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L Chen

Publications and source records attributed to L Chen.

At least 1,081 records · Page 60Linked to original sources
PubMed1995

Probing the active sites of rat and human cytochrome P450 2E1 with alcohols and carboxylic acids.

Cytochrome P450 2E1 (P450 2E1) catalyzes the biotransformation of many low-molecular-weight compounds including industrial solvents, indoor pollutants, alcohol, and drugs. In order to understand the nature of the P450 2E1 active site, we studied the competitive inhibition of the P450 2E1-catalyzed N-nitrosodimethylamine demethylation by alcohols and carboxylic acids with different alkyl chain lengths. Using microsomes from acetone-treated rats as the enzyme source of P450 2E1, the Ki value for each compound was measured. With primary alcohols and secondary alcohols, the Ki decreased with the increase in the carbon chain length until the carbon number reached 6 or 7; the free energy increment of binding was 0.28 kcal/mol CH2 group. Similar inhibitory effects were also observed with human P450 2E1 heterologously expressed in Hep G2 cells. These results suggest that both rat and human P450 2E1 contain a pocket with hydrophobicity that serves as a binding site for low-molecular-weight substrates. Among a series of carboxylic acids and omega-hydroxycarboxylic acids investigated, dodecanoic acid was the strongest inhibitor (Ki = 22 microM), and 12-hydroxydodecanoic acid had the lowest Ki (320 microM) within the series of omega-hydroxycarboxylic acids. The free energy increment of binding for carboxylic acids was 0.35 kcal/mol CH2 group. 1,10-Decanedicarboxylic acid, which contains a carboxylic group at each end, showed a Ki > 20 mM. We suggest that for optimal interaction of a carboxylic acid moiety with the active site of P450 2E1, the hydrocarbon end of the molecule binds to the substrate binding site, leaving the carboxylic acid group outside of a proposed substrate access channel. The length of optimal substrates such as dodecanoic acid may reflect the length of the substrate access channel and the size of the active site. We estimate that the distance from the opening of the access channel to the oxygenation site is about 15 A. Based on the structural features of P450 2E1 substrates and competitive inhibitors, we propose a conceptual model to illustrate the binding of these molecules in the active site of P450 2E1.

Alcohols↗
PubMed1995

The opioid peptide dynorphin directly blocks NMDA receptor channels in the rat.

1. The actions of dynorphin on N-methyl-D-aspartate (NMDA) responses were examined in acutely dissociated trigeminal neurons in rat. Whole-cell and single-channel currents were recorded using the patch clamp technique. 2. Dynorphins reduced NMDA-activated currents (INMDA). The IC50 was 0.25 microM for dynorphin (1-32), 1.65 microM for dynorphin (1-17) and 1.8 microM for dynorphin (1-13). 3. The blocking action of dynorphin is voltage independent. 4. The inhibitory action of dynorphin cannot be blocked by high concentration of the non-selective opioid receptor antagonist naloxone, nor by the specific kappa-opioid receptor antagonist nor-Binaltorphimine (nor-BNI). 5. Single-channel analyses indicate that dynorphin reduces the fraction of time the channel is open without altering the channel conductance. 6. We propose that dynorphin acts directly on NMDA receptors.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1995

Intratumoral activation and enhanced chemotherapeutic effect of oxazaphosphorines following cytochrome P-450 gene transfer: development of a combined chemotherapy/cancer gene therapy strategy.

Cyclophosphamide and its isomer ifosfamide are cell cycle-nonspecific alkylating agents that undergo bioactivation catalyzed by liver cytochrome P-450 enzymes. The therapeutic efficacy of these oxazaphosphorine anticancer drugs is limited by host toxicity resulting from the systemic distribution of activated drug metabolites formed in the liver. Since tumor cells ordinarily do not have the capacity to activate oxazaphosphorines, we examined whether introduction into tumor cells of a cDNA encoding CYP2B1, a major catalyst of oxazaphosphorine activation, sensitizes the cells to the cytotoxic effects of cyclophosphamide and ifosfamide. Here we show that 9L gliosarcoma cells stably transfected with a cDNA encoding rat CYP2B1 are highly sensitive to cyclophosphamide and ifosfamide cytotoxicity as compared to parental 9L cells or 9L cells transfected with an Escherichia coli beta-galactosidase gene. The CYP2B1 enzyme inhibitor metyrapone protects the CYP2B1-expressing 9L cells from oxazaphosphorine cytotoxicity, demonstrating that the chemosensitivity of these cells is a direct consequence of intracellular prodrug activation. Moreover, CYP2B1-expressing 9L cells potentiate the cytotoxic effects of cyclophosphamide and ifosfamide toward cocultured CYP2B1-negative 9L tumor cells. This "bystander effect" does not require cell-cell contact, and therefore may have the therapeutic advantage of distributing cytotoxic drug metabolites to a wide area within a solid tumor mass. In vivo experiments using Fischer 344 rats implanted s.c. with CYP2B1-expressing 9L tumor cells demonstrated that intratumoral expression of the CYP2B1 gene provides a substantial therapeutic advantage over that provided by liver cytochrome P-450-dependent drug activation alone; cyclophosphamide treatment resulted in complete growth inhibition of CYP2B1-positive tumors, whereas only a modest growth delay effect was obtained with CYP2B1-negative tumors. These studies establish that drug-activating CYP genes may be useful for the development of novel combined chemotherapy/gene therapy strategies for cancer treatment utilizing established cancer chemotherapeutic agents.

Animals↗
PubMed1995

Chimeric human calcitonin and glucagon receptors reveal two dissociable calcitonin interaction sites.

Two chimeric receptors were constructed by transposing the coding regions for the putative N-terminal domains of the human calcitonin (hCTR) and glucagon (hGGR) receptors. These receptors were stably expressed as glycosylated proteins with molecular masses of 80 kDa for the calcitonin receptor N-terminus chimera (NtCTr) and 65 kDa for the glucagon receptor N-terminus chimera (NtGGr). The NtCTr chimera binds salmon calcitonin (sCT) with an apparent Kd of 12 nM relative to 0.3 nM for the native hCTR. However, this chimera does not mediate a cAMP response even with a transfectant expressing 1.8 x 10(6) cell surface receptors. Stable transfectants expressing the NtGGr chimera show no detectable binding of 125I-sCT or 125I-human glucagon. Surprisingly, adenylate cyclase is activated through the NtGGr chimera by sCT, pCT, and hCT with half-maximal activation at 2.2 +/- 0.6, 5.8 +/- 2.1, and 810 +/- 151 nM, respectively, and the maximum response is similar to that induced by 25 microM forskolin. The rank-order of competition for sCT binding to the NtCTr chimera is similar to the hCTR (sCT > pCT > hCT), but the concentrations required for half-maximal competition are 100- to > 2000-fold higher. In addition, salmon calcitonin binds with a much more rapid on-rate and off-rate to the NtCTr chimera relative to the hCTR which binds hormone irreversibly. Cross-linking of 125I-sCT to the NtCTr chimera with bis(sulfosuccinimidyl) suberate is much greater than to the hCTR, suggesting unique conformations for the two receptor-hormone complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1995

Hyaluronan on the surface of tumor cells is correlated with metastatic behavior.

In the present study, we examined the metastatic potential of tumor cells expressing different levels of cell surface hyaluronan. We used flow cytometry to isolate subsets of the B16-F1 mouse melanoma cell line that expressed either high (HA-H) or low (HA-L) amounts of hyaluronan on their surfaces. These two subsets of cells showed a 32-fold difference in the amount of cell surface hyaluronan, due to its rate of synthesis. However, these cell lines did not differ from each other with regard to their in vitro growth rates, susceptibility to natural killer-mediated cytotoxicity, or the expression of the cell surface proteins CD44, ICAM-1, and GMP-140. When these cells were injected s.c., they both formed s.c. tumors of approximately the same size. However, when injected into the tail vein of mice, the HA-H cells formed a greater number of nodules in the lungs and caused a faster rate of mortality than the HA-L cells. The presence of hyaluronan did enhance the interaction of the HA-H cells with cultured endothelial cells that expressed CD44. Thus, it is possible that enhanced interactions between hyaluronan and CD44 promoted the formation of tumor embolisms which, in turn, increased the chances that the tumor cells would be trapped in the lungs. Taken together, these results suggest that hyaluronan may play a critical role in the process of tumor metastasis.

Animals↗
PubMed1995

Glycine 122 is essential for cooperativity and binding of Mg2+ to porcine fructose-1,6-bisphosphatase.

Site-directed mutagenesis of an amino acid residue in the substrate binding site of porcine fructose-1,6-bisphosphatase was carried out based on the crystal structure of the enzyme (Zhang, Y., Liang, J.-Y., Huang, S., Ke, H., and Lipscomb, W. N. (1993) Biochemistry 32, 1844-1857). A mutant enzyme form of fructose-1,6-bisphosphatase, G122A, was purified and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectrometry (CD), and initial rate kinetics. There were no discernible differences between the secondary structures of the wild-type and the mutant enzyme on the basis of CD data. Altering Gly-122 to alanine caused a significant decrease in the enzyme's activity and affinity for Mg2+. The kcat for this mutant enzyme was only about 5% of that of wild-type fructose-1,6-bisphosphatase, and the Ka for Mg2+ was about 20-fold higher than that of the wild-type enzyme. The Ki for AMP was increased 77-fold in the case of the mutant enzyme; however, the Hill coefficient was unaltered. Most importantly, it was observed that replacement of Gly-122 with alanine caused the total loss of cooperativity for Mg2+. It is concluded that Gly-122 is essential for Mg2+ cooperativity and important for binding of Mg2+ and AMP as well as for enzyme activity.

Animals↗
PubMed1995

Melatonin administration prevents lipopolysaccharide-induced oxidative damage in phenobarbital-treated animals.

The protective effect of melatonin on lipopolysaccharide (LPS)-induced oxidative damage in phenobarbital-treated rats was measured using the following parameters: changes in total glutathione (tGSH) concentration, levels of oxidized glutathione (GSSG), the activity of the antioxidant enzyme glutathione peroxidase (GSH-PX) in both brain and liver, and the content of cytochrome P450 reductase in liver. Melatonin was injected intraperitoneally (ip, 4mg/kg BW) every hour for 4 h after LPS administration; control animals received 4 injections of diluent. LPS was given (ip, 4 mg/kg) 6 h before the animals were killed. Prior to the LPS injection, animals were pretreated with phenobarbital (PB), a stimulator of cytochrome P450 reductase, at a dose 80 mg/kg BW ip for 3 consecutive days. One group of animals received LPS together with Nw-nitro-L-arginine methyl ester (L-NAME), a blocker of nitric oxide synthase (NOS) (for 4 days given in drinking water at a concentration of 50 mM). In liver, PB, in all groups, increased significantly both the concentration of tGSH and the activity of GSH-PX. When the animals were injected with LPS the levels of tGSH and GSSG were significantly higher compared with other groups while melatonin and L-NAME significantly enhanced tGSH when compared with that in the LPS-treated rats. Melatonin alone reduced GSSG levels and enhanced the activity of GSH-PX in LPS-treated animals. Additionally, LPS diminished the content of cytochrome P450 reductase with this effect being largely prevented by L-NAME administration. Melatonin did not change the content of P450 either in PB- or LPS-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1995

Acute appendicitis secondary to metastatic nasopharyngeal carcinoma.

This is a review of a rare case of acute appendicitis secondary to metastatic nasopharyngeal carcinoma (NPC) that had never before been reported in the literature. The clinical presentation did not differ from usual cases of acute appendicitis, but the pathology caused us to re-evaluate the NPC stage of the patient.

Acute Disease↗
PubMed1995

Crystallization and preliminary crystallographic data for class I deoxyribose-5-phosphate aldolase from Escherichia coli: an application of reverse screening.

X-ray quality crystals of class I-deoxyribose-5-phosphate aldolase from Escherichia coli have been obtained for the unliganded enzyme and in complex with its substrate, 2-deoxyribose-5-phosphate. The enzyme catalyzes the reversible cleavage of 2-deoxyribose-5-phosphate to acetaldehyde and D-glyceraldehyde-3-phosphate. The unliganded and complex crystals are prismatic long rods and belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 183.1 A, b = 61.4 A, c = 49.3 A and a = 179.2 A, b = 60.5, A, c = 49.1 A, respectively. Two molecules in the asymmetric unit are related by a noncrystallographic 2-fold axis. The crystals are stable in the X-ray beam and diffract to at least 2.6 A. A new method, reverse screening, designed to minimize protein utilization during the screening process was used to determine supersaturation and crystallization conditions.

Aldehyde-Lyases↗
PubMed1995

Malate metabolism by Desulfovibrio gigas and its link to sulfate and fumarate reduction: purification of the malic enzyme and detection of NAD(P)+ transhydrogenase activity.

Malate metabolism was investigated in lactate grown cells of Desulfovibrio gigas ; 3 mol of malate are converted into 2 mol succinate and 1 mol acetate. The malic enzyme (L-malate:NADP+ oxidoreductase) was purified to homogeneity and partially characterized. The enzyme is monomeric with molecular weight of 45 kDa. Its spectrum has no visible absorption and the activity is stimulated by K+ and Mg2+. The presence of an NAD(P)+ transhydrogenase, the observation of partial reduction of adenylylsulfate reductase by NADH (via NADH-rubredoxin oxidoreductase) and evidence for NADH-linked fumarate reductase activity support the involvement of pyridine nucleotides in the electron pathway toward the reduction of sulfur compounds and/or fumarate. An electron transfer chain to fumarate is proposed, taking into consideration these results and the stoichiometry of end-products derived from malate dismutation.

Journal Article↗
PubMed1995

Preparation of antisera to recombinant, soluble N-acetylglucosaminyltransferase V and its visualization in situ.

N-Acetylglucosaminyltransferase V (GlcNAc-T V) is a glycosyltransferase which transfers N-acetylglucosamine in beta(1,6) linkage to the alpha(1,6)-linked mannose residue of Asn-linked oligosaccharides. This enzyme is characterized by several unusual properties: GlcNAc-T V is the largest lumenal Golgi glycosyltransferase described thus far, and its multiple mRNA transcripts range from 4.5 to about 9.5 kb; GlcNAc-T V mRNA and activity are regulated by the src tyrosine kinase signalling pathway; in brain tissue, large levels of GlcNAc-T V mRNA are present, but only relatively low levels of catalytic activity can be detected; a lectin-resistant cell line, Lec4A, expresses active GlcNAc-T V which is mislocalized intracellularly. In addition, the cell surface oligosaccharide products of this enzyme have been hypothesized to regulate intercellular adhesion. In order to devise specific inhibitors of this enzyme it is necessary to understand its physical structure and how structural changes can influence its activity and localization. We have expressed milligram amounts of a soluble form of recombinant rat GlcNAc-T V, purified it from CHO cell-conditioned media, and used it to prepare specific antisera. This antisera binds selectively to GlcNAc-T V and has been used to visualize B-16 mouse melanoma cell GlcNAc-T V on immunoblots after SDS-PAGE. When the antisera was used in immunofluorescence microscopy experiments on permeabilized B-16 and baby hamster kidney cells, intense, specific staining was observed in intracellular structures which appear to correspond to the Golgi apparatus.

Animals↗
PubMed1995

Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: functional and structural considerations.

Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca(2+) ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following enzyme phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca2+ binding site are thereby changed, permitting vectorial dissociation of bound Ca2+ against a concentration gradient. A long range linkage between phosphorylation and Ca2+ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.

Animals↗
PubMed1995

Calcium mitigation in bioprosthetic tissues by iron pretreatment: the challenge of iron leaching.

Preliminary studies in our laboratory have shown that iron pretreatment of glutaraldehyde-preserved tissues inhibited calcification. The present study was designed to further investigate this finding and to optimize the technique. Porcine valve tissue and bovine pericardium preserved in glutaraldehyde and pretreated by iron at different concentrations and incubation times were implanted either subcutaneously in rats or as a mitral valve substitute in sheep. Compared with control groups, calcification was markedly inhibited in the groups pretreated with Fe3+. There was a direct correlation between the degree of calcification and the iron content within the tissue. A minimal iron content within the tissue of 0.5% for porcine valve tissue and 0.2% for pericardial tissue was found to be necessary for calcium mitigation. A progressive leaching of Fe3+ was observed that could be stabilized by using a higher concentration of iron in the solution and or by additional pretreatment in glutaraldehyde at high temperature, but only for subcutaneously implanted tissues. By contrast, leaching was faster and more complete in the iron-pretreated tissue valves placed in the circulating blood environment, therefore resulting in calcified valves. Pretreatment in glutaraldehyde at high temperature (50 degrees C) alone was able to mitigate calcification in both subcutaneous and circulating blood environments, a new finding that may lead to an improved method for glutaraldehyde preservation of tissues.

Animals↗
PubMed1995

Effects of kanamycin ototoxicity and hair cell regeneration on the DC endocochlear potential in adult chickens.

High doses of aminoglycoside antibiotics cause massive damage to the avian basilar papilla. The resulting functional loss could conceivably arise from the reduction in the DC endocochlear potential (EP) due to impairment of the tegmentum vasculosum (TV) or to shunting of current through the damaged sensory epithelium. To test this hypothesis, the EP was measured in adult chickens after destroying hair cells in the basal half of the cochlea with a high dose (400 mg/kg per day for 10 days) of kanamycin (KM). KM treatment caused an increase in the steady-state EP from +18.1 to +23.3 mV and a decrease in the magnitude of the negative EP from -42.0 to -19.2 mV. The EP showed almost no change between 1 and 2 days and 1 week post-KM treatment. After 4 weeks of recovery, most hair cells had regenerated; however, the steady-state EP was still elevated by 13% and the negative EP was depressed by 37%. These results suggest that functional loss as shown by the large reduction in cochlear microphonic (CM) and the elevated thresholds of compound action potential (CAP) following KM treatment is not due to a reduction in the EP but may arise from functional deficits in the hair cells and/or the auditory nerve.

Animals↗
PubMed1995

Rapid angiographic progression of "target" and "nontarget" stenoses in patients awaiting coronary angioplasty.

OBJECTIVES: Our aim was to compare the short-term evolution of "target" versus "nontarget" stenoses in patients awaiting coronary angioplasty. BACKGROUND: Coronary angioplasty is effective therapy for angina pectoris, but coronary events occur after successful angioplasty that are caused by both restenosis and progression of mild preexisting nontarget stenoses. METHODS: We prospectively studied 161 consecutive patients with stable angina (124 men and 37 women). After diagnostic angiography, target stenoses for angioplasty and nontarget lesions were identified. Patients were put on a routine waiting list and followed up regularly until repeat coronary arteriography was performed (mean +/- SD 7 +/- 3 months), either immediately before angioplasty (138 patients) or soon after an acute coronary event (23 patients), if one occurred. Stenosis diameter was measured by using computerized arteriography. Progression of disease was defined as > or = 20% lesion diameter reduction, new total occlusion or development of a "new" stenosis > or = 30%. RESULTS: At study entry, the mean diameter of target (n = 207) and nontarget (n = 184) lesions was 68 +/- 9% and 38 +/- 9%, respectively (p < 0.001). Disease progression occurred in 33 patients (20%). Seven new lesions (one total occlusion) developed. Eighteen target (9%) and 15 nontarget (8%) stenoses progressed. The power of the study to detect a difference of 1% between the risks of progression of target and nontarget stenoses with a 90% probability was < 0.1. Total occlusion developed in 15 (83%) of the 18 target and 6 (40%) of the 15 nontarget stenoses (p = 0.03). During follow-up, a myocardial infarction developed in 3 patients (2%) and unstable angina in 20 (12%). These coronary events were associated with progression of target stenoses in 10 patients and nontarget stenoses in 7 and with the development of new lesions in 1. In five patients coronary events were not associated with stenosis progression. CONCLUSIONS: Despite differences in baseline severity, a similar proportion of target and nontarget lesions progressed rapidly. However, target stenoses were more likely than nontarget lesions to progress to total occlusion. Progression of nontarget stenoses may contribute to recurrence of angina and new coronary events after successful angioplasty and should be considered when developing strategies aimed at improving outcome after angioplasty.

Aged↗