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Biomedical subjects

H H Tai

Publications and source records attributed to H H Tai.

At least 55 records · Page 3Linked to original sources
PubMed1996

Stimulation of prostaglandin E2 synthesis by interleukin-1beta is amplified by interferons but inhibited by interleukin-4 in human amnion-derived WISH cells.

Human amnion-derived WISH cells synthesize little prostaglandin E2 at the basal state. However, the cells were stimulated greatly to synthesize prostaglandin E2 by interleukin-1beta in a dose-dependent manner. Stimulation by interleukin-1beta was synergistically increased by pretreatment of the cells with interferon alpha or gamma, which alone were inactive. Synergism by interferons was dose-dependent. Stimulation by interleukin-1beta, on the contrary, was inhibited by preincubation of the cells with interleukin-4. Inhibition by interleukin-4 was also dose-dependent. Regulation of prostaglandin E2 synthesis by cytokines was further examined at the m-RNA level of cyclooxygenase-2. Quantitative reverse transcription-polymerase chain reaction indicated that the m-RNA level was not increased by interferon-gamma but was synergistically increased by interferon-gamma plus interleukin-1beta. Furthermore, the m-RNA level increased by interleukin-1beta was attenuated by interleukin-4. These results indicate that regulation of interleukin-1beta-stimulated prostaglandin E2 synthesis by interferons and interleukin-4 is controlled at the m-RNA level of cyclooxygenase-2.

Amnion↗
PubMed1996

Oxygen radicals in the nonvagal component of noncholinergic airway constriction.

To test the hypothesis that oxygen radicals play an important role in the nonvagal component of the noncholinergic bronchoconstriction in vivo, 37 guinea pigs weighing 329 +/- 8 g were randomly divided into five groups: group 1, vagotomy; group 2, vagotomy + CAT (catalase); group 3, vagotomy + SOD (superoxide dismutase); group 4, vagotomy + PBN (alpha-phenyl-N-tert-butyl nitrone); and group 5, capsaicin pretreatment. CAT, SOD, and PBN are antioxidants. Each animal was anesthetized, paralyzed, artificially ventilated, and pretreated with atropine and phenoxybenzamine. Immediately after acute capsaicin challenge, animals in group 1 exhibited decreases in maximal expiratory flow, dynamic respiratory compliance, and total lung capacity, as well as an increase in functional residual capacity, indicating noncholinergic airway constriction. The bronchoconstriction was significantly ameliorated by SOD and PBN, and it was almost abolished by capsaicin pretreatment. Thirty minutes after acute capsaicin challenge, there was a significant decrease in airway NEP activity and an increase in lung substance P level in group 1 but not in other groups. These results indicate that nonvagal component of noncholinergic bronchoconstriction is partially modulated by oxygen radicals.

Animals↗
PubMed1996

Identification of metabolites of azaperone in horse urine.

Two metabolites of the tranquilizer azaperone were extracted from alkalinized horse urine after treatment with beta-glucuronidase/sulfatase from limpets (Patella vulgata). The metabolites were identified by a combination of independent chemical synthesis and GC/MS and 1H NMR analysis. The metabolites were identified as 1-(fluorophenyl)-4-[4-(5-hydroxy-2-pyridinyl)-1-piperazinyl]-1-butanol, designated as 5'-hydroxy-azaperol, and 1-(fluorophenyl)-4-[4-(5-hydroxy-2-pyridinyl)-1-piperazinyl]-1-butanone, designated as 5'-hydroxyazaperone. A TLC screening test was developed for detecting both metabolites in basic extracts of horse urine treated with beta-glucuronidase/sulfatase. The screening test was used to detect azaperone metabolites in extracts of horse urine collected for 24 h after intravenous administration of azaperone. The administration of azaperone to horses was confirmed by GC/MS identification of 5'-hydroxyazaperone and 5'-hydroxyazaperol from basic extracts of horse urine treated with beta-glucuronidase/sulfatase. The extracted metabolites were treated with bis(trimethylsilyl)acetamide to produce trimethylsilyl (TMS) ether derivatives, and mass spectra and retention times were compared to those of the synthesized metabolites treated in the same manner.

Animals↗
PubMed1996

Enzyme immunoassay of two nicergoline metabolites, 10 alpha-methoxy-9, 10-dihydrolysergol (MDL) and 1-methyl-10 alpha-methoxy-9, 10-dihydrolysergol (MMDL).

Specific and sensitive enzyme immunoassays for two nicergoline metabolites, 10 alpha-methoxy-9, 10-dihydrolysergol (MDL) and 1-methyl-10 alpha-methoxy-9, 10-dihydrolysergol (MMDL) have been developed. The hydroxyl group of hydroxymethyl at position 8 of either MDL or MMDL was carboxymethylated to introduce a carboxyl group for protein conjugation. Antibodies generated from O-carboxymethyl MDL or MMDL recognized the spacer arm between the hapten and the carrier protein and the molecular domain near the conjugation site as well. A heterologous bridge strategy was used to improve the affinity of the hapten-enzyme conjugate to the antibodies. The sensitivity of both assays was greatly increased by using such an approach. Both antibodies are specific for their own haptens. Little cross reactivity was observed with nicergoline and other metabolites. Determination of MDL and MMDL from both spiked plasma and urine showed nearly quantitative recovery. Detection of MDL and MMDL can be as sensitive as 10 pg/ml.

Animals↗
PubMed1995

Delayed elevation of platelet activating factor in ischemic hippocampus.

We used in vivo microdialysis to define the chronological relationship between release of thromboxane and platelet activating factor (PAF) into the extracellular space of ischemic hippocampus. The thromboxane level peaked after 20 min of postischemic reperfusion, followed by a delayed PAF response 120 min later. We conclude that cerebral ischemia causes delayed elevation of PAF in the extracellular space, long after the immediate synthesis and release of thromboxane metabolites.

Analysis of Variance↗
PubMed1995

Bacterial expression of functional membrane-bound thromboxane synthase having intact sequence and truncated N-terminal hydrophobic segment.

A full-length cDNA for human placental thromboxane synthase and a shortened cDNA lacking the sequence corresponding to the N-terminal 2-29 amino acids were expressed in Escherichia coli using a pCW expression vector. Both intact and truncated recombinant enzyme were found in the membrane fraction and were catalytically active. These results suggest that the N-terminal hydrophobic segment, a proposed membrane anchor for P-450 enzymes, is not solely responsible for attachment of thromboxane synthase to the membrane and is not required for the proper protein folding or the enzyme activity.

Amino Acid Sequence↗
PubMed1995

Expression and site-directed mutagenesis of mouse prostaglandin E2 receptor EP3 subtype in insect cells.

A cDNA encoding for mouse prostaglandin E2 (PGE2) receptor EP3 subtype was cloned from a mouse kidney cDNA library by PCR using terminal primers derived from the known sequence of mouse lung EP3 receptor cDNA. The cloned cDNA was confirmed by sequencing and was expressed in Trichoplusia ni (MG1) insect cells using a baculovirus expression system. A specific protein of 60 kDa was detected by immunoblot with antibodies generated against a unique decapeptide sequence present in the second extracellular loop of the EP3 receptor. Specific binding of [3H]PGE2 with a Kd of 3 nM was also found in the membrane fraction of the insect cells. Ligand binding of the receptor was further studied by site-directed mutagenesis. Arg-309 of the receptor was separately mutated to lysine, glutamate and valine. cDNAs of the wild-type and mutant EP3 receptors were respectively expressed and studied in MG1 insect cells. Binding studies indicated that both glutamate and valine mutant EP3 receptors had no binding of [3H]PGE2. On the contrary, the lysine mutant receptor exhibited an even tighter binding (Kd = 1.3 nM) than the wild-type EP3 receptor. Immunoblot studies indicated that these receptors were expressed in a comparable amount in MG1 insect cells. These results suggest that Arg-309 of EP3 receptor may be essential in ligand binding through ionic interaction.

Amino Acid Sequence↗
PubMed1995

Altered hepatic eicosanoid concentrations in rats treated with the peroxisome proliferators ciprofibrate and perfluorodecanoic acid.

Several hypolipidemic drugs, plasticizers, and other chemicals induce hepatic peroxisome proliferation and hepatocellular carcinomas in rodents. These agents induce and promote hepatocarcinogenesis by unknown mechanisms, since most studies have not found them to be genotoxic. Peroxisome proliferators increase the expression of several genes, including those for the enzymes of the peroxisomal beta-oxidation pathway and the cytochrome P-450 4A family, which metabolize lipids, including eicosanoids and their precursor fatty acids. The peroxisome proliferators ciprofibrate and perfluorodecanoic acid (PFDA) were therefore examined for their ability to alter hepatic eicosanoid concentrations. Rats received injections of 3 or 10 mg PFDA/kg body weight every 14 days or were fed 0.01% ciprofibrate for 10 days, 24 days, 6 weeks, 26 weeks, or 54 weeks. The activity of the peroxisomal enzyme fatty acyl CoA oxidase was significantly increased by both ciprofibrate and PFDA at all times. Hepatic concentrations of prostaglandins E2 and F2a (PGE2, PGF2a), thromboxane B2 (TXB2), and leukotriene C4 (LTC4) were measured by immunoassay. Concentrations of PGE2, PGF2a, and TXB2 were decreased in livers of rats receiving ciprofibrate or PFDA compared to livers of control rats, with ciprofibrate exerting a greater effect than PFDA at the doses used. Hepatic LTC4 concentrations were significantly increased by ciprofibrate at 10 days and PFDA at 54 weeks, and significantly decreased by PFDA at 26 weeks. These alterations in eicosanoid concentrations may be important in the natural history of peroxisome proliferator-induced hepatocarcinogenesis.

Acyl-CoA Oxidase↗
PubMed1995

Age-dependent mechanism in guinea pig bronchoconstriction induced by exsanguination.

Age-related mechanism in exsanguination-induced bronchoconstriction (EIB) was studied in guinea pigs. We used a total of 36 guinea pigs divided into three age categories (immature, adolescent, and mature). Each age category was separated into two groups: control and catalase (CAT). CAT is an antioxidant. Both saline (the control group) and CAT were instilled intratracheally 30 min before exsanguination. The animals were anesthetized, sternotomized and exsanguinated. Maximal expiratory flow (Vmax) was measured both before and 1-30 min after exsanguination. In the control group, exsanguination caused gradual decreases in both total lung capacity (TLC) and Vmax at 50% baseline TLC, and an increase in minimal lung volume, indicating EIB. EIB occurred faster in the immature than in the adolescent and mature guinea pigs, and it was significantly ameliorated by CAT. In 36 additional animals tested before exsanguination, there was no age-related change in either airway neutral endopeptidase (NEP) activity or lung tissue substance P level. Thirty minutes after exsanguination in the control groups, airway NEP activity decreased significantly in all age categories, while lung tissue substance P level increased significantly only in the immature category. CAT prevented most of these NEP and substance P changes. These data suggest that exsanguination increases production of oxygen radicals, which inactivate NEP and enhance breakdown of substance P precursor; the increased tachykinin levels induce EIB. Furthermore, our data indicate that this chain reaction is age-dependent, with a faster and more severe reaction in immature guinea pigs.

Aging↗
PubMed1995

15-Hydroxyprostaglandin dehydrogenase.

Relatively little is known about how 15-PGDH activity is regulated. Changes in 15-PGDH activity have been reported in response to physiological changes brought about by aging, pregnancy, hormonal changes, hypertension and smoking. In addition a large number of drugs have been shown to affect 15-PGDH activity both in vivo and in vitro. The availability of the 15-PGDH cDNA will be a valuable tool for studying how this enzyme is regulated. Isolation of the genomic DNA with its promoter regions has not yet been reported.

Amino Acid Sequence↗
PubMed1995

A sensitive enzyme immunoassay for cyclosporin A using antibodies generated against a novel hapten.

A simple and sensitive enzyme immunoassay for cyclosporin A has been developed. Cyclosporin A (CsA) antibodies were generated in rabbits using a novel hapten (CsA-C6) derived from ozonolysis of a double bond at residue 1 followed by reaction with 6-amino hexanoic acid. The antibodies generated from CsA-C6 recognized the spacer arm between the hapten and the carrier protein. A heterologous bridge strategy for haptenenzyme conjugate was used to improve the sensitivity. A hemisuccinyl ester of cyclosporin A alcohol (CsA-C4) was synthesized, conjugated to horseradish peroxidase and used as the enzyme labeled hapten. A high sensitivity enzyme immunoassay for CsA was developed. The concentration that gave 50% inhibition of binding was 1 ng/ml (8.3 x 10(-10)M) of CsA. The specificity of the antibodies was examined against various metabolites. Metabolites with structural modifications at distal residues 4, 8 and 9 showed the least crossreactivity, whereas metabolites derived from residue 1 exhibited full crossreaction as expected.

Animals↗
PubMed1994

Bacterial expression and site-directed mutagenesis of two critical residues (tyrosine-151 and lysine-155) of human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the first step in the catabolic pathway of the prostaglandins. This enzyme oxidizes the 15-hydroxyl group of prostaglandins to produce 15-keto metabolites which are usually biologically inactive. In this study the cDNA for human placental 15-PGDH was expressed in Escherichia coli and the recombinant enzyme was purified to homogeneity and characterized. The N-terminus of the recombinant protein was sequenced and found to be identical with the known amino-acid sequence of 15-PGDH. Determinations of Km and Vmax values for a number of the prostaglandins and NAD+ indicate that the recombinant enzyme does not appear to be kinetically different from the human placental enzyme. Site-directed mutagenesis was used to examine the importance of two residues which are highly conserved in the short-chain dehydrogenases which are known to be related to 15-PGDH. Tyrosine-151 was changed to phenylalanine and serine while lysine-155 was changed to glutamine and leucine. Western blot analysis indicated that the mutant and wild-type proteins were expressed at the similar levels. However, all of the mutant proteins were found to be inactive. These results indicate that both tyrosine-151 and lysine-155 are required for 15-PGDH activity.

Amino Acid Sequence↗
PubMed1994

The porcine thromboxane synthase-encoding cDNA: sequence, mRNA expression and enzyme production in Sf9 insect cells.

A full-length cDNA encoding porcine thromboxane synthase (TS) was isolated and sequenced. The open reading frame encodes a 534-amino acid (aa) protein (M(r) 60,451) which shares more than 75% identity with TS from other species and is 30% homologous to several enzymes of the cytochrome P-450 III family. Sequence comparison among porcine (p), human (h), and murine (m) TS indicated conservation of eight Cys residues and one putative N-glycosylation site. Several highly conserved regions were identified at the near N terminus, middle and C terminus. The most divergent region lies at aa residues 290-325, within which a Lys308 residue was unique to pTS. Between aa residues 70 and 90, considerable divergence was observed in mTS. Northern analysis showed that the pTS gene was expressed as a 2.3-kb transcript primarily in lung, kidney and thymus. A high-titer recombinant (re-) baculovirus containing pTS cDNA was developed to conduct a time course study of enzyme production in Spodoptera frugiperda (Sf9) cells. TS activity was detectable in the microsomes of Sf9 cells 12-h post-infection and reached maximum by 48 h. The produced TS resembles purified pTS in catalysis, as well as inhibition by a substrate analog inhibitor.

Amino Acid Sequence↗
PubMed1994

Xist is expressed in female embryonal carcinoma cells with two active X chromosomes.

The Xist gene resides on the X chromosome and is expressed in female but not male somatic cells. In female cells, only the Xist allele on the inactive X chromosome is transcribed. We investigated the expression of Xist in diploid P10 female embryonal carcinoma cells that have two active X chromosomes. Xist RNA was present in these P10 cells. The X chromosomes in P10 cells carry different Xist alleles whose transcripts can be distinguished by restriction digestion of their cDNAs. Both alleles were expressed. Clones of P10 cells that had lost an X chromosome did not express Xist from the remaining allele. Thus Xist is expressed in cultured cells developmentally arrested prior to X chromosome inactivation, indicating that the Xist transcript is not always derived from an inactive X chromosome. Therefore, Xist expression per se cannot be a sufficient signal to inactivate an X chromosome.

Alleles↗
PubMed1994

An 125I-labeled N6-substituted azido analog of NAD+ for the photoaffinity labeling of NAD(+)-linked enzymes.

125I-N6-(N-[6-N-(5-iodo-4-azidosalicyl)-aminohexyl]- aminocarbamoylmethyl)-nicotinamide adenine dinucleotide (125I-N6-I-ASA-AH-NAD+) was synthesized by coupling N6-([6-aminohexyl]-carbamoylmethyl)-NAD+ with 4-azidosalicylic acid N-hydroxysuccinimide ester followed by radioiodination. The utility of 125I-N6-I-ASA-AH-NAD+ as an effective site-directed photoprobe was demonstrated by the photolabeling of both glutamate dehydrogenase and 15-hydroxyprostaglandin dehydrogenase. Both enzymes can be saturated with labeled probe with apparent dissociation constants comparable to those reported for NAD+. Photoincorporation of the probe into both enzymes was found to be protected specifically by NAD+. These results indicate that 125I-N6-I-ASA-AH-NAD+ can be a specific photoprobe for NAD(+)-linked enzymes.

Affinity Labels↗
PubMed1994

Immunohistochemical localization, messenger ribonucleic acid abundance, and activity of 15-hydroxyprostaglandin dehydrogenase in placenta and fetal membranes during term and preterm labor.

Type 1 15-hydroxyprostaglandin dehydrogenase (PGDH) is the main enzyme responsible for the metabolism of prostaglandin E2 (PGE2) and PGF2 alpha. To examine the possibility that a deficiency of PGDH might contribute to preterm labor, we measured localization of immunoreactive (IR-) PGDH, PGDH mRNA, and PGDH enzyme activity in chorio-decidua, placenta, and amnion in patients after term elective cesarean section (n = 9), after spontaneous vaginal term delivery (n = 10), and at idiopathic preterm labor (PTL) in the absence of infection (< 36 weeks gestation; n = 11). Localization of IR-PGDH was determined in additional specimens of membranes after PTL with infection (n = 13) and without (n = 37). IR-PGDH was localized in syncytiotrophoblast and intermediate trophoblasts in placenta and in the trophoblast layer of extraplacental chorion, but was absent from amnion in all patient groups. In chorion, the number of IR-positive trophoblasts was significantly reduced in the idiopathic PTL group compared to those in the other groups. The relative abundance of PGDH mRNA in the chorio-decidua, but not the placenta, from spontaneous labor and PTL was significantly less than that after cesarean section. PGDH mRNA in chorio-decidua from preterm patients correlated with PGDH enzyme activity. Undetectable or low IR-PGDH in chorionic trophoblasts was also associated with low enzyme activity. These results suggest that there exists a subset of patients that present in PTL because of reduced PGDH expression in chorionic trophoblasts. We suggest that this relative deficiency would allow PGs synthesized in the amnion or chorion to escape metabolism in the chorion and thereby contribute to the stimulus to idiopathic PTL.

Amnion↗