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Biomedical subjects

H H Tai

Publications and source records attributed to H H Tai.

At least 37 records · Page 2Linked to original sources
PubMed1998

Thromboxanes: synthase and receptors.

Thromboxane A2 is a biologically potent arachidonate metabolite through the cyclooxygenase pathway. It induces platelet aggregation and smooth muscle contraction and may promote mitogenesis and apoptosis of other cells. Its roles in physiological and pathological conditions have been widely documented. The enzyme that catalyzes its synthesis, thromboxane A2 synthase, and the receptors that mediate its actions, thromboxane A2 receptors, are the two key components critical for the functioning of this potent autacoid. Recent molecular biological studies have revealed the structure-function relationship and gene organizations of these proteins as well as genetic and epigenetic factors modulating their gene expression. Future investigation should shed light on detailed molecular signaling events specifying thromboxane A2 actions, and the genetic underpinning of the enzyme and the receptors in health and disease.

Alternative Splicing↗
PubMed1998

Interleukin-1 beta and dexamethasone regulate gene expression of prostaglandin H synthase-2 via the NF-kB pathway in human amnion derived WISH cells.

Interleukin-1beta (IL-1beta) stimulated PGE2 synthesis in human amnion derived WISH cells, whereas dexamethasone blocked IL-1beta-mediated stimulation of PGE2 production. Sequence analysis of the 5'-flanking region of the human prostaglandin H synthase-2 (PGHS-2) gene indicates two putative NF-kB binding sites. Mutation of a single site or both sites resulted in significantly decreased activity of the PGHS-2 promoter. IL-1beta treatment increased significantly the native promoter activity and this increase was attenuated by using the NF-kB-mutant promoter. Dexamethasone treatment also decreased the IL-1beta mediated stimulation of the PGHS-2 native promoter but not the NF-kB mutant promoter. Furthermore, the involvement of the NF-kB was supported by electrophoretic mobility shift assay which revealed an increased nuclear binding of the NF-kB probe upon IL-1beta induction and a decreased nuclear binding of the NF-kB probe upon dexamethasone pre-treatment. These results provide convincing evidence that NF-kB may mediate the IL-1beta stimulation of PGHS-2 gene expression as well as the dexamethasone inhibition of the IL-1beta induction process in WISH cells.

5' Untranslated Regions↗
PubMed1998

Prostaglandin E2 receptor EP3alpha subtype: the role of N-glycosylation in ligand binding as revealed by site-directed mutagenesis.

Functional mouse prostaglandin E2 (PGE2) receptor EP3alpha subtype has been expressed in insect cells using a baculovirus system (Huang C. and Tai H.-H. Biochem J 1995; 307: 493-498). EP3alpha receptor has two potential sites (Asn-X-Ser/Thr), Asn 16 and Asn 193, for N-glycosylation. The role of glycosylation in ligand binding of the EP3alpha receptor was investigated by site-directed mutagenesis. Asn was mutated to Gln in each of the two potential glycosylation sites in the EP3alpha receptor. Recombinant wild-type and mutant EP3alpha receptors were expressed in insect cells using baculovirus. Ligand binding assay indicated that the affinity of PGE2 binding was reduced by 50% in the Gln 193 mutant EP3alpha receptor, while the specificity of ligand binding was unaltered. The affinity for PGE2 binding was not affected in the Gln 16 mutant EP3alpha receptor. However, its specificity was partially changed as the EP3-specific agonist became less effective in displacing the [3H]-PGE2 binding to the mutant receptor. These results indicated that N-glycosylation of the EP3alpha receptor could partially affect the affinity and specificity of the ligand binding.

Animals↗
PubMed1998

Prostaglandin E2 alterations during sepsis are partially mediated by endotoxin-induced inhibition of prostaglandin 15-hydroxydehydrogenase.

Prostaglandin E2 (PGE2) is significantly elevated in the plasma of septic or injured patients and is thought to be a component of the resultant immune suppression associated with augmented rates of infection and mortality. Many studies have examined the effect of burn injury and sepsis on PGE2 synthesis. However, the effect of sepsis or burn injury on the expression of prostaglandin 15-hydroxydehydrogenase (PGDH), the key enzyme responsible for PGE2 degradation, has not been explored. The aim of this study was to examine the effect of endotoxin treatment and/or burn injury on the expression of PGDH. Male BDF1 mice were assigned to four groups (n = 4/group): sham, lipopolysaccharide (LPS) (2.5 mg/kg, Escherichia coli LPS, i.p.), burn (15% body surface area scald injury), and burn + LPS (15% body surface area + 2.5 mg/kg LPS, i.p.). Lung tissue was harvested at specific time points after treatment and subsequently was processed for total RNA and protein. Northern and Western blot analyses were used to examine differences in PGDH protein and mRNA expression. Total RNA was probed with the riboprobe for murine PGDH, and the 100,000 g protein fraction was immunoblotted using an rabbit antimurine PGDH antibody. PGDH was expressed in lung at t = 0 in both the saline and LPS-treated animals. A decrease in mRNA expression was initially observed at 2 hours after LPS treatment. The decrease was also significant (p < 0.05) at 3 hours after LPS and maximal decrease in mRNA and protein expression was observed at 6 hours. At 24 hours after LPS administration, the PGDH mRNA and protein expression was still significantly depressed to 49% of control expression. PGDH expression was similar and not statistically different in both burn and burn + LPS treatment at t = 0. At 2 hours after LPS, PGDH mRNA expression in the burn + LPS treatment group had significantly decreased to 47% in comparison with the burn alone group. Maximal decrease in PGDH mRNA and protein expression in lung from burn + LPS was observed at 6 hours after LPS treatment. This change represents a 73% decrease in mRNA in comparison with the time-matched burn control. At 24 hours after LPS administration, PGDH mRNA but not protein expression in the lung from burn + LPS treated mice was still significantly decreased. In summary, LPS treatment alters PGDH mRNA expression at the transcriptional and protein levels. Consequently, sepsis-induced increases in PGE2 levels may not be only due to increased PGE2 synthesis but also due to decreased PGDH expression and, hence, PGE2 degradation.

Animals↗
PubMed1998

Aspirin-triggered 15-epi-lipoxin A4 (ATL) generation by human leukocytes and murine peritonitis exudates: development of a specific 15-epi-LXA4 ELISA.

Aspirin (ASA) triggers the formation of 15-epi-lipoxins (15-epi-LXs or ATL [ASA-triggered LX]), which are potent bioactive eicosanoids that may contribute to the therapeutic impact of ASA. To elucidate the role of these new compounds in vivo, it is essential to establish quick and sensitive detection methods. To this end, we prepared an enzyme-linked immunosorbent assay specific for 15-epi-LXA4 that proved to be highly sensitive (IC50 approximately 50 pg, minimum detection approximately 3.5 pg) and stereoselective. The amounts of 15-epi-LXA4 generated by human neutrophils from peripheral blood of healthy volunteers using this enzyme-linked immunosorbent assay were in agreement with those values obtained by liquid chromatography. Formation of 15-epi-LXA4 was cell ratio-dependent during THP-1 (a monocytic leukemia cell line)-neutrophil interactions with ASA-treated cells, and 15-epi-LXA4 was not detected with either cell type alone. Generation of 15-epi-LXA4 was also examined in murine peritonitis with ASA administration. Exudates from ASA-treated mice showed increased production of 15-epi-LXA4 that was diminished by indomethacin, a blocker of ASA-dependent acetylation of prostaglandin G/H synthase. A cytochrome P450 inhibitor administered in the presence of ASA did not prevent 15-epi-LXA4 formation, which suggests that P450 does not significantly contribute to formation of 15-epi-LXA4 in this murine model. These results indicate that the new enzyme-linked immunosorbent assay is both sensitive and selective for 15-epi-LXA4 and that 15-epi-LXA4 is produced by human leukocyte-leukocyte interactions. In addition, 15-epi-LXA4 is generated by inflammatory exudates when ASA is administered during murine peritonitis and when prostaglandin G/H synthase is upregulated and acetylated. This assay should provide rapid means to investigate 15-epi-LXA4 generation in both cellular and animal models.

Animals↗
PubMed1997

Characterization of the genomic structure and promoter of the mouse NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene.

The mouse NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) gene and its 5'-flanking region was cloned from a 129 mouse ES bacteriophage P1 genomic library. The gene contains 7 exons and 6 introns and is 11.3 kb in length. The transcription initiation site was mapped at 35 bases upstream from the ATG start codon. The nucleotide sequence of the 1.6 kb promoter region contains two TATA boxes and a number of potential regulatory elements including Sp1, CRE, GRE, AP1, AP2, NF-IL6 and estrogen receptor binding site. Studies of the promoter's activity showed that the first 400 nucleotides of 5'-flanking region efficiently drove the transcription of the luciferase reporter gene in U936 cells upon stimulation with a phorbol ester.

Animals↗
PubMed1997

Cloning and expression of the cDNA for rat NAD+-dependent 15-hydroxyprostaglandin dehydrogenase.

The cDNA for rat NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) was cloned from an intestinal cDNA library. The sequence of this cDNA was found to be identical to that of the reverse transcription-polymerase chain reaction (RT-PCR) product obtained using rat lung RNA as a template. The cDNA contains a 798-bp open reading frame that codes for a protein of 266 amino acids (M(r) 28,775) which shares 88.7% identity with the human 15-PGDH and 92.1% identity with the mouse 15-PGDH protein. The regions that are believed to be the NAD+ binding domain and the active site are conserved in the enzymes from the three different species. However, the sequence of the C-terminal 9 amino acids appears to be significantly different. The authenticity of the rat cDNA was confirmed by the expression of an enzymatically active 15-PGDH in E. coli.

Amino Acid Sequence↗
PubMed1997

Expression of rat steroid 5 alpha-reductase (isozyme-1) in Spodoptera frugiperda, SF21, insect cells: expression of rat steroid 5 alpha-reductase.

The enzyme steroid 5 alpha-reductase (5 alpha R) catalyzes the reduction of testosterone (T) to 5 alpha-dihydrotestosterone (DHT). In this study, the baculovirus expression system was used to overexpress rat 5 alpha R type I isozyme (r5 alpha R 1). The full length of r5 alpha R1 cDNA was inserted into the Autographa californica nuclear polyhedrosis virus (Ac-MNPV) genome and expressed in Spodoptera frugiperda, Sf 21, insect cells. The expressed recombinant r5 alpha-R1 showed maximal enzymatic activity when the infected cells were harvested on day 3 of post-transfection. The K(m) values for NADPH and T were 17 microM and 2.7 microM, respectively. Inhibition of the recombinant r5 alpha R1 by N,N diethyl-4-aza-4-methyl-3-oxo-5 alpha-androstane-17 beta-carboxamide (4MA) was competitive with respect to the substrate (T), and a Ki of 3 nM was obtained. The enzyme was located primarily in the nuclear fraction, and the maximum velocity for the recombinant r5 alpha R1 in this fraction was 60 nmoles DHT/min/mg. Immunoblot analysis indicated a single immunoreactive band at 26 kDa, which corresponds to the molecular weight of r5 alpha R1. Photoaffinity labeling by [2'-32P]-2-azido-NAD P+ ([2'-32P]2N3-NAD P+) and [1,2(3)H] N-(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5 alpha androstane-17 beta-carboxamide ([3H]-4MABP) also showed a labeled protein band at 26 kDa.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1997

Analysis of lupin seed protein digestibility using gel electrophoresis and immunoblots.

Proteins from the seeds of 12 cultivars of three lupin species were analyzed by gel electrophoresis. Similarities between cultivars of the same species were noted. Antibodies raised against the three major globular proteins, conglutin alpha, beta, and gamma, of Lupinus albus cv. Ultra were used to probe immunoblots of crude extracts. The immunoblots revealed variations between cultivars not previously resolved and identified which protein-subunits were derived from which conglutin. In vitro digestibility studies were done on four of the lupin cultivars. During the digestion of these cultivars, the large protein units were shown to be degraded to smaller intermediates with specific molecular sizes. Some of the intermediate protein subunits were identified as being derived from conglutin beta. The digestibility of the four cultivars, based on the amount of identifiable protein in the ruminal fluid digest at 9 and 24 h, showed Ultra > Primorski > Juno > Danja. From this study a novel system of analyzing protein digestibility was devised.

Animals↗
PubMed1997

Intramolecular ADP-ribose transfer reactions and calcium signalling. Potential role of 2'-phospho-cyclic ADP-ribose in oxidative stress.

Intramolecular ADP-ribose transfer reactions result in the formation of cyclic ADP-ribose (cADPR) and 2'-phospho-cyclic ADP-ribose (P-cADPR) from NAD and NADP, respectively. The potent Ca2+ releasing activity of these cyclic nucleotides has led to the postulation that they function as second messengers of Ca2+ signalling. The synthesis and hydrolysis of cADPR and P-cADPR are catalyzed by NAD(P) glycohydrolases, but the metabolic signals that regulate their metabolism are poorly understood. To investigate the physiological roles of cADPR and P-cADPR, it is essential to have methods that allow the routine measurement of these nucleotides in cellular systems. As described here, a sensitive and selective radioimmunoassay (RIA) for cADPR has been adapted to search for the natural occurrence of P-cADPR in mammalian tissues. Perchloric acid extracts prepared from bovine tissues and purified by anion exchange chromatography were found to contain immunoreactive material which was identified as P-cADPR. P-cADPR may play an important role in oxidative stress as a link between NADP(H) metabolism and alteration of intracellular Ca2+ homeostasis.

Adenosine Diphosphate Ribose↗
PubMed1996

Cloning and expression of the cDNA for mouse NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

The cDNA for mouse NAD+ dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) was isolated from a lung cDNA library. The cDNA contains a 798 bp open reading frame that codes for a protein of 266 amino acids (M(r) 28775) which shares 87% identity with the human 15-PGDH protein. The regions that are believed to form the NAD+ binding site and the active site are conserved in the mouse and human enzymes. The authenticity of the mouse cDNA was confirmed by expression of an active 15-PGDH in Escherichia coli. Northern blot analysis demonstrated that 15-PGDH mRNA is expressed primarily in lung, intestine, stomach and liver.

Amino Acid Sequence↗
PubMed1996

The Escherichia coli-derived Fab fragment of the IgM/kappa antibody IN-1 recognizes and neutralizes myelin-associated inhibitors of neurite growth.

A recombinant Fab fragment was prepared from the monoclonal IgM/kappa antibody IN-1, which neutralizes central nervous system myelin-associated neurite growth inhibitors both in vitro and in vivo. The variable domain gene sequences were amplified and cloned after cDNA synthesis from the hybridoma RNA. After insertion into the tet promoter vector pASK85, which provided the constant domains of class IgG1/kappa, equipped with a His6 tag, large amounts of the Fab fragment were produced in Escherichia coli by medium cell density fermentation. The Fab fragment was purified to homogeneity by immobilized metal-affinity chromatography and its biochemical activity was compared with the original IN-1 antibody. In an assay for neurite outgrowth and fibroblast spreading, the Fab fragment showed a similar neutralizing effect on inhibitory substrate properties of central nervous system myelin as the unpurified IgM, although an approximately tenfold higher concentration was necessary. Immunoprecipitation experiments revealed a more selective antigen-binding behaviour for the Fab fragment. The Fab fragment was also successfully applied for antigen detection in immunohistochemical analyses. Therefore, the recombinant Fab fragment of IN-1 shows full functionality in vitro and appears to be well suited for replacing the monoclonal IgM in investigations on fiber tract regeneration in vivo.

3T3 Cells↗
PubMed1996

Site-directed mutagenesis of cysteinyl and serine residues of human thromboxane A2 receptor in insect cells.

A thromboxane A2 receptor cDNA was isolated from a human placenta library by polymerase chain reaction (PCR) and was expressed in insect (Sf21) cells using baculovirus system. The recombinant receptor exhibited [3H]-SQ29548 and [125I]-BOP binding activities with Kd values of 1.01 +/- 0.09 nM and 1.63 +/- 0.23 nM, respectively. The receptor binding activity was inhibited by dithiothreitol in a time- and concentration-dependent manner, indicating the involvement of disulfide linkage in ligand binding. The role of the four conserved cysteinyl residues in ligand binding was further examined by site-directed mutagenesis. Each of the four cysteinyl residues was respectively mutated to a serine residue. C102S, C105S, and C183S mutants exhibited no ligand binding activity although successful expression was achieved as revealed by immunoblot analysis, whereas C257S mutant retained most of the binding activity. Homology analysis of all prostanoid receptors indicates that Cys-105 (first extracellular loop) and Cys-183 (second extracellular loop) are conserved and are presumed to form a disulfide bond for receptor stability as suggested by the inhibition of ligand binding by dithiothreitol reduction. Loss of binding activity by C102S mutant revealed that the sulfhydryl group of Cys-102 must play an essential role in ligand binding. Molecular modeling proposed that the Ser-201 is involved in interacting with TXA2 by forming hydrogen bonding. Point mutations of both Ser-201 and a conserved Ser-255 did not affect the ligand binding specificity and affinity for [3H]-SQ29548, but have significantly altered Kd values for [125I]-BOP. These results indicate that various cysteinyl and serine residues of thromboxane A2 receptor may play different roles in ligand binding.

Amino Acid Sequence↗
PubMed1996

Cysteine 182 is essential for enzymatic activity of human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

Evidence suggests that one or more cysteine residues may be important for the activity of human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). All of these four cysteines (Cys 45, Cys 63, Cys 152, and Cys 182) are found in areas which are believed to be important for the functioning of the enzyme. Site-directed mutagenesis was used to examine the role of the four cysteine residues found in 15-PGDH. Each cysteine was individually changed to an alanine and to phenylalanine. The C182A mutant protein was completely inactive, while the other three alanine mutants retained full activity. When all of the cysteines were individually changed to phenylalanine, only the C45F mutant retained full activity. The C63F mutant enzyme retained only about 10% of the wild-type activity while the C152F and C182F mutants were inactive. From these results it appears that only C182 is necessary for enzyme activity. Mutagenesis of Cys 63 and Cys 152 to phenylalanine lends support to the suggestion that these two residues are located in critical parts of the enzyme.

Base Sequence↗
PubMed1996

Site-directed mutagenesis of the conserved serine 138 of human placental NAD+-dependent 15-hydroxyprostaglandin dehydrogenase to an alanine results in an inactive enzyme.

Human placental NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a member of the short-chain dehydrogenase family of enzymes. It has been proposed that a highly conserved serine residue (corresponding to serine 138 of 15-PGDH) may be involved in the catalytic mechanism of many of these enzymes. Site-directed mutagenesis was used to change serine 138 of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase to an alanine. The mutant protein was then expressed in E. coli. Western blot analysis indicated that the S138A mutant protein was expressed at levels similar to the wild type enzyme; however, the mutant protein was found to be inactive. These results support the proposed role of this highly conserved serine in enzyme activity.

Alanine↗
PubMed1996

Ser-268 plays an important role in ligand binding of prostaglandin E2 receptor EP3alpha subtype.

Functional mouse prostaglandin E2 (PGE2) receptor EP3alpha subtype has been expressed in insect cells using the baculovirus system (C. Huang and H. H. Tai, 1995,Biochem . J. 307, 493-498). Site directed mutagenesis was carried out at Thr-221, Ser-268, and Ser-272. These hydroxy amino acid residues are highly conserved among prostaglandin receptors of many species and could potentially form hydrogen bonds with the hydroxyl group or the keto group of the receptor ligand PGE2. The mutant EP3alpha receptors again were expressed in insect cells and were studied by [3h]PGE2 binding assay. The results indicated that (1) replacement of Ser-272 by alanine did not cause any significant change in PGE2-binding activity, (2) replacement of Thr-221 by alanine also did not cause any significant change in PGE2-binding activity, and (3) replacement of Ser-268 by alanine almost abolished the PGE2-binding activity, while replacement by a threonine did not cause significant change in the PGE2-binding activity but did alter subtype specificity. These results suggest that Ser-268 of EP3alpha receptor plays an important role in ligand binding.

Amino Acid Sequence↗