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Biomedical subjects

H Xia

Publications and source records attributed to H Xia.

At least 109 records · Page 6Linked to original sources
PubMed1996

In vitro and in vivo activity of 16,17-dehydro-epipregnanolones: 17,20-bond torsional energy analysis and D-ring conformation.

PURPOSE: Certain neuroactive pregnane steroids (also known as "epalons") are allosteric modulators of the GABA, receptor and have been shown to be potent anticonvulsants, anxiolytics, sedative/hypnotics, and anesthetic agents. The purpose of this study was to calculate the structural consequences of introduction of a double bond in the 16,17-position and to determine if this modification would selectively reduce sedative activity, but maintain the potent anticonvulsant activity of neuroactive steroids. METHODS: We have studied the biochemical and behavioral effects of introducing a 16,17 double bond into the naturally occurring neuroactive steroids, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-P) and 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha,5 beta-P) and three synthetic neuroactive steroid derivatives, 3 alpha-hydroxy-3 beta-methyl-5 alpha-pregnan-20-one (3 alpha,3 beta Me,5 alpha-P), 3 alpha-hydroxy-5 alpha-androstane (3 alpha, 5 alpha-A), and alphaxalone (3 alpha,5 alpha-11-one-P). RESULTS: The 16-ene analogs of most of these neuroactive steroids were found to be 7- and 16-fold less potent in inhibiting [35S]TBPS binding to GABAA receptors and in a similar fashion, had reduced anticonvulsant and sedative potency in proportional amounts. The exception was the androstane (3 alpha,5 alpha-A) without a 17-acetyl group, that had virtually identical IC50 and ED50 values for the saturated and unsaturated derivatives. Calculation of the torsional energy profile for each of the 17-acetyl side chain conformations showed that the conformational energy minima found in the alpha,beta-unsaturated keto systems, produce an orientation of the 20-keto group that is rotated by 165 degrees when compared to the non-conjugated acetyl group (determined by X-ray crystallography and its minimum energy conformation). CONCLUSIONS: The modified orientation of the 20-keto group of neuroactive steroids containing a 16-ene, provides an explanation for their decreased biological activity overall, but did not lead to an enhanced protective index.

Animals↗
PubMed1996

The elimination of diazepam in Chinese subjects is dependent on the mephenytoin oxidation phenotype.

1. The disposition of diazepam and desmethyldiazepam was studied in 21 healthy male Chinese subjects who were phenotyped with mephenytoin. Four poor metabolizers (PM) were identified by phenotyping with mephenytoin and by genotyping for CYP2C19. 2. Serum diazepam and desmethyldiazepam concentrations were measured by high performance liquid chromatography in samples drawn up to 24 days after administration. 3. The plasma elimination half-lives of diazepam (100.8 +/- 32.3 h) and desmethyldiazepam (219.9 +/- 62.7 h) in PMs were significantly longer than those (34.7 +/- 23.0 h for diazepam, 103.1 +/- 25.9 h for desmethyldiazepam) of the 17 phenotyped extensive metabolizers (EM), and those (30.8 +/- 24.9 h for diazepam, 103.1 +/- 27.5 h for desmethyldiazepam) of the five genotyped EMs. 4. The mephenytoin S/R ratios were significantly correlated with the plasma half-lives of diazepam (r = 0.543, P < 0.05) and desmethyldiazepam (r = 0.522, P < 0.05), and with the clearance (r = -0.524, P < 0.05) of diazepam in 21 subjects. 5. These results are compatible with the conclusion that both diazepam and desmethyldiazepam are metabolized by cytochrome P450 CYP2C19 in the Chinese population. 6. The mephenytoin S/R ratios in nine EMs who drank alcohol frequently were significantly higher than those of seven EMs who were non-drinkers, but the plasma kinetics of diazepam and desmethyldiazepam were not significantly different between the two groups. The explanation for these finding is not clear.

Asian People↗
PubMed1996

Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori using short oligonucleotide probes containing repetitive sequences.

The ability of oligonucleotide probes containing short repetitive sequence motifs to differentiate between isolates of Helicobacter pylori was investigated. Genomic DNA preparations from H. pylori were digested with the restriction enzyme HindIII, electrophoresed in agarose gels and transferred to nylon filters. Five separate oligonucleotide probes were tested for hybridization sequentially to fingerprint the digested DNA from a panel of 29 clinical isolates and one type strain of H. pylori, and their relative discriminatory abilities were assessed. Four probes, (GACA)4, (GT)8, (GTG)5 and (GGAT)4, were each shown to yield highly informative hybridization band profiles allowing differentiation of H. pylori isolates. The DNA fingerprints of individual isolates obtained with each probe were distinct and reproducible. Direct comparison with ribotyping revealed that oligonucleotide fingerprinting had far superior discriminatory power. Computer-assisted similarity analysis of (GGAT)4-generated hybridization profiles of pairwise combinations of H. pylori isolates revealed that there was no correlation between ribotype and oligonucleotide fingerprint patterns. The results of this study demonstrate that oligonucleotide probes containing microsatellite sequences provide a new and powerful tool for isolate discrimination of H. pylori.

DNA Fingerprinting↗
PubMed1996

Site-directed mutagenesis of amino acids 33-44 of the common alpha-subunit reveals different structural requirements for heterodimer expression among the glycoprotein hormones and suggests that cyclic adenosine 3',5'-monophosphate production and growth promotion are potentially dissociable functions of human thyrotropin.

Amino acid residues 33-44 of the common alpha-subunit of the glycoprotein hormones have been implicated in heterodimerization as well as high affinity receptor binding of human (h) CG. In the present study, we compared the role of specific amino acids within this region for glycoprotein hormone heterodimer formation, using a transient transfection system to coexpress different mutant alpha-subunit constructs with the beta-subunit of either hTSH, hCG, or hFSH. Our results identified a crucial role for alpha Pro38 in the heterodimer expression of hTSH as well as hFSH, similiar to what had been described for hCG. In contrast, alpha Ala38, which had been critical for hCG, was not essential for hTSH heterodimer expression and less important for hFSH, whereas alpha Phe33 and alpha Arg35 appeared uniquely important for hFSH. Furthermore, we assessed the role of these residues for bioactivity and receptor binding of hTSH. Mutation of the surface-exposed residues alpha Arg42-Ser43-Lys44, which form part of a unique alpha-helical structure, to Ala42-Ala43-Ala44, decreased TSH receptor binding using porcine thyroid membranes as well as rat FRTL-5 cells. Residues alpha Phe33 and alpha Arg35, in contrast, were not important for high affinity binding of hTSH. In the signal transduction of hTSH, alpha Ala36 was necessary for efficient growth induction in FRTL-5 cells but not for cAMP production in either FRTL-5 cells or Chinese hamster ovary cells expressing the human TSH receptor (JP09). Similarly, residues alpha Arg42-Ser43-Lys44 were more important for hTSH-mediated induction of cell growth than cAMP production. Mutating alpha Arg35 to Ala reduced cAMP induction but not receptor binding of hTSH. In summary, using site-directed mutagenesis, we identified a domain, residues 33-44 of the common alpha-subunit, important in heterodimer expression, receptor binding, and activation of hTSH. The comparison of the relative roles of specific amino acids within this region in hTSH with hCG and hFSH highlights previously unrecognized differences in the structural requirements for heterodimer expression among the members of the glycoprotein hormone family. Moreover, our findings revealed a novel role for residues alpha 33-44 in triggering different postreceptor events, suggesting that cAMP production and growth promotion may, at least in part, be dissociable functions of hTSH.

Amino Acid Sequence↗
PubMed1996

Mutations of Arg68 of the human chorionic gonadotrophin beta subunit lead to reduced secretion.

The Arg68-Leu69 sequence is invariant in the beta subunits of chorionic gonadotrophin and luteinizing hormone from a variety of species. Using site-directed mutagenesis of the human chorionic gonadotrophin (hCG)-beta cDNA, several replacements of Arg68, an Ala replacement of Leu69, and a multiple replacement with Ala-Ala-Ala-Ala of the tetrapeptide sequence, Arg68-Leu69-Pro70-Gly71, were prepared and characterized. The wild-type and mutant cDNAs were subcloned into a pRSV expression vector and transiently transfected into CHO cells containing a stably integrated gene for bovine a. Concentrations of secreted wild-type and mutant hCG-beta subunit and holoprotein were determined using radioimmunoassays; potencies, i.e. the ratio of biologic to immunologic activity, of several of the mutant heterodimers were measured in vitro via gonadotrophin-mediated steroidogenesis in transformed murine Leydig cells (MA-10). The Leu69-->Ala mutant formed a mutant holoprotein that was essentially equipotent with wild-type hormone in the steroidogenesis assay. The Arg68 replacements with Lys, Ala, and Leu were poorly secreted by the cells, e.g. < 10% that of wild-type hCG; however, sufficient quantities of mutant holoproteins containing Lys68 and Ala68 were obtained for biological assays, and both exhibited greater apparent potencies than wild-type hormone. Likewise, a mutant holoprotein containing the Arg68-Leu69-Pro70-Gly71-->Ala-Ala-Ala-Ala multiple replacement was apparently more potent than wild-type hormone, but it too was secreted at lower levels than wild-type. These results establish that replacements of Arg68 in hCG-beta diminish secretion, but the small amount of holoprotein that is formed and secreted appears to be of somewhat greater potency than wild-type hormone.

Animals↗
PubMed1996

[Protective effect of angiotensin coverting enzyme inhibitor on renal function in normotensive non-insulin dependent diabetes mellitus patients with early diabetic nephropathy and microalbuminuria].

Fourty six cases of non-insulin dependent diabetes mellitus (NIDDM) with normotension and microalbuminuria (< 30 mg/24 h) were divided into groups A and B, and observed for about 2 years. Only group A was treated with perindopril 2 mg a day. We found that 2 years later glomerular filtration rate (GFR), effective renal plasma flow (ERPF) and filtration fraction (FF) in the two groups decreased markedly, but GFR and FF decreased more markedly in group B than in group A. Meanwhile, in group A urinary albumin excretion rate (UAER) and insulin sensitivity index (SI) decreased significantly and blood pressure was stable, but in group B UAER and blood pressure elevated significantly and SI was only slightly ameliorated (P > 0.05). The levels of serum triglycerides and cholesterol were not markedly different between the two groups during the 2 years. These results indicate that ACEI may play a role in protecting the renal function in diabetic nephropathy, reduce the risk factors of atherosclerosis and improve SI. It is beneficial to retard the development of diabetic nephropathy and to protect in renal function with small dose of ACEI.

Adult↗
PubMed1996

[Effect of bone xenograft transplantation on total T lymphocyte and its subset counts in mouse blood and spleen].

Fresh calf cancellous bone xenograft (FX), antigen free cancellous bone carrier (BC) and reconstituted bone xenograft (RBX) were inplanted in the thigh muscle pouches of Balb/c mice. Histological examinations were done at 7, 14 and 28 days postoperation, and indirect immunofluorescence was used to determine the positive counts of Thy1, L3T4, Lyt2, and Tac T lymphocyte of the blood and spleen at the same time, with the normal mouse lymphocytes as control. There was no significant difference between the BC implanted group and the normal (control) group, while the FX group had a significant increase rates of Thy1, L3T4 and Lyt2, especially of Tac lymphocytes. The RBX implanted group had no increase of Tac but increase of Thy1 and Lyt2. Th-Ts ratio decreased, and it was probably caused by bovine bone morphogenetic protein (bBMP) in the RBX. Histologically, intense immune rejection was noted in FX implanted group but not in the other two groups. Heterotopic ossification was noted in the RBX implanted group. FX increased intense immunologic rejection in the host, bat RBX did not, that might be due to the decrease of Th/Ts caused by bBMP. Tac lymphocyte count may indicate the immunologic rejection of bone xenograft transplantation.

Animals↗
PubMed1995

Modulation of cisplatin sensitivity and accumulation by interferon alpha-2A in human squamous carcinoma cell lines.

This study was undertaken to elucidate the mechanism(s) of potentiation of cisplatin (CDDP) cytotoxicity by interferon alpha-2a (IFN alpha-2a) in human squamous carcinoma cell lines SCC-25 and SCC-4. IFN alpha-2a treatment significantly increased the cytotoxicity of CDDP in both cell lines in a dose-dependent manner. In SCC-25 cells, the cytotoxicity of CDDP was increased by about 2- and 4-fold, respectively, by treating the cells with 400 and 800 IU/ml IFN alpha-2a. Sensitivity of SCC-4 cells to CDDP was increased by about 3- and 7-fold, respectively, by 400 and 800 IU/ml IFN alpha-2a treatment. Drug uptake experiments revealed approximately 1.4- to 5-fold higher platinum accumulation in IFN alpha-2a-treated cells as compared to respective controls. Cellular levels of glutathione (GSH) and GSH transferase, which have been suggested to be important determinants of tumor cell sensitivity to CDDP, were not altered by IFN alpha-2a treatment in either of the cell lines. Northern blot analysis showed a moderate increase (about 30-40%) in the level of MT-IIA mRNA by IFN alpha-2a treatment in these cells. Our results suggest that IFN alpha-2a-mediated sensitization of SCC-25 and SCC-4 cell lines to CDDP in vitro may be due to an increase in intracellular platinum accumulation.

Antineoplastic Agents↗
PubMed1995

Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy.

Nitric oxide (NO) is synthesized in skeletal muscle by neuronal-type NO synthase (nNOS), which is localized to sarcolemma of fast-twitch fibers. Synthesis of NO in active muscle opposes contractile force. We show that nNOS partitions with skeletal muscle membranes owing to association of nNOS with dystrophin, the protein mutated in Duchenne muscular dystrophy (DMD). The dystrophin complex interacts with an N-terminal domain of nNOS that contains a GLGF motif. mdx mice and humans with DMD evince a selective loss of nNOS protein and catalytic activity from muscle membranes, demonstrating a novel role for dystrophin in localizing a signaling enzyme to the myocyte sarcolemma. Aberrant regulation of nNOS may contribute to preferential degeneration of fast-twitch muscle fibers in DMD.

Amino Acid Oxidoreductases↗
PubMed1995

Effect of eradication of Helicobacter pylori infection on gastric epithelial cell proliferation.

Helicobacter pylori infection has been linked with gastric carcinoma. Epithelial cell proliferation is an indicator of cancer risk. The aim of this study was to assess gastric epithelial cell proliferation before and after eradication therapy and to assess the efficacy of treatment of H. pylori infection using lanzoprazole and clarithromycin. Twenty-three patients with H. pylori-associated gastritis were treated with lanzoprazole 30 mg daily for four weeks and clarithromycin 500 mg three times a day for two weeks. Antral mucosal biopsies were taken for gastric epithelial cell proliferation analysis using the in vitro bromodeoxyuridine (BrdU) immunohistochemical technique before and four weeks after eradication therapy. Labeling index percent (LI%) was calculated as the percent ratio of proliferating cells to the total number of cells in the gastric pit. Efficacy of treatment was assessed in 16 subjects. Eight were negative for H. pylori infection 28 days after therapy and in eight patients H. pylori infection was not eradicated. The eradication rate for the regime was 50%. Cell kinetics were assessed in 19 subjects who completed treatment. Patients with H. pylori infection had a significantly higher LI% compared to normal (N = 19, LI%: 5.01 +/- 0.3 vs 3.2 +/- 0.2, N = 29). Eradication of H. pylori infection significantly reduced epithelial cell proliferation (N = 9, LI%: 5.2 +/- 0.4 to 3.2 +/- 0.8, P < 0.001), whereas it was unaltered in those whose infection was not eradicated (N = 10, LI%: 4.8 +/- 0.4 to 5.5 +/- 0.5, P = 0.18).(ABSTRACT TRUNCATED AT 250 WORDS)

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1995

Genetic analysis of the S-mephenytoin polymorphism in a Chinese population.

The 4'-hydroxylation of S-mephenytoin exhibits a polymorphism in humans, with the poor metabolizer phenotype exhibiting a lower frequency in white (3% to 5%) than in Oriental populations (13% to 23%). Two mutations in CYP2C19 (CYP2C19m1 and CYP2C19m2) have recently been described that account for approximately 85% of white and 100% of Japanese poor metabolizers. This study examines whether these mutations account for the poor metabolizer phenotype in the Chinese population. The metabolism of S-mephenytoin exhibited a bimodal distribution in 244 unrelated Chinese subjects, although the distribution of the two phenotypes overlapped. In 75 selected Chinese subjects, CYP2C19 genotype analysis predicted the phenotype with 100% accuracy. The frequency of the poor metabolizer phenotype was approximately 11% (95% confidence interval 7% to 15%). The frequency of the CYP2C19m1 allele was 0.289, whereas that of CYP2C19m2 was 0.044. Homozygous extensive metabolizers had slightly lower ratios of S/R-mephenytoin compared with heterozygous extensive metabolizers, showing a gene-dosage effect. These data show the advantages of genotype analysis in investigations of the mephenytoin phenotype in Oriental subjects.

Adolescent↗
PubMed1994

Identification of conserved amino acid residues in the beta subunit of human choriogonadotropin important in holoprotein formation.

The beta subunits of mammalian glycoprotein hormones contain 20 invariant amino acid residues, including 12 Cys residues that form six disulfide bonds, as well as other highly conserved residues. Such stringent conservation suggests an important role for those residues in chain folding, holoprotein formation, or receptor binding/activation, but not receptor specificity. Using site-directed mutagenesis, we have prepared and characterized replacements of 14 conserved non-Cys residues in human choriogonadotropin beta for which there is little, if any, information available: Pro7, Thr32, Tyr37, Thr40, Val56, Tyr59, Phe64, Pro70, Gly71, Ser81, Tyr82, Val84, Ala85, and Ser87. The cDNAs were subcloned into a pRSV mammalian expression vector and transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. Holoprotein formation was assessed by radioimmunoassays, and in vitro competitive binding and steroidogenic assays were used to determine potencies. Our results demonstrated that Tyr37, Thr40, Tyr59, and Ala85 participate directly or indirectly in holoprotein formation, as may Ser81. Replacements of the other conserved amino acid residues resulted in no significant change in subunit assembly or receptor binding/activation as measured, although an Arg at position 81 or 82 reduced secretion. This study has identified several amino acid residues of the beta subunit that are important in alpha binding and has raised interesting questions on the constraints that maintain conservation of amino acid residues during evolution.

Amino Acid Sequence↗
PubMed1994

Standardization of disk diffusion test and its clinical significance for susceptibility testing of metronidazole against Helicobacter pylori.

Susceptibilities of 121 clinical Helicobacter pylori strains to metronidazole were determined by both a 5-micrograms metronidazole disk diffusion test and a plate dilution method in duplicate and after different periods of incubation. The distribution of MICs of metronidazole against H. pylori among the strains was found to be bimodal. The diameters of inhibitory zones obtained by the disk diffusion test and the MICs obtained by the plate dilution method correlated well, especially after 4 days of incubation (r = 0.77). An inhibitory zone diameter of 20 mm was found to correspond to a MIC of 8 micrograms/ml and is recommended as a suitable zone for differentiating susceptibility and resistance with a 5-micrograms metronidazole disk. Three interpretive categories of susceptibility results were defined; strains with inhibitory zone diameters of more than 26 mm were defined as susceptible (MIC, < 4 micrograms/ml), strains with zone diameters of 20 to 26 mm were deemed intermediate (MIC, 4 to 8 micrograms/ml), and those with zone diameters of less than 20 mm were deemed resistant (MIC, > 8 micrograms/ml). Furthermore, 76 H. pylori-positive patients with duodenal ulcers or nonulcer dyspepsia were treated with a 1 week of triple therapy (colloidal bismuth subcitrate, metronidazole, and tetracycline). H. pylori strains were isolated before treatment from antral biopsies from those patients, and the metronidazole susceptibilities of the strains were determined by the disk diffusion test. H. pylori status was evaluated again 4 weeks after completion of treatment. The eradication rates for susceptible, intermediate, and resistant strains were 95.9% (47 of 49), 62.5% (5 of 8), and 52.6% (10 of 19), respectively. It is included that the 5-micrograms disk diffusion test is easy to perform and gives final results similar to those of the plate dilution method. The three interpretive categories of susceptibility may be of benefit for clinical choice of chemotherapy in eradicating H. pylori.

Adolescent↗
PubMed1994

A region in the human glycoprotein hormone alpha-subunit important in holoprotein formation and receptor binding.

Using site-directed mutagenesis of the human glycoprotein hormone alpha-subunit, we have shown that single replacements of Ala36 and Pro38 with Glu and Asp, respectively, result in mutant subunits that do not bind significantly to hCG beta. In contrast, the replacement of Lys44 with Ala did not interfere with hCG beta binding, but the resulting holoprotein failed to exhibit high affinity binding to the LH/CG receptor. These results in conjunction with other data suggest that the region of human alpha between positions 33-45 contains several amino acid residues that participate in subunit binding and others that function in receptor binding.

Amino Acid Sequence↗
PubMed1994

Characterization of polyketide ketoreductase gene (MPKR) from midecamycin-producing strain (Streptomyces mycarofaciens 1748).

This paper presents the results about the expression of the polyketide ketoreductase gene from midecamycin-producing strain (S. mycarofaciens 1748), gene localization and nucleotide sequences analysis. A BamHI-BamHI 4.0 kg fragment isolated from a genomic library of midecamycin-producing strains containing the actIII homologous DNA was inserted into E. coli-Streptomyces shuttle vector pWHM3. A recombinant plasmid pCB4 was obtained and introduced into a 2-hydroxyaklavinone producer S. galilaeus ATCC 31671 that was a polyketide ketoreductase gene deficient mutant. The transformant produced aklavinone according to TLC and HPLC analyses. The BamHI-BamHI 4.0-kb fragment was inserted into pWHM3 in the reverted orientation with pCB4 and a recombinant plasmid pCBR4 was obtained. Introduction pCBR4 into S. galilaeus ATCC 31671 also resulted in the production of aklavinone. Thus we demonstrated that this gene encoded a polyketide ketoreductase which results in deoxygenation of 2-hydroxyaklavinone in C-2 position and that this gene has its own promoter. Restriction map analysis of pCB4 indicated that there were no sites for EcoRI and HindIII, but there were sites for BssHII, SalI, SphI and XhoI on the cloned gene fragment. The polyketide ketoreductase gene was located on a BssHII-BamHI 1.3-kb fragment from Southern hybridization result, using actIII gene as a probe, and that was confirmed by gene expression in S. galilaeus ATCC 31671. The nucleotide sequence analysis showed that the BssHII-BamHI 1.3-kb fragment contained an open reading frame (ORF). The protein coding region was assumed to start with an ATG start codon, end at an TAG stop codon. There was a 5nt (GGAGG) SD sequence at the upstream of start codon. A presumptive promoter consisted of 7 nt AACCGGA at the -10 region and 5 nt TTCGA at the -35 region. The deduced amino acid sequences of MPKR gene consisted of 261 aa residues. Its amino acid were compared with actIII gene coding protein sequences. The similarity was 77.4% and the identity was 66.7%.

Amino Acid Sequence↗