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Biomedical subjects

J H Lin

Publications and source records attributed to J H Lin.

At least 181 records · Page 10Linked to original sources
PubMed1997

In vitro metabolism of indinavir in the human fetal liver microsomes.

In vitro microsomal formation of primary metabolites of indinavir (CRIXIVAN, MK-0639, L-735,524), an HIV protease inhibitor, were qualitatively similar among the different developmental stages in humans, although the fetal liver had a lower capability to form the metabolites than the pediatric and adult liver. The lower activity of fetal liver was mainly owing to a decrease in the Vmax values. The Vmax value in the fetus was about one-third of that in the adult human, while no significant difference was found in Km values between groups. The liver microsomes were also characterized using P450 markers to examine the development-associated alteration in P450 functional activities. Debrisoquine 4-hydroxylase activity was comparable among the three age groups. In contrast, tolbutamide methyl hydroxylase activity, as well as the CYP3A marker, testosterone 6beta-hydroxylase activity, in the fetal liver microsomes was much lower than in the pediatric and adult by more than 40-fold. However, the difference in testosterone 2beta-hydroxylase and nifedipine N-oxidase activities between fetus and adult was markedly smaller. The ratio of indinavir metabolism in pediatric or adult liver to fetus was 1.7 for pediatric and 3.6 for adult liver microsomes. Similarly, testosterone 2beta-hydroxylase and nifedipine N-oxidase activities showed smaller differences between adult (or pediatric) and fetal liver microsomes than testosterone 6beta-hydroxylase activity. The reason for the observed marked differences in the development-associated alteration may lie in the differences of substrate specificities between CYP3A isoforms.

Adult↗
PubMed1997

Metabolic profiles of montelukast sodium (Singulair), a potent cysteinyl leukotriene1 receptor antagonist, in human plasma and bile.

Montelukast sodium [1-([(1(R)-(3-(2-(7-chloro-2-quinolinyl)-(E)- ethenyl)phenyl)-3-(2-(1-hydroxy-1-methylethyl)phenyl)propyl)thio]methyl)cyclopropylacetic acid sodium salt] (MK-476, Singulair) is a potent and selective antagonist of the cysteinyl leukotriene (Cys-LT1) receptor and is under investigation for the treatment of bronchial asthma. To assess the metabolism and excretion of montelukast, six healthy subjects received single oral doses of 102 mg of [14C]montelukast, and the urine and feces were collected. Most of the radioactivity was recovered in feces, with </=0.2% appearing in urine. Based on these results and the reported modestly high oral bioavailability of montelukast, it could be concluded that a major part of the radioactivity was excreted via bile. A second clinical study was conducted to identify biliary metabolites of montelukast. The bile was aspirated using a modified procedure involving a nasogastric tube placed fluoroscopically near the ampulla of Vater, after an oral dose of 54.8 mg of [14C]montelukast. This technique appears to be a new application for drug metabolism studies. The study was conducted with fasted and nonfasted subjects, with the bile being aspirated continuously under suction over periods of 2-8 hr and 8-12 hr after the dose, respectively. Two hours before the end of the collection procedure, cholecystokinin carboxyl-terminal octapeptide was administered iv to stimulate gallbladder contraction. Plasma samples also were collected periodically over 10 hr. Due to the nature of the collection procedure and the limited sampling time, recovery of radioactivity in bile was incomplete and varied from 3 to 20% of the dose. Radiochromatographic and LC-MS/MS analyses of bile showed the presence of one major and several minor metabolites, along with small amounts of unchanged parent drug. The minor metabolites were identified, by LC-MS/MS comparison with synthetic standards or by NMR, as acyl glucuronide (M1), sulfoxide (M2), 25-hydroxy (a phenol, M3), 21-hydroxy (diastereomers of a benzylic alcohol, M5a and M5b), and 36-hydroxy (diastereomers of a methyl alcohol, M6a and M6b) analogs of montelukast. The major metabolite was characterized as a dicarboxylic acid (M4), a product of further oxidation of the hydroxymethyl metabolite M6. Chiral LC-MS/MS analyses of M4 revealed that this diacid, like M5 and M6, was formed in both diastereomeric forms. The levels of metabolites in the systemic circulation were low in the fed as well as fasted subjects, with <2% of the circulating radioactivity being due to metabolites M5a, M5b, M6a, and M6b. Overall, this bile aspiration technique, which is less invasive than either T-tube drainage or fine-needle percutaneous puncture, provided a convenient and expedient means of identifying the biliary metabolites of montelukast, relatively free of contributions from colonic microflora.

Acetates↗
PubMed1997

Intramural coronary artery disease in swine with naturally occurring hypertrophic cardiomyopathy.

Intramural coronary artery disease (ICAD) has been reported in myocardium affected with hypertrophic cardiomyopathy (HCM), but has never been studied in detail with respect to the cell type or lipid infiltration involved in the wall-thickening. The lack of heart samples may be one of the rationales to hamper the progress in investigating this disease. Recently, the discovery of naturally occurring HCM in swine has provided an excellent opportunity for the study of ICAD because of the high prevalence of ICAD in this animal. The present study provides a detailed structure feature in the thickened arterial wall of ICAD by both histologic and electron microscopic means. Morphologically, the feature of ICAD is due primarily to the neointimal thickening. Smooth muscle cells (SMC) and extracellular matrix (collagen and elastic fibers) are the major components responsible for the thickened neointima. Fragmentation of the internal elastic membrane is associated with the migration and proliferation of SMC from the media to the intima. Therefore, pigs with HCM may be a potential animal model not only for the study of the mechanism by which SMC migrate and proliferate into intima, but also for the future investigation of interventions in coronary artery occlusion.

Animals↗
PubMed1996

Induction of vascular cell adhesion molecule-1 by low-density lipoprotein.

Low-density lipoprotein (LDL) is a well-established risk factor for atherosclerosis. When endothelial cells are incubated with this lipoprotein in pathophysiologic amounts, the cells are activated. Among the documented cellular responses to LDL is increased recruitment of monocytes, which are believed to play a major role in promoting intimal plaque formation. The findings presented here link an atheogenic lipoprotein, LDL, with the induction of an adhesion molecule important in atherogenesis Human LDL induces the vascular cell adhesion molecule-1 (VCAM-1) transcriptionally with an increase in mRNA levels through activation of the VCAM promoter. This effect is blocked by anti-VCAM antibodies. After a 2-day incubation in LDL, the binding of NF-kappa B, which is believed to be a key oxidative-stress sensor for VCAM regulation, remains at basal level. In contrast, the binding activities of AP-1 and GATA, on the other hand, are increased by LDL. Thus, a component of LDL-enhanced endothelial recruitment of monocytes is attributed to VCAM-1 expression, which appears to be mediated through AP-1 and GATA. These data identify LDL as a VCAM-inducer possibly distinct from cytokines and endotoxin.

Arteriosclerosis↗
PubMed1996

Inhibition of nitric oxide synthase or cyclo-oxygenase pathways in organum vasculosum laminae terminalis attenuates interleukin-1 beta fever in rabbits.

Fever was induced in rabbits by i.v. administration of lipopolysaccharide (LPS) or administration of interleukin-1 beta (IL-1 beta) into the organum vasculosum laminae terminalis (OVLT). Intra-OVLT injection of IL-1 receptor antagonist (IL-lra), 1 h before LPS or IL-1 beta injection, inhibited the LPS- or IL-1 beta-induced fever. Dexamethasone (a potent inhibitor of the transcription of inducible nitric oxide synthase, iNOS), L-N5-(1-iminoethyl)ornithine (an irreversible NOS inhibitor), aminoguanidine (a specific iNOS inhibitor), or indomethacin (an inhibitor of cyclo-oxygenase, COX) also inhibited IL-1 beta-induced fever when injected into the OVLT 1 h before IL-1 beta injection. These results suggest that iNOS or COX pathways in the OVLT mediate the IL-1 beta-induced fever in rabbits.

Animals↗
PubMed1996

Improved oral absorption of L-365,260, a poorly soluble drug.

L-365,260, a specific and potent nonpeptide antagonist of the CCKB receptor, is under investigation for its potential utility in the treatment of anxiety and panic disorders. This drug has a very low aqueous solubility (< 2 micrograms mL-1). When L-365,260 was given orally as a suspension in 0.5% methylcellulose (5 mg kg-1), the absorption was rapid but incomplete. The peak concentrations occurred in about 30-40 min, and the bioavailability was 8.6% for the dog and 13.6% for the rat. The poor bioavailability could be attributed to poor absorption or extensive first-pass metabolism. By comparing the drug concentrations in the systemic circulation during portal and femoral (or cephalic) vein infusion, the hepatic first-pass metabolism was estimated to be 0.30 for the rat and 0.14 for the dog, suggesting that first-pass metabolism is not the main reason for the low bioavailability of the drug in rats and dogs. The limited bioavailability is, therefore, more likely due to its poor absorption as a result of its poor aqueous solubility and slow dissolution rate. However, while the absorption was substantially improved when the drug was given orally as a solution in PEG 600 solution, the bioavailability increased threefold to fourfold in rats and sixfold to sevenfold in dogs. Although the underlying mechanism for the improved absorption is unknown, PEG 600 may have exerted a cosolubilizing effect which enhances the dissolution rate of L-365,260 in the GI tract, resulting in better absorption. Kinetic analysis by a deconvolution technique revealed that PEG 600 increased both the extent and rate of absorption. These results are consistent with the notion that absorption of L-365,260 is rate limited by its dissolution rate. The data from these animal studies provides valuable information in selecting the formulation for clinical trials.

Absorption↗
PubMed1996

Synergistic activation of cAMP and calcium on cAMP-response-element-mediated gene expression in GH3 pituitary tumor cells.

Signals responsible for expression of the vasoactive intestinal peptide (VIP)-stimulated prolactin gene in GH3 pituitary tumor cells were examined. Transfection with a deoxyribonucleic acid (DNA) construct containing the chloramphenicol acetyltransferase (CAT) gene fused to the 2.5-kb prolactin 5'-upstream regulatory sequence indicated that VIP stimulated CAT expression. However, this effect could not be mimicked by 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), and was inhibited by the L-type Ca(2+)-channel blocker verapamil. While KCl had little effect on CAT activity, combined treatment with KCl and 8-Br-cAMP synergistically activated CAT expression. Potentiation between KCl and 8-Br-c-AMP was also seen with c-fos messenger ribonucleic acid (mRNA) expression. In addition, KCl and 8-Br-cAMP synergistically activated cAMP response element (CRE)-mediated CAT expression, and the synergism was abolished by verapamil. In the presence of okadaic acid, cAMP had no significant activation on CRE-driven CAT expression, whereas KCl-stimulated CAT expression was greatly potentiated. These results indicate that cAMP and Ca2+ synergistically activated CRE-driven gene expression through non-overlapping phosphorylation events in GH3 cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Bisphosphonates: a review of their pharmacokinetic properties.

Bisphosphonates are a unique class of drugs. As a family, they are characterized pharmacologically by their ability to inhibit bone resorption, whereas, pharmacokinetically, they are classified by their similarity in absorption, distribution, and elimination. Although all bisphosphonates have similar physicochemical properties, their antiresorbing activities differ substantially. Activity is dramatically increased when the amino group is contained in the aliphatic carbon chain. For example, alendronate, an aminobisphosphonate, is approximately 700-fold more potent than etidronate, both in vitro and in vivo. In general, bisphosphonates are poorly absorbed from the gastrointestinal tract as a result of their poor lipophilicity. In vitro and in vivo studies have shown that bisphosphonates are absorbed from the gastrointestinal tract via paracellular transport. Systemically available bisphosphonates disappear very rapidly from plasma, and are partly taken up by the bone and partly excreted by the kidney. The relative contribution of these two processes to overall plasma elimination differs significantly among bisphosphonates. To date, all bisphosphonates studied show no evidence of metabolism. Renal excretion is the only route of elimination. Studies with alendronate in rats indicate that the drug is actively secreted by an uncharacterized renal transport system, and not by the anionic or cationic renal transport systems. Bisphosphonates bind preferentially to bones which have high turnover rates, and their distribution in bone is not homogeneous. After bone uptake, the bisphosphonates are liberated again only when the bone in which they are deposited is resorbed. Thus, the half-life of bisphosphonates in bone is very long, ranging among different species from 1 to 10 years, depending largely on the rate of bone turnover.

Animals↗
PubMed1996

In vitro studies on the metabolic activation of the furanopyridine L-754,394, a highly potent and selective mechanism-based inhibitor of cytochrome P450 3A4.

L-754,394, a furanopyridine derivative, is an experimental anti-HIV agent which has been shown to be an unusually potent and selective inhibitor of cytochrome P450 3A enzymes in a number of mammalian species. In the present studies, L-754,394 was demonstrated to undergo NADPH-dependent metabolic activation in hepatic microsomal preparations from rats, dogs, rhesus monkeys, and humans to electrophilic intermediates which became bound covalently to cellular proteins. The extent of binding was species-dependent, the highest levels being observed with liver microsomes from rhesus monkeys. Inclusion in incubation media of the nucleophilic trapping agents glutathione, cysteine, or methoxyamine led to a modest (15-25%) decrease in the covalent binding, while trichloropropylene oxide, an inhibitor of epoxide hydrolase, had no effect. When L-754,394 was incubated with monkey liver microsomes, the corresponding dihydrofurandiol was identified as a metabolite by liquid chromatography-tandem mass spectrometry. In contrast, when incubations were carried out in the presence of methoxyamine, the O-methyloxime derivative of the ring-opened dihydrodiol tautomer was formed, while inclusion of glutathione or N-acetylcysteine led to the formation of S-linked conjugates of a putative furan epoxide. Collectively, these results are taken to indicate that L-754,394 undergoes cytochrome P450-dependent oxidation of the fused furan ring system, leading to the formation of chemically-reactive intermediates. One or more of these electrophilic species may be responsible for the autocatalytic destruction of cytochrome P450 enzymes which accompanies L-754,394 metabolism in vitro and in vivo.

Animals↗
PubMed1996

A modified procedure for caseinophosphopeptide analysis.

A modified procedure is established for analyzing caseinophosphopeptides. The sodium caseinate hydrolysate is first treated by immobilized metal ion affinity chromatography to enrich the phosphopeptides. Because of the formation of Fe(3+)-peptide complexes, ethylenediaminetetraacetic acid is added to the bound fraction eluted with the immobilized metal ion affinity chromatography to disintegrate the complexes. Thus, the subsequent high-performance liquid chromatographic analysis is facilitated. A stepwise gradient elution is also suggested to enhance the resolution of caseinophosphopeptides during high-performance liquid chromatographic analysis.

Amino Acid Sequence↗
PubMed1996

Substantial decrease of heat shock protein 90 in ventricular tissues of two sudden-death pigs with hypertrophic cardiomyopathy.

We recently developed a pig model with naturally occurring hypertrophic cardiomyopathy (HCM). To further characterize the biochemical features of high frequency of sudden death affected by this disease, the protein profiles of ventricular tissues were analyzed on normal, HCM, and HCM with sudden-death pigs. By sodium dodecylsulfate-polyacrylamide gel electrophoresis, a protein corresponding to an apparent molecular mass of 90 kDa (p90) was found to be markedly decreased in the HCM-affected tissues of sudden-death pigs, but not in HCM or normal pigs. Further study showed that the primary decrease of p90 in HCM pigs with sudden death was located mainly in the interventricular septum. As determined by the molecular mass and isoelectric point on 2-dimensional gels and Western immunoblot with a specific 90 kDa heat shock protein (HSP90) monoclonal antibody, the unknown protein was identified as HSP90. Our findings indicate for the first time that substantially decreased heart HSP90 is associated with HCM pigs with sudden death. Although the role that HSP90 may play in protecting pigs with HCM from sudden death is still nuclear, the model itself may provide further insight into understanding the role of heat shock proteins in cardiac sudden death.-Lee, W.-C., Lin, K.-Y., Chiu, Y.-T., Lin, J.-H., Cheng, H.-C., Huang, H.-C., Yang, P.-C., Liu, S.-K., Mao, S. J. T. Substantial decrease of heat shock protein 90 in ventricular tissues of two sudden-death pigs with hypertrophic cardiomyopathy.

Animals↗
PubMed1996

Characterization of the NTPase activity of Japanese encephalitis virus NS3 protein.

Japanese encephalitis (JE) virus NS3 protein and two N-terminally truncated (delta 1-148 and delta 1-323) forms of NS3 were engineered and expressed in E. coli as fusion proteins with a histidine tag at the N terminus. The purified recombinant proteins his-NS3 and his-NS3(delta 1-148) were found to possess NTPase activity which was stimulated by single-stranded RNA, whereas NS3(delta 1-323) did not. The requirements for MgCl2 and MnCl2 and the salt and pH ranges necessary for optimal activity of the enzyme were determined and shown to be slightly different from those of the NTPases of other flaviviruses. Poly(U) and poly(C) were better than poly(A) at stimulating the NTPase activities, in contrast to other flaviviral NTPases. The substrate preference was in the order GTP > ATP >> UTP > CTP. Interestingly, we found that Ca2+ could not substitute for Mg2+; on the contrary, it inhibited NTPase activity. The removal of the N-terminal 148 amino acids enhanced NTPase activity, but further deletion of the region (amino acids 148-323) completely abolished the activity. Therefore, amino acids 148-323 contain a critical region required for NTPase activity.

Acid Anhydride Hydrolases↗
PubMed1996

Nitric oxide synthase-cyclo-oxygenase pathways in organum vasculosum laminae terminalis: possible role in pyrogenic fever in rabbits.

1. Fever was induced in rabbits by administration of Escherichia coli endotoxin (lipopolysaccharide; LPS; 0.001-10 micrograms) into the organum vasculosum laminae terminalis (OVLT). Deep body temperature was evaluated over a period of 7 h. 2. The LPS-induced febrile response was mimicked by intra-OVLT injection of the nitric oxide (NO) donors, S-nitroso-acetylpenicillamine (SNAP, 1-10 micrograms), sodium nitroprusside (SNP, 50 micrograms), or hydroxylamine (10 micrograms), the cyclic GMP analogue 8-bromo-cyclic GMP (8-Br-cyclic GMP, 10-100 micrograms), or prostaglandin E2 (PGE2, 0.2 micrograms). 3. Dexamethasone (Dex, a potent inhibitor of the transcription of inducible NO synthase, iNOS, 10 micrograms), anisomycin (a protein synthesis inhibitor, 100 micrograms), L-N5-(1-iminoethyl)ornithine (L-NIO; an irreversible NOS inhibitor, 10-200 micrograms), aminoguanidine (a specific iNOS inhibitor, 1000 micrograms), or NG-methyl-L-arginine acetate (L-NMMA, a NOS inhibitor, 100 micrograms) inhibited fever induced by LPS when injected into the OVLT 1 h before LPS injection. An intra-OVLT dose of 1000 micrograms of NG-nitro-L-arginine methyl ester (L-NAME, a potent inhibitor of constitutive NOS) did not exhibit antipyretic effects. 4. Methylene blue (an inhibitor of NOS and soluble guanylate cyclase, 1-10 micrograms), 6-(phenylamino)-5,8-quinolinedione (LY-83583; an inhibitor of soluble guanylate cyclase and NO release, 20 micrograms), or indomethacin (an inhibitor of cyclo-oxygenase, COX, 400 micrograms) inhibited fever induced by LPS when injected into the OVLT 1 h before LPS injection. Pretreatment with methylene blue or haemoglobin (a NO scavenger, 100 micrograms) attenuated the fever induced by intra-OVLT injection of SNAP. 5. The PGE2-induced fever was potentiated, rather then attenuated, by pretreatment with an intra-OVLT dose of animoguanidine (1000 micrograms), L-NMMA (100 micrograms) or L-NIO (200 micrograms). 6. These results suggest that iNOS-COX pathways in the OVLT represent an important mechanism for modulation of pyrogenic fever in rabbits.

Animals↗
PubMed1996

The retrotransposon Tf1 assembles virus-like particles that contain excess Gag relative to integrase because of a regulated degradation process.

The retrotransposon Tf1, isolated from Schizosaccharomyces pombe, contains a single open reading frame with sequences encoding Gag, protease, reverse transcriptase, and integrase (IN). Tf1 has previously been shown to possess significant transposition activity. Although Tf1 proteins do assemble into virus-like particles, the assembly does not require readthrough of a translational reading frame shift or stop codon, common mechanisms used by retroelements to express Gag in molar excess of the polymerase proteins. This study was designed to determine if Tf1 particles contain equal amounts of Gag and polymerase proteins or whether they contain the typical molar excess of Gag. After using two separate methods to calibrate the strength of our antibodies, we found that both S. pombe extracts and partially purified Tf1 particles contained a 26-fold molar excess of Gag relative to IN. Knowing that Gag and IN are derived from the same Tf1 primary translation product, we concluded that the excess Gag most likely resulted from specific degradation of IN. We obtained evidence of regulated IN degradation in comparisons of Tf1 protein extracted from log-phase cells and that extracted from stationary-phase cells. The log-phase cells contained equal molar amounts of Gag and IN, whereas cells approaching stationary phase rapidly degraded IN, leaving an excess of Gag. Analysis of the reverse transcripts indicated that the bulk of reverse transcription occurred within the particles that possess a molar excess of Gag.

Base Sequence↗
PubMed1996

Native LDL increases endothelial cell adhesiveness by inducing intercellular adhesion molecule-1.

Native LDL (n-LDL) increased human umbilical vein endothelial cell (EC) adherence of mononuclear cells. Such phenotypic changes suggest that n-LDL alters the usual expression of cell adhesion molecules to enhance the adhesive properties of the endothelium. To investigate n-LDL mechanisms governing adherence, ECs were exposed to n-LDL in concentrations up to 240 mg/dL for 2 and 4 days. n-LDL-treated ECs bound nearly threefold more phorbol myristate acetate (PMA)-stimulated U937 cells than control ECs but did not bind unstimulated U937 cells. Anti-cellular adhesion molecule-1 (ICAM-1) antibodies blocked PMA-stimulated U937 cell binding to control and n-LDL-treated ECs by more than 80%, suggesting that increases in ICAM-1 may be involved in this increased adherence. Although increases in PMA-stimulated U937 cell binding developed with respect to time and concentration, statistically significant increases were achieved only when n-LDL concentrations exceeded 180 mg cholesterol/dL at day 4. n-LDL increased endothelial adherence of freshly isolated human monocytes more than twofold and neutrophils by almost twofold. Fluorescent-linked immunoassays revealed that n-LDL increased ICAM-1 protein expression by twofold, which corresponded with increased ICAM-1 message levels. n-LDL also appeared to increase E-selectin and vascular cell adhesion molecule-1 message levels, but these changes did not translate into statistically significant differences in protein levels. Taken together, these data indicate that n-LDL increases ICAM-1 expression to enhance the adhesive properties of the endothelium. Such perturbations in EC function likely represent a proinflammatory response to protracted n-LDL exposure and one of the early steps in atherogenesis.

Base Sequence↗
PubMed1996

Microstructure and segregation behavior of palladium in silver-copper-palladium alloys.

It has been reported that the addition of palladium can modify the microstructure and improve the properties of Ag-Cu eutectic alloy as well as admixed Cu-rich amalgam. The purpose of this work was to study the microstructure and segregation behavior of palladium in a series of Ag-Cu-Pd alloys. All microstructural and microchemical results consistently indicated a strong tendency for palladium to form the ordered Cu3Pd superlattice in the copper-rich phase of the present ternary alloys. Transmission electron microscopic examination indicated that, in addition to the large Cu-rich particles, numerous small (typically tens of nanometers) Cu-rich particles were distributed in the Ag-rich phase. In the alloys containing 10 and 15 wt% Pd, the Cu3Pd superlattice had an L1(2)-type crystal structure. In the alloy containing 20 wt% Pd, the Cu3Pd had a periodic (regular) APB structure. The solubility of palladium in the Cu-rich phase was always much larger than that in the Ag-rich phase. The ratio of the palladium concentration in the copper-rich phase to that in the silver-rich phase decreased with the overall palladium content.

Chemical Phenomena↗