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Biomedical subjects

S Liu

Publications and source records attributed to S Liu.

At least 739 records · Page 41Linked to original sources
PubMed1997

[Study on the cytotoxic and DNA damaging effects in oral mucosal fibroblasts by areca nut extract].

An aqueous areca nut extract (ANE) was tested for its cytotoxic effects on cultured human embryo oral mucosal fibroblasts (HE-OMF) in vitro by using the trypan blue and thiazolyl blue (MTT) assay. The ANE decreases the cell survival rate in a dose-dependent manner (P < 0.05). So was the extract in inducing damage on the cellular DNA of HE-OMF in vitro examined by the nick translation assay. The increase in counts per minute (CPM) values was significant (P < 0.05) for comparing all four concentration groups tested, The results suggests that aqueous ANE is highly cytotoxic and capable to induce DNA damage on cultured HE-OMF. It may have potential carcinogenic effect on the oral mucosal membrane of whom habitually chewing the areca nut frequently for quite a long time. Futher study is required to illustrate the detail process and study the mechanism of these effects.

Areca↗
PubMed1997

[A preliminary evaluation on transcranial Dopplor ultrasonic scanning for differentiation of symptoms and signs in patients with ganyang shangkangzheng].

Intracranial main artery of Ganyang Shangkangzheng and its related syndrome were determined by Transcranial Doppler (TCD) Ultrasonography. The results showed that the blood flow speed in the mid-cerebral artery of 32 cases with Ganyang Shangkangzheng was obviously higher than that of the healthy group, Ganhuo Shangyan group and Gan-shenyinxu group, similar change was found in three kinds of parameters in the blood flow speed. Therefore, the results suggest that TCD in determinating the blood flow speed of middle cerebral artery can be thought as one of the diagnostic assays in differentiation of symptom and sign of Ganyang Shangkangzheng.

Adult↗
PubMed1997

[The preventing function of garlic on experimental oral precancer and its effect on natural killer cells, T-lymphocytes and interleukin-2].

In order to study the effect and mechanism of garlic on preventing oral precancer, we divided randomly 32 Wistar rats into two groups. The garlic group was painted with garlic solution on the hard palatal mucosae. The control group was applied with distilled water that is equal in quantity. Then, chemical carinogen 4-nitroquinoline 1-oxide (4NQO) was painted on the same sites for both groups, three times weekly. Eight rats were randomly killed in the 10th, 13th week. The hard palatal mucosae were examined with light microscope. Meanwhile, lymphocytes were isolated from the rat spleens. The activation of natural killer (NK) cells and T-lymphocytes, and level of interleukin-2 were determined by radioimmunoassay. The results revealed that garlic effectively prevented oral precancer induced by 4NQO. This effect may be related to the following factors that garlic can improve the activation of NK cells, the function of T-lymphocytes, and the level of IL-2.

Animals↗
PubMed1996

Activated function of the pyruvate dehydrogenase phosphatase through Ca2+-facilitated binding to the inner lipoyl domain of the dihydrolipoyl acetyltransferase.

Micromolar Ca2+ facilitates approximately 10-fold enhancement of pyruvate dehydrogenase phosphatase (PDP) activity by aiding the association of PDP with the dihydrolipoyl acetyltransferase (E2) component. Connected by linker regions, E2 consists of two lipoyl domains, the NH2-lipoyl domain (L1) and the interior lipoyl domain (L2), and a pyruvate dehydrogenase component binding domain surrounding a 60-mer inner core. Using recombinant constructs of L1 or L2, E2-enhanced PDP activity was markedly decreased by L2 but not by L1, effectively competing with intact E2 in Ca2+-dependent binding of PDP (half-maximal reduction at 2.0 microM L2 versus 6.7 microM E2 subunit). Using L2 fused to glutathione S-transferase resulted in direct Ca2+-dependent binding of PDP to L2 (Kd, approximately 1.7 microM L2). Affinity-bound glutathione S-transferase-L2 was used to purify PDP to homogeneity by selective binding and elution by Ca2+ chelation. The large activity enhancement of PDP by E2 was eliminated by enzymatic removal of lipoates from E2 and restored by their enzymatic reintroduction. The critical role of the L2 lipoate is not in binding of PDP to E2, since PDP was still bound by delipoylated L2, and delipoylated L2 inhibited E2-enhanced PDP activity, although lipoylated L2 was more effective in each of these tests. Thus, pyruvate dehydrogenase complex activity is increased by enhanced availability of PDP to its E2-bound, phosphorylated pyruvate dehydrogenase substrate as a consequence of the Ca2+-facilitated interchange of PDP among the mobile L2 domains and an essential (undetermined) step engaging the L2 lipoate.

Acetyltransferases↗
PubMed1996

Inactivation of S-adenosyl-L-homocysteine hydrolase by amide and ester derivatives of adenosine-5'-carboxylic acid.

S-Adenosyl-L-homocysteine (AdoHcy) hydrolase has been shown to have (5'/6') hydrolytic activity with vinyl (5') or homovinyl (6') halides derived from adenosine (Ado). This hydrolytic activity is independent of its 3'-oxidative activity. The vinyl (or homovinyl) halides are converted into 5'(or 6')-carboxaldehydes by the hydrolytic activity of the enzyme, and inactivation occurs via the oxidative activity. Amide and ester derivatives of Ado-5'-carboxylic acid were prepared to further probe the hydrolytic capability of AdoHcy hydrolase. The oxidative activity (but not the hydrolytic activity) is involved in the mechanism of inhibition of the enzyme by the ester and amide derivatives of Ado-5'-carboxylic acid, in contrast to the inactivation of this enzyme by adenosine-derived vinyl or homovinyl halide analogues during which both activities are manifested.

Adenosine↗
PubMed1996

Interaction of ATP binding sites in the ArsA ATPase, the catalytic subunit of the Ars pump.

The ArsA ATPase is the catalytic subunit of the Ars pump that catalyzes arsenical extrusion in Escherichia coli, thus providing resistance. The active form of ArsA is a homodimer with four nucleotide binding sites, two from each monomer. The codons for Gly-15 in the N-terminal consensus nucleotide binding sequence and Gly-334 in the C-terminal sequence were individually mutated to cysteine codons. Cells expressing an arsAG334C mutation retained arsenite resistance, while an arsAG15C mutation resulted in substantial reductions in arsenite resistance, transport, and ATPase activity. Selection for suppression of the G15C mutation that restored arsenite resistance yielded an A344V substitution. Ala-344 is located adjacent to the C-terminal nucleotide binding sequence. The second site mutation did not suppress the loss of resistance resulting from G18D, G20S, or T22I substitutions in the N-terminal nucleotide binding site. Cells expressing the G15C/A344V double mutant regained arsenite extrusion. These results suggest a spatial proximity of Gly-15 and Ala-344 and support a model for interaction of the nucleotide binding sites in ArsA.

Adenosine Triphosphatases↗
PubMed1996

Phosphatidylinositol 4,5-bisphosphate binding to the pleckstrin homology domain of phospholipase C-delta1 enhances enzyme activity.

The pleckstrin homology (PH) domain is a newly recognized protein module believed to play an important role in signal transduction. While the tertiary structures of several PH domains have been determined, some co-complexed with ligands, the function of this domain remains elusive. In this report, the PH domain located in the N terminus of human phospholipase C-delta1 (PLCdelta1) was found to regulate enzyme activity. The hydrolysis of phosphatidylinositol (PI) was stimulated by phosphatidylinositol 4,5-bisphosphate (PIP2) in a dose-dependent manner with an EC50 = 1 microM (0.3 mol%), up to 9-fold higher when 5 microM (1.5 mol%) of PIP2 was incorporated into the PI/phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles (30 microM of PI with a molar ratio of PI:PS:PC = 1:5:5). Stimulation was specific for PIP2, since other anionic phospholipids including phosphatidylinositol 4-phosphate had no stimulatory effect. PIP2-mediated stimulation was, however, inhibited by inositol 1,4, 5-triphosphate (IP3) in a dose-dependent manner, suggesting a modulatory role for this inositol. When a nested set of PH domain deletions up to 70 amino acids from the N terminus of PLCdelta1 were constructed, the deletion mutant enzymes all catalyzed the hydrolysis of the micelle forms of PI and PIP2 with specific activities comparable with those of the wild type enzyme. However, the stimulatory effect of PIP2 was greatly diminished when more than 20 amino acid residues were deleted from the N terminus. To identify the specific residues involved in PIP2-mediated enzyme activation, amino acids with functional side chains between residues 20 and 40 were individually changed to glycine. While all these mutations had little effect on the ability of the enzyme to catalyze the hydrolysis of PI or PIP2 micelles, the catalytic activity of mutants K24G, K30G, K32G, R38G, or W36G was markedly unresponsive to PIP2. Analysis of PIP2-stimulated PI hydrolysis by a dual substrate binding model of catalysis revealed that the micellar dissociation constant (Ks) of PLCdelta1 for the PI/PS/PC vesicles was reduced from 558 microM to 53 microM, and the interfacial Michaelis constant (Km) was reduced from 0.21 to 0.06 by PIP2. The maximum rate of PI hydrolysis (Vmax) was not affected by PIP2. These results demonstrate that a major function of the PH domain of PLCdelta1 is to modulate enzyme activity. Further, our results identify PIP2 as a functional ligand for a PH domain and suggest a general mechanism for the regulation of other proteins by PIP2.

Binding Sites↗
PubMed1996

The rat liver glutathione S-transferase Ya subunit gene: characterization of the binding properties of a nuclear protein from HepG2 cells that has high affinity for the antioxidant response element.

A nuclear protein from HepG2 cells (YABP) that binds the antioxidant response element (ARE), which is required for activation of the rat glutathione S-transferase (GST) Ya subunit gene by planar aromatic compounds and phenolic antioxidants, was further characterized by quantitative competition binding experiments and DNA mutational analysis. The apparent dissociation constant of the YABP-ARE complex was estimated as < or = 0.77 nM, suggesting that the YABP has very high affinity for the ARE. There is no difference in the affinity of the YABP for the ARE when HepG2 cells are treated with inducers that transcriptionally activate the GST Ya subunit gene. Quantitative competition binding analyses in conjunction with mutagenesis of the ARE revealed that an 11-nucleotide region in the 41-nucleotide ARE, 5'-GGTGACAAAGC-3', is responsible for binding to the YABP. Eight nucleotides of this core sequence are in close proximity to the YABP, indicating that there is a broader spectrum of protein contact points than those required for the transcriptional activation. van't Hoff analysis of effects of temperature on binding has revealed that the binding reaction is governed mainly by entropy changes, which could result from conformational changes in the YABP and/or the ARE upon the formation of the complex. In addition, the native molecular weight of the YABP was determined to be 74 300 using gel filtration chromatography. These data together with previous UV cross-linking data suggest that the YABP exists as a heterodimer.

Animals↗
PubMed1996

Effects of aging on Na(+)-pump current in rat ventricular myocytes.

This study was designed to determine if aging affects ouabain-sensitive Na(+)-pump current (Ip), an electrophysiological measure of Na,K-ATPase activity, in rat heart. Ventricular myocytes were enzymatically isolated from hearts of young adult (4-6 months of age) and aged (28-36 months of age) male Long-Evans rats. Ip was monitored using conventional whole-cell patch-clamp techniques under both maximally stimulating (85 mM intracellular Na+ with 10.8 mM extracellular K+) and 'physiological' (10 mM intracellular Na+ with 5.4 mM extracellular K+) conditions. Values were expressed relative to membrane capacitance to account for the larger cell size of the aged myocytes. Results indicated that maximal Ip is smaller (e.g. approximately 23% at the holding potential of -40 mV) in myocytes isolated from aged as compared to young adult rats. However, neither the voltage-dependence of maximal Ip nor the pump current monitored under 'physiological' conditions were affected by age in general, these data support results of previous biochemical and ion flux studies which demonstrated that aging is associated with a decline in the Na(+)-pump capacity of the myocardium.

Aging↗
PubMed1996

PSK protects macrophages from lipoperoxide accumulation and foam cell formation caused by oxidatively modified low-density lipoprotein.

In previous works, it has been evidenced that lipoperoxidative injury to macrophages caused by oxidatively modified low-density lipoprotein (O-LDL) plays an important role in foam cell formation, and that PSK, a protein bound polysaccharide extracted from the class Basidiomycetes Coriolus Versicolor, can protect macrophages from lipoperoxidative injury induced by tert-butyl hydroperoxide (tbOOH). In this paper PSK protection of macrophages from lipoperoxide (LPO) accumulation and foam cell formation caused by O-LDL and its action mechanism were further studied. The LPO accumulation was determined by using ACAS 570. Dynamic assay of the LPO level in eight single cells after adding O-LDL or determination of the average LPO content in a lot of cells incubated in advance with O-LDL for 12 h, both indicated that O-LDL might induce LPO accumulation in macrophages and the effects of O-LDL could be prevented by PSK. O-LDL might cause the changes of morphological structure in macrophages and the transformation of macrophages into foam cells, and the effects could also be prevented by PSK. The determination of selenium-dependent glutathione peroxidase (SeGSHPx) activities and mRNA contents of macrophages and changes of SeGSHPx activity and mRNA content after incubation with tbOOH showed that PSK might increase the SeGSHPx activity of macrophage and the enhanced SeGSHPx activity may occur at the level of gene transcription.

Animals↗
PubMed1996

Time, voltage and ionic concentration dependence of rectification of h-erg expressed in Xenopus oocytes.

The rapid delayed rectifier, IKr, is believed to have h-erg (human ether-à-go-go related gene) as its molecular basis. A recent study has shown that rectification of h-erg involves a rapid inactivation process that involves rapid closure of the external mouth of the pore or C-type inactivation. We measured the instantaneous current to voltage relationship for h-erg channels using the saponin permeabilized variation of the cut-open oocyte clamp technique. In contrast to C-type inactivation in other voltage-gated K+ channels, the rate of inactivation was strongly voltage dependent at depolarized potentials. This voltage dependence could be modulated independently of activation by increasing [K+]0 from 2 to 98 mM. These results suggest that inactivation of h-erg has its own intrinsic voltage sensor.

Animals↗
PubMed1996

Sex- and tissue-specific expression of a cytochrome P450 2C2-luciferase transgene.

To study the transcriptional regulation of the rabbit cytochrome P450 2C2 gene (CYP2C2), transgenic mice were generated using a DNA fragment containing the CYP2C2 5'-flanking region fused to a luciferase reporter gene. In one of three transgenic lines, this transgene was expressed in the liver and at a lower level in the kidney, similar to the tissue-specific pattern of this gene in rabbits. Unlike expression in rabbits, the transgene was expressed in a male-specific pattern in liver and kidney, and in the brain of both sexes. Expression of the transgene in male mice was repressed after castration, while expression in females was stimulated after testosterone treatment and periodic injection of rat growth hormone (GH). The expression of this transgene was also regulated developmentally in the liver. These results suggest that this 3500 bp of CYP2C2 5'-flanking region contains the basal transcriptional regulation elements for tissue-specific and developmental expression. Although a position effect can not be ruled out, this transgene may also include the cis-regulatory element for sexual dimorphic expression.

Animals↗
PubMed1996

Aristeromycin-5'-carboxaldehyde: a potent inhibitor of S-adenosyl-L-homocysteine hydrolase.

In an earlier study, Liu et al. (Bioorg. Med. Chem. Lett. 1992, 2, 1741-1744) showed that both the E and Z isomers of 4',5'-didehydro-5'-fluoroaristeromycin were very potent irreversible inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase. However, it was unclear from a mechanistic standpoint whether these vinyl fluorides were themselves type-I mechanism-based inhibitors causing reduction of enzyme-bound NAD+ or whether they were prodrug for aristeromycin-5'-carboxaldehyde, which was the ultimate type-I inhibitor. To elucidate this mechanism of enzyme inhibition, (4'S)- and (4'R)-aristeromycin-5'-carboxaldehydes (1a,b) were synthesized in this study and shown to be potent type-I mechanism-based inhibitors of AdoHcy hydrolase with k2/Ki values of 4.4 x 10(6) and 8.2 x 10(4)M-1min-1, respectively. However, Using 19F NMR and HPLC, it was shown that (4'S)-4,5'-dedehydro-5'-fluoraristeromycin in the presence of AdoHcy hydrolase did not release fluoride ion or generate aristeromycin-5'-carboxaldehyde (1a,b). These results suggest that the E and Z isomers of 4',5'-didehydro-5'-fluoroaristeromycin are inactivating AdoHcy hydrolase by directly reducing NAD+ to NADH and not using the hydrolytic activity of the enzyme to generate aristeromycin-5'-carboxaldehyde.

Adenosine↗
PubMed1996

cDNA cloning and expression of HIP, a novel cell surface heparan sulfate/heparin-binding protein of human uterine epithelial cells and cell lines.

Heparan sulfate proteoglycans and their corresponding binding sites have been suggested to play an important role during the initial attachment of murine blastocysts to uterine epithelium and human trophoblastic cell lines to uterine epithelial cell lines. Previous studies on RL95 cells, a human uterine epithelial cell line, had characterized a single class of cell surface heparin/heparan sulfate (HP/HS)-binding sites. Three major HP/HS-binding peptide fragments were isolated from cell surfaces by tryptic digestion, and partial amino-terminal amino acid sequence for each peptide fragment was obtained (Raboudi, N., Julian, J., Rohde, L. H., and Carson, D. D. (1992) J. Biol. Chem. 267, 11930-11939). In the current study, using approaches of reverse transcription-polymerase chain reaction and cDNA library screening, we have cloned and expressed a novel, cell surface HP/HS-binding protein, named HP/HS interacting protein (HIP), from RL95 cells. The full-length cDNA of HIP encodes a protein of 159 amino acids with a calculated molecular mass of 17,754 Da and pI of 11.75. Transfection of HIP full-length cDNA into NIH-3T3 cells demonstrated cell surface expression and a size similar to that of HIP expressed by human cells. Predicted amino acid sequence indicates that HIP lacks a membrane spanning region and has no consensus sites for glycosylation. Northern blot analysis detected a single transcript of 1.3 kilobases in both total RNA and poly(A+) RNA. Examination of human cell lines and normal tissues using both Northern blot and Western blot analyses revealed that HIP is expressed at different levels in a variety of human cell lines and normal tissues but absent in some cell lines and some cell types of normal tissues examined. HIP has relatively high homology (approximately 80% both at the levels of nucleotide and protein sequence) to a rodent ribosomal protein L29. Thus, members of the L29 family may be displayed on cell surfaces where they may participate in HP/HS binding events.

3T3 Cells↗
PubMed1996

First-sphere and second-sphere electrostatic effects in the active site of a class mu gluthathione transferase.

The activation of the thiol of glutathione (GSH) bound in the active site of the class mu glutathione transferase M1-1 from rat involves a hydrogen-bonding network that includes a direct (first-sphere) interaction between the hydroxyl group of Y6 and the sulfur of GSH and second-sphere interactions involving a hydrogen bond between the main-chain amide N-H of L12 and the hydroxyl group of Y6 and an on-face hydrogen bond between the hydroxyl group of T13 and the pi-electron cloud of Y6 (i.e., T13-OH---pi-Y6-OH--- -SG). The functions of these hydrogen bonds have been examined with a combination of site-specific mutagenesis and X-ray crystallography. The hydroxyl group of Y6 has a normal pKa of about 10 even though it is shielded from solvent and is in a largely hydrophobic environment. The apparent pKa of GSH in the binary Y6F.GSH complex is increased by 1.6 log units, and the reactivity of the enzyme-bound nucleophile is reduced. The catalytic properties of the Y6L mutant are identical to those of Y6F, suggesting that the weakly polar on-edge interaction between the aromatic ring and sulfur has no influence on catalysis. The refined three-dimensional structure of the Y6F mutant in complex with GSH shows no major structural perturbation of the protein other than a change in the coordination environment of the sulfur. Removal of the second-sphere influence of the on-face hydrogen bond between the hydroxyl groups T13 as in the T13V and T13A mutants elevates the pKa of enzyme-bound GSH by about 0.7 pKa units. Crystal structures of these mutants show that structural changes in the active site are minor and suggest that the changes in pKa of E.GSH are due to the presence or absence of the on-face hydrogen bond. The T13S mutant has a completely different side-chain hydrogen-bonding geometry than T13 in the native enzyme and catalytic properties similar to the T13A and T13V mutants consistent with the absence of an on-face hydrogen bond. The gamma-methyl group of T13 is essential in enforcing the on-face hydrogen bond geometry and preventing the hydroxyl group from forming more favorable conventional hydrogen bonds.

Animals↗
PubMed1996

Identification of glutamic acid 381 as a candidate active site residue of Pseudomonas aeruginosa exoenzyme S.

Exoenzyme S of Pseudomonas aeruginosa (ExoS) is a member of the family of bacterial ADP-ribosylating exotoxins (bAREs). Site-directed mutagenesis of glutamic acids within the catalytic domain of ExoS (termed delta N222) allowed the identification of the preferential inactivation of ADP-ribosyltransferase activity by alanine substitution of E381. The specific activity of E381A mutant was 0.02% of wild-type delta N222. Delta N222(E381A) retained the requirement of factor activating exoenzyme S (FAS) activation for the expression of ADP-ribosyltransferase activity. In contrast, E387A, E399A, and E414A mutants possessed ADP-ribosyltransferase activity similar to that of wild-type delta N222. Kinetic evaluation of E381A and two other mutants, E381D and E381S, showed that their primary defect was a lower kcat in the ADP-ribosylation of soybean trypsin inhibitor (SBTI). The Km for NAD and SBTI and activation by FAS varied 2- and 10-fold relative to delta N222. In addition, the E381 mutants possessed identical protease patterns during thrombin and trypsin digestion as delta N222, which indicated that E381 mutants had retained their overall conformation. Together, these data identify E381 as contributing to the catalytic activity of exoenzyme S.

ADP Ribose Transferases↗