology. A comparison of baseline and posttreatment measurements of cochlear blood flow and mean arterial blood pressure in guinea pigs revealed no significant changes. Auditory brainstem response measurements revealed no significant changes in auditory threshold shifts when compared to controls at the 2-, 4-, 6-, and 8-week time points. Furthermore, poststudy surface preparations of the organs of Corti and cytocochleograms from leupeptin-treated ears and controls revealed no significant hair cell losses. These data suggest that the prolonged administration of leupeptin (1 mg/ml at a rate of 0.5 microliter/hr for 8 weeks) to the round window membrane is not ototoxic. This study may serve as a basis for future clinical trials of leupeptin administration for the prevention or treatment of noise-induced hearing loss and the management of tinnitus.

Animals↗
PubMed2000

Asymmetric synthesis of quaternary centers. Total synthesis of (-)-malyngolide.

[structure] The deracemization of 3-nonyl-3,4-epoxybut-1-ene with Pd(0) in the presence of chiral ligands using p-methoxybenzyl alcohol as a nucleophile proceeds regio- and enantioselectively to form the monoprotected vinylglycidol in 99% ee. This chiral building block was converted in seven steps to (-)-malyngolide, an antibiotic showing significant activity against Mycobacterium smegmatis and Streptococcus pyogenes. An interesting aspect involves controlling the diastereoselectivity of protonation of an enolate via a distal hydroxyl group.

Alkylation↗
PubMed2000

Macrophages deficient in CTP:Phosphocholine cytidylyltransferase-alpha are viable under normal culture conditions but are highly susceptible to free cholesterol-induced death. Molecular genetic evidence that the induction of phosphatidylcholine biosynthesis in free cholesterol-loaded macrophages is an adaptive response.

Macrophages in atherosclerotic lesions accumulate excess free cholesterol (FC) and phospholipid. Because excess FC is toxic to macrophages, these observations may have relevance to macrophage death and necrosis in atheromata. Previous work by us showed that at early stages of FC loading, when macrophages are still healthy, there is activation of the phosphatidylcholine (PC) biosynthetic enzyme, CTP:phosphocholine cytidylyltransferase (CT), and accumulation of PC mass. We hypothesized that this is an adaptive response, albeit transient, that prevents the FC:PC ratio from reaching a toxic level. To test this hypothesis directly, we created mice with macrophage-targeted disruption of the major CT gene, CTalpha, using the Cre-lox system. Surprisingly, the number of peritoneal macrophages harvested from CTalpha-deficient mice and their overall health under normal culture conditions appeared normal. Moreover, CT activity and PC biosynthesis and in vitro CT activity were decreased by 70-90% but were not absent. As a likely explanation of this residual activity, we showed that CTbeta2, a form of CT that arises from another gene, is induced in CTalpha-deficient macrophages. To test our hypothesis that increased PC biosynthesis is an adaptive response to FC loading, the viability of wild-type versus CTalpha-deficient macrophages under control and FC-loading conditions was compared. After 5 h of FC loading, death increased from 0.7% to only 2.0% in wild-type macrophages but from 0. 9% to 29.5% in CTalpha-deficient macrophages. These data offer the first molecular genetic evidence that activation of CTalpha and induction of PC biosynthesis in FC-loaded macrophages is an adaptive response. Furthermore, the data reveal that CTbeta2 in macrophages is induced in the absence of CTalpha and that a low level of residual CT activity, presumably due to CTbeta2, is enough to keep the cells viable in the peritoneum in vivo and under normal culture conditions.

Animals↗
PubMed2000

Conformational changes and cleavage by the homing endonuclease I-PpoI: a critical role for a leucine residue in the active site.

The homing endonuclease I-PpoI severely bends its DNA target, resulting in significant deformations of the minor and major groove near the scissile phosphate groups. To study the role of conformational changes within the protein catalyst and the DNA substrate, we have determined the structure of the enzyme in the absence of bound DNA, performed gel retardation analyses of DNA binding and bending, and have mutagenized a leucine residue that contacts an adenine nucleotide at the site of cleavage. The structure of the L116A/DNA complex has been determined and the effects of the mutation on affinity and catalysis have been measured. The wild-type protein displays a rigid-body rotation of its individual subunits upon DNA binding. Homing site DNA is not detectably bent in the absence of protein, but is sharply bent in both the wild-type and L116A complexes. These results indicate that binding involves a large distortion of the DNA and a smaller change in protein conformation. Leucine 116 is critical for binding and catalysis: it appears to be important for forming a well-ordered protein-DNA complex at the cleavage site, for maximal deformation of the DNA, and for desolvation of the nucleotide bases that are partially unstacked in the enzyme complex.

Amino Acid Substitution↗
PubMed2000

Secreted and membrane attractin result from alternative splicing of the human ATRN gene.

Attractin, initially identified as a soluble human plasma protein with dipeptidyl peptidase IV activity that is expressed and released by activated T lymphocytes, also has been identified as the product of the murine mahogany gene with connections to control of pigmentation and energy metabolism. The mahogany product, however, is a transmembrane protein, raising the possibility of a human membrane attractin in addition to the secreted form. The genomic structure of human attractin reveals that soluble attractin arises from transcription of 25 sequential exons on human chromosome 20p13, where the 3' terminal exon contains sequence from a long interspersed nuclear element-1 (LINE-1) retrotransposon element that includes a stop codon and a polyadenylation signal. The mRNA isoform for membrane attractin splices over the LINE-1 exon and includes five exons encoding transmembrane and cytoplasmic domains with organization and coding potential almost identical to that of the mouse gene. The relative abundance of soluble and transmembrane isoforms measured by reverse transcription-PCR is differentially regulated in lymphoid tissues. Because activation of peripheral blood leukocytes with phytohemagglutinin induces strong expression of cell surface attractin followed by release of soluble attractin, these results suggest that a genomic event unique to mammals, LINE-1 insertion, has provided an evolutionary mechanism for regulating cell interactions during an inflammatory reaction.

Alternative Splicing↗