Modulation of nuclear REST by alternative splicing: a potential therapeutic target for Huntington's disease.

Huntington's disease (HD) is caused by a genetically mutated huntingtin (mHtt) protein with expanded polyQ stretch, which impairs cytosolic sequestration of the repressor element-1 silencing transcription factor (REST), resulting in excessive nuclear REST and subsequent repression of neuronal genes. We recently demonstrated that REST undergoes extensive, context-dependent alternative splicing, of which exon-3 skipping (∆E3 )-a common event in human and nonhuman primates-causes loss of a motif critical for REST nuclear targeting. This study aimed to determine whether ∆E3 can be targeted to reduce nuclear REST and rescue neuronal gene expression in mouse striatal-derived, mHtt-expressing STHdhQ111/Q111 cells-a well-established cellular model of HD. We designed two morpholino antisense oligos (ASOs) targeting the splice sites of Rest E3 and examined their effects on ∆E3 , nuclear Rest accumulation and Rest-controlled gene expression in STHdhQ111/Q111 cells. We found that (1) the ASOs treatment significantly induced ∆E3 , reduced nuclear Rest, and rescued transcription and/or mis-splicing of specific neuronal genes (e.g. Syn1 and Stmn2) in STHdhQ111/Q111 cells; and (2) the ASOs-induced transcriptional regulation was dependent on ∆E3 induction and mimicked by siRNA-mediated knock-down of Rest expression. Our findings demonstrate modulation of nuclear REST by ∆E3 and its potential as a new therapeutic target for HD and provide new insights into environmental regulation of genome function and pathogenesis of HD. As ∆E3 is modulated by cellular signalling and linked to various types of cancer, we anticipate that ∆E3 contributes to environmentally tuned REST function and may have a broad range of clinical implications.

MdTPR16, an apple tetratricopeptide repeat (TPR)-like superfamily gene, positively regulates drought stress in apple.

The Tetratricopeptide repeat (TPR)-like superfamily with TPR conserved domains is widely involved in the growth and abiotic stress in many plants. In this report, the gene MdTPR16 belongs to the TPR family in apple (Malus domestica). Promoter analysis reveal that MdTPR16 incorporated various stress response elements, including the drought stress response elements. And different abiotic stress treatments, drought especially, significantly induce the response of MdTPR16. Overexpression of MdTPR16 result in better drought tolerance in apple and Arabidopsis by up-regulating the expression levels of drought stress-related genes, achieving a higher chlorophyll content level, more material accumulation, and overall better growth compared to WT in the drought. Under drought stress, the overexpressed MdTPR16 also mitigate the oxidative damage in cells by reducing the electrolyte leakage, malondialdehyde content, and the H2O2 and O2- accumulation in apples and Arabidopsis. In conclusion, MdTPR16 act as a beneficial regulator of drought stress response by regulating the expression of related genes and the cumulation of reactive oxygen species (ROS).

Advances in tryptophan hydroxylase-2 gene expression regulation: new insights into serotonin-stress interaction and clinical implications.

Serotonin (5-HT) modulates the stress response by interacting with the hormonal hypothalamic-pituitary-adrenal (HPA) axis and neuronal sympathetic nervous system (SNS). Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in 5-HT biosynthesis, and the recent identification of a second, neuron-specific TPH isoform (TPH2) opened up a new area of research. While TPH2 genetic variance has been linked to numerous behavioral traits and disorders, findings on TPH2 gene expression have not only reinforced, but also provided new insights into, the long-recognized but not yet fully understood 5-HT-stress interaction. In this review, we summarize advances in TPH2 expression regulation and its relevance to the stress response and clinical implications. Particularly, based on findings on rhesus monkey TPH2 genetics and other relevant literature, we propose that: (i) upon activation of adrenal cortisol secretion, the cortisol surge induces TPH2 expression and de novo 5-HT synthesis; (ii) the induced 5-HT in turn inhibits cortisol secretion by modulating the adrenal sensitivity to ACTH via the suprachiasmatic nuclei (SCN)-SNS-adrenal system, such that it contributes to the feedback inhibition of cortisol production; (iii) basal TPH2 expression or 5-HT synthesis, as well as early-life experience, influence basal cortisol primarily via the hormonal HPA axis; and (iv) 5'- and 3'-regulatory polymorphisms of TPH2 may differentially influence the stress response, presumably due to their differential roles in gene expression regulation. Our increasing knowledge of TPH2 expression regulation not only helps us better understand the 5-HT-stress interaction and the pathophysiology of neuropsychiatric disorders, but also provides new strategies for the treatment of stress-associated diseases.

Normal thermoregulatory responses to 3-iodothyronamine, trace amines and amphetamine-like psychostimulants in trace amine associated receptor 1 knockout mice.

3-Iodothyronamine (T1AM) is a metabolite of thyroid hormone. It is an agonist at trace amine-associated receptor 1 (TAAR1), a recently identified receptor involved in monoaminergic regulation and a potential novel therapeutic target. Here, T1AM was studied using rhesus monkey TAAR1 and/or human dopamine transporter (DAT) co-transfected cells, and wild-type (WT) and TAAR1 knock-out (KO) mice. The IC(50) of T1AM competition for binding of the DAT-specific radio-ligand [(3)H]CFT was highly similar in DAT cells, WT striatal synaptosomes and KO striatal synaptosomes (0.72-0.81 microM). T1AM inhibition of 10 nM [(3)H]dopamine uptake (IC(50): WT, 1.4 + or - 0.5 microM; KO, 1.2 + or - 0.4 microM) or 50 nM [(3)H]serotonin uptake (IC(50): WT, 4.5 + or - 0.6 microM; KO, 4.7 + or - 1.1 microM) in WT and KO synaptosomes was also highly similar. Unlike other TAAR1 agonists that are DAT substrates, TAAR1 signaling in response to T1AM was not enhanced in the presence of DAT as determined by CRE-luciferase assay. In vivo, T1AM induced robust hypothermia in WT and KO mice equivalently and dose dependently (maximum change degrees Celsius: 50 mg/kg at 60 min: WT -6.0 + or - 0.4, KO -5.6 + or - 1.0; and 25 mg/kg at 30 min: WT -2.7 + or - 0.4, KO -3.0 + or - 0.2). Other TAAR1 agonists including beta-phenylethylamine (beta-PEA), MDMA (3,4-methylenedioxymethamphetamine) and methamphetamine also induced significant, time-dependent thermoregulatory responses that were alike in WT and KO mice. Therefore, TAAR1 co-expression does not alter T1AM binding to DAT in vitro nor T1AM inhibition of [(3)H]monoamine uptake ex vivo, and TAAR1 agonist-induced thermoregulatory responses are TAAR1-independent. Accordingly, TAAR1-directed compounds will likely not affect thermoregulation nor are they likely to be cryogens.

The effect of rearing experience and TPH2 genotype on HPA axis function and aggression in rhesus monkeys: a retrospective analysis.

Gene-environment (GxE) interactions contribute to the development of many neuropsychiatric disorders. Tryptophan hydroxylase-2 (TPH2) synthesizes neuronal serotonin and is closely related to the hypothalamic-pituitary-adrenal (HPA) axis, while early life experience is a critical environmental factor programming the HPA axis response to stress. This retrospective study investigated GxE interaction at the TPH2 locus in rhesus monkeys. Twenty-eight adult, male rhesus monkeys of Indian origin, either mother-reared or peer-reared as infants, were involved in this study. These monkeys have been previously genotyped for the functional A2051C polymorphism in rhTPH2, and had been physiologically and behaviorally characterized. rhTPH2 A2051C exerted a significant main effect (CC>AA&AC) on the cerebrospinal fluid (CSF) level of 5-hydroxyindole-3-acetic acid (5-HIAA; F((1,14))=6.42, p=0.024), plasma cortisol level in the morning (F((1,18))=14.63, p=0.002) and cortisol response to ACTH challenge (F((1,17))=6.87, p=0.018), while the rearing experience showed a significant main effect (PR>MR) on CSF CRH (F((1,20))=11.66, p=0.003) and cage shaking behavior (F((1,27))=4.45, p=0.045). The effects of rhTPH2 A2051C on the afternoon cortisol level, plasma ACTH level, dexamethasone suppression of urinary cortisol excretion, and aggression were dependent upon the rearing experience. These results were not confounded by the functional C77G polymorphism in the mu-opioid receptor (MOR). The present study supports the hypothesis that rearing experience and rhTPH2 A2051C interact to influence central 5-HT metabolism, HPA axis function, and aggressive behaviors. Our findings strengthen the involvement of G x E interactions at the loci of serotonergic genes and the utility of the nonhuman primate to model G x E interactions in the development of human neuropsychiatric diseases.

[Herbs for calming liver and suppressing liver-yang in treatment of migraine with hyperactive liver-yang syndrome and its effects on lymphocyte protein expression: a randomized controlled trial].

OBJECTIVE: To observe the efficacy of herbs for calming liver and suppressing liver-yang in treatment of migraine patients with hyperactivity of liver-yang syndrome and to investigate its effects on the lymphocyte protein expression. This approach may lay a foundation for the further investigation of pathogenic mechanisms in migraine with hyperactive liver-yang syndrome and the curative mechanisms of calming liver and suppressing liver-yang treatment. METHODS: A total of 32 migraine patients with hyperactivity of liver-yang syndrome were randomly divided into treatment group (16 cases) and control group (16 cases). The patients in the treatment group were treated with herbs for calming liver and suppressing liver-yang in accordance with traditional Chinese medicine (TCM) theory and the patients in the control group were treated with Flunarizine Capsules for two courses of treatment. The therapeutic effects, the score of TCM symptom and the changes of headache attack were observed in both groups before and after the treatment. The side effects were also observed in both groups. The level of differential protein expression was analyzed by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: The herbs for calming liver and suppressing liver-yang had better effects on headache improvement than the Flunarizine Capsules (P<0.01). The cure rates in the treatment and control groups were 87.5% and 75.0% respectively. Vertigo, restlessness and tantrum, and prosopo-heat in the treatment group were also improved as compared with those in the control group (P<0.05). After treatment, the score of TCM symptom in the two groups were all decreased (P<0.01), and there was a significant difference between the treatment group and the control group (P<0.01). The herbs for calming liver and suppressing liver-yang had no side effects. The average protein spots in the blood lymphocyte of normal people, migraine patients with hyperactivity of liver-yang syndrome in the treatment group before and after the treatment were (534+/-42), (552+/-54) and (529+/-55) spots respectively. Six down-regulated protein expressions and 14 up-regulated protein expressions were obtained in the treatment group. Four strengthened protein expressions in the six down-regulated proteins and 11 low protein expressions in the 14 up-regulated proteins were also obtained after treatment. Ten of the total 12 differential protein spots were successfully identified by MALDI-TOF-MS. The functions of these proteins were involved in metabolism, energy generation, transportation, antioxidation, signal transduction and immune, etc. According to information provided by NCBI and MSDB database, there were some proteins closely related to migraine with hyperactivity of liver-yang syndrome, such as peroxiredoxin 2, heat shock protein 27 and annexin A1. CONCLUSION: Herbs for calming liver and suppressing liver-yang is effective in treating migraine, and can improve TCM symptoms. The effects on migraine patients with hyperactivity of liver-yang syndrome may be related to regulating the blood lymphocyte protein expression.

Functional characterization of the human TPH2 5' regulatory region: untranslated region and polymorphisms modulate gene expression in vitro.

Tryptophan hydroxylase-2 (TPH2) is a recently identified TPH isoform responsible for neuronal serotonin (5-HT) synthesis, and TPH2 polymorphisms are associated with a range of behavioral traits and psychiatric disorders. This study characterized cis-acting elements and three common polymorphisms (-703G/T, -473T/A, and 90A/G) in the 5' regulatory region of human TPH2 by using luciferase reporter assay, quantitative real-time PCR, and electrophoretic mobility shift assay (EMSA). The core promoter of human TPH2 was localized to the region between -107 and +7, and the segment of +8 to +53 within the 5'-UTR was found to exert a potent inhibitory effect on gene expression at both transcriptional and post-transcriptional levels. In both RN46A and HEK-293 cell lines, the TTA (-703T/-473T/90A) haplotype of the three polymorphisms showed the lowest gene expression compared with other haplotypes, and the -703G/T and -473T/A polymorphisms tended to exert a synergic effect on gene expression dependent upon the sequence of the 5'-UTR. In RN46A, the 90A/G polymorphism significantly increased luciferase activity and mRNA level irrespective of the other two polymorphisms, while in HEK-293 cells the effect of 90A/G was dependent on the alleles at loci -703 and -473. EMSA showed that all the three polymorphisms potentially alter DNA-protein interactions, while the 90A/G polymorphism predictably alters the 5'-UTR secondary structure of mRNA and influences RNA-protein interactions. In conclusion, our present study demonstrates that both the 5'-UTR and common polymorphisms (especially the 90A/G) in the 5' regulatory region of human TPH2 have a significant impact on gene expression.

[Effect of calming the liver and suppressing the hyperactive YANG drugs on lymphocyte protein in hypertension patients with hyperactivity of liver-YANG].

OBJECTIVE: To explore the effect of calming the liver and suppressing the hyperactive YANG drugs on the lymphocyte protein and clinical efficacy in the hypertension patients with hyperactivity of liver-YANG, and to identify the therapy. METHODS: Twenty-six hypertension patients with hyperactivity of liver-YANG were treated by calming the liver and suppressing the hyperactive YANG drugs for 2 courses. Symptoms of Chinese medicine and blood pressure were observed, and the separated lymphocyte total protein of normal and hypertensions before and after the treatment were examined by the solid-state pH gradient 2-dimensional gel electrophoresis. The differences of the protein expression were analyzed by ImageMaster 2DE analysis software with two-way patterns. RESULTS: The total efficiency rate of calming the liver and suppressing the hyperactive YANG drugs was 88.5%, and the drugs could significantly relieve the symptoms, such as headache, dizziness, dry mouth, irritability, etc. Calming the liver and suppressing the hyperactive YANG drugs could also remarkably reduce the blood pressure,with significant different between pre-treatment and post-treatment (P<0.05). The average spots of lymphocyte gel proteins in the normal and the hypertension patients with syndrome of hyperactivity of liver-YANG before and after the treatment were 527+/-41,559+/-62, and 543+/-59, respectively. Compared with normal people, the expression of 15 proteins was down-regulated, and 10 up-regulated in the hypertension patients with syndrome of hyperactivity of liver-YANG. Compared with the pre-treatment, the expression of 12 proteins was increased in the 15 down-regulated proteins, and 6 decreased in the 10 up-regulated proteins after the treatment in the hypertension patients with syndrome of the hyperactivity of liver-YANG. CONCLUSION: Calming the liver and suppressing the hyperactive yang drugs may mildly depress the blood pressure and improve the symptoms of Chinese medicine. The effect of drugs in treating hypertension may probably be associated with regulating the expression of some proteins in lymphocytes.

Perturbations of Neuron-Restrictive Silencing Factor Modulate Corticotropin-Releasing Hormone Gene Expression in the Human Cell Line BeWo.

Stress exacerbates disease, and understanding its molecular mechanisms is crucial to the development of novel therapeutic interventions to combat stress-related disorders. The driver of the stress response in the hypothalamic-pituitary-adrenal axis (HPA) is corticotropin-releasing hormone (CRH), a neuropeptide synthesized in the paraventricular nucleus of the hypothalamus. Evidence supports that CRH expression is epigenetically modified at the molecular level by environmental stimuli, causing changes in the stress response. This effect is mediated by a concert of factors that translate environmental change into alterations in gene expression. An important regulator and epigenetic modulator of CRH expression is neuron-restrictive silencing factor (NRSF). Previously, our lab identified numerous splice variants of NRSF that are specific to humans and predictive of differential regulatory effects of NRSF variants on targeted gene expression. The human cell line BeWo has endogenous CRH and NRSF expression providing an in vitro model system. Here, we show that manipulation of NRSF expression through siRNA technology, overexpression by plasmid vectors, and direct cAMP induction that CRH expression is linked to changes in NRSF expression. Accordingly, this epigenetic regulatory pathway in humans might be a critical mechanism involved in the regulation of the stress response.

[Immune response to HSP70-HBcAg(18-27) complex in HBV transgenic mice].

OBJECTIVES: To study the cellular immune response to HSP70-HBcAg(18-27) complex in HBV transgenic mice. METHOD: HSP70-HBcAg(18-27) complex was reconstituted in vitro, then it was injected into HBV transgenic mice to observe the cellular immune response. At the same time, we investigated whether HSP70-HBcAg(18-27) complex could generate antigen specific cytotoxic T lymphocyte responses in spleen cells. RESULTS: Our results demonstrated that HSP70-HBcAg(18-27) complex increased levels of CD4+ and CD8+ T cells in the spleens and peripheral blood of HBV transgenic mice, and the complex also activated dendritic and natural killer cells. CONCLUSION: HSP70-HBcAg(18-27) complex has an immunological antigenicity in raising the immunoresponse to chronic HBV infection in HBV transgenic mice. HSP70-HBcAg(18-27) complex might be considered as a candidate for further studies on its role as a therapeutic vaccine against chronic HBV infection in humans.

[Effect of tumor antigen specific CTL induced by dendritic cells on a model of human hepatocellular carcinoma in nude mice (LCI-D20)].

OBJECTIVES: To investigate the cure effect of tumor antigen specific CTL on a model of human hepatocellular carcinoma in nude mice LCI-D20. METHODS: Dendritic cells (DCs) were induced from peripheral blood mononuclear cells of healthy people in vitro by using recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) and interleukin-4 (rhIL-4) and were pulsed with tumor antigen from hepatocellular carcinoma cell line MHCC97H. Then tumor antigen specific cytotoxic T lymphocytes (CTLs) were induced. By intraperitoneal injection of tumour antigen specific CTLs into the LCI-D20, the preventive and therapeutic effects of these CTLs to HCC in the LCI-D20 model were assessed. Cytokine-induced killer (CIK) cells and phosphate buffer solution were used as controls at the same time. RESULTS: The weights of tumors in the tumor antigen specific CTL group, in the CIK cell group and in the blank group were (1.11+/-0.63), (1.12+/-0.36) and (2.68+/-0.53) grams respectively (t = 5.18, t = 6.06, P < 0.01). The amount of blood alpha fetal protein in the tumor antigen specific CTL and CIK groups were (52.1+/-9.7) microg/L and (48.6+/-5.2) microg/L, and was (82.2+/-7.2) microg/L in the blank group (t = 17.26, t = 22.07, P < 0.01 respectively). The metastasis rates in livers were 16.7%, 16.7% and 58.3% in the tumor antigen specific CTL, CIK cell and blank control groups respectively (chi2= 4.44, P < 0.01). The survival time of the mice in the tumor antigen specific CTL group was (79.0+/-5.02) days, (73.3+/-7.0) days in the CIK group, and (52.3+/-5.2) days in the blank group (t = 14.56, t = 17.54, P < 0.01). CONCLUSION: Tumor antigen specific CTLs may prevent metastasis in the LCI-D20 model and prolong the survival time.

CYP3A5*3 and CYP3A4*18 single nucleotide polymorphisms in a Chinese population.

BACKGROUND: Human cytochrome P450 3A evolved to catalyze the metabolism of numerous common therapy drugs and endogenous molecules. Members of the CYP3A are the majority expressed in human liver and intestine, and there are marked interindividual differences in their protein expression and activity. The activity of CYP3A enzyme in Chinese is highly variable, exceeding 14-fold, and contributes greatly to variation in oral bioavailability and systemic clearance of CYP3A substrates. The genetic factors play an important role in the interindividual variability in CYP3A activity. Detection of CYP3A5 and CYP3A4 variant alleles and knowledge about their allelic frequency in specific ethnic groups are important to lead to individualized drug dosing and improved therapeutics. METHODS: We determined the allelic frequency of the CYP3A5*3 and CYP3A4*18 in a group of 302 Chinese subjects by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays. RESULTS: In the group of 302 unrelated individuals, the frequency of the CYP3A5*3 and CYP3A4*18 variant allele in Chinese population were 0.778 (95% CI: 0.754, 0.802) and 0.01 (95% CI: 0, 0.02), respectively. CONCLUSIONS: We developed a simple assay for the detection of the CYP3A4*18 allele and showed that in a Chinese population, CYP3A4*18 and CYP3A5*3 allelic frequencies are similar to that reported previously in Chinese resident in Taiwan. The frequency of the CYP3A5*3 allele in Chinese population is similar to the Japanese but lower than Caucasians. Meanwhile, our findings suggest that an approximate 62% of the Chinese population carrying CYP3A5*3/*3 genotype may appear not to express CYP3A5 protein.

MDR1 genotype do not influence the absorption of a single oral dose of 100 mg talinolol in healthy Chinese males.

OBJECTIVE: We investigated the linkage between SNPs in exon 12 (C1236T), exon 21 (G2677T/A) and exon 26 (C3435T) of MDR1, and explored the effect of linked polymorphism on the absorption of talinolol after a single oral dose of 100 mg. METHODS: The genotype of 192 healthy Chinese volunteers was determined using PCR-RFLP with respect to the MDR1 alleles of interest, C1236T, G2677T/A and C3435T. Linkage disequilibrium was analyzed using PHASE software. Consecutive eligible subjects received a single oral dose of 100 mg talinolol. Venous blood samples were taken at intervals up to 60 h post dose for HPLC analysis of plasma concentration of talinolol to obtain a pharmacokinetic profile. RESULTS: Linkage disequilibrium existed between exon 21 (G2677T/A) and exon 26 (C3435T), exon 12 (C1236T) and exon 21 (G2677T/A), but not between exon 12 (C1236T) and exon 21 (G2677T/A). AUC (0,3 h), AUC (0, infinity), Cmax and Cmax/AUC (0, infinity), used as indices of talinolol absorption, were not significantly different between the genotype groups of 2677GG/3435TT, 2677TT/3435TT, 2677GT/3435CT and 2677AT/3435CT. For these 4 groups, AUC(0,3 h) were 436.8 +/- 50.1, 510.1 +/- 86.3, 466.1 +/- 77.8 and 437.2 +/- 73.4 (microg x h/l) and the Cmax/AUC (0, infinity) were 0.097 +/- 0.018, 0.093 +/- 0.022, 0.105 +/- 0.014 and 0.102 +/- 0.027 (h(-1)), respectively. (P > 0.05). CONCLUSIONS: The linked MDR1 polymorphisms in exon 21 G2677T/A and exon 26 C3435T apparently did not contribute to the absorptive pharmacokinetics of a single oral dose of 100 mg talinolol.

Gene-gene interactions among CX3CL1, LEPR and IL-6 related to coronary artery disease in Chinese Han population.

OBJECTIVE: The purpose of this study was to investigate the impact of the interactions among CX3CL1 (rs170364 and rs614230), LEPR (rs6700896), and IL-6 (rs2066992) polymorphisms on the risk of coronary artery disease (CAD) in Chinese Han population. METHODS: 120 CAD patients and 109 healthy controls were enrolled in the study. Polymerase chain reaction (PCR) and direct sequencing methods were used to analyze the genotypes of CX3CL1, LEPR, and IL-6 polymorphisms. Multifactor dimensionality reduction (MDR) software was utilized to analyze gene-gene interactions. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were used for evaluating the association between gene polymorphisms or gene-gene interactions and CAD risk. RESULTS: In the study, TT genotype of rs170364 in CX3CL1 might decrease the CAD risk (OR=0.39, 95% CI=0.16-0.98). No significant correlation was found between T allele of rs170364 and CAD risk (P>0.05). CC genotype and C allele in rs614230 (CX3CL1) were significantly related with decreased risk of CAD (OR=0.38, 95% CI=0.17-0.86; OR=0.66, 95% CI=0.45-0.97). For IL-6 rs2066992 polymorphism. GG genotype could increase the risk of CAD (OR=2.32, 95% CI=1.04-5.17). Whereas, no significant correlation was observed between LEPR rs6700896 and CAD susceptibility. MDR analysis showed that CX3CL1, LEPR and IL-6 genes might jointly promote the occurrence of CAD. CONCLUSIONS: The interactions of CX3CL1, LEPR and IL-6 genes might increase the risk of CAD.

Histamine N-methyltransferase gene polymorphisms in Chinese and their relationship with enzyme activity in erythrocytes.

The aim of this study was to identify polymorphisms in the histamine N-methyltransferase (HNMT) gene in Chinese and to assess their relationship with HNMT activity. One hundred and ninety-two unrelated subjects were recruited. HNMT polymorphisms were screened by direct sequencing with purified polymerase chain reaction products comprising all six exons, plus splice junctions, as well as approximately 2 kb of the 5'-flanking region (5'-FR). Erythrocyte HNMT activity was previously measured by radiochemical microassay. A total of 11 single nucleotide polymorphisms (SNPs) were identified, among which six SNPs had variant allele frequencies greater than 5%. Of the six common SNPs, three (-1637T>C, -463T>C and -411C>T) were located in 5'-FR, one (314C>T) in coding exons, and two (939A>G and 1097A>T) in the 3'-untranslated region (3'-UTR). Most of these common SNPs were in linkage disequilibrium. Genotype-phenotype correlation analyses were performed for those common SNPs in 5'-FR and 3'-UTR. In males, no significant association was found between HNMT activity and these non-coding SNPs. However, in females, the -1637T>C or -463T>C tended to be associated with decreased HNMT activity, whereas the 939A>G or 1097A>T appeared to be correlated with increased enzymatic activity. HNMT polymorphisms differ considerably between Chinese and American. The common SNPs in 5'-FR (-1637T>C and -463T>C) and 3'-UTR (939A>G and 1097A>T) might conditionally regulate the activity of HNMT, or might be genetically linked to unknown mutation(s) underlying the HNMT phenotypic variance.

The influence of St John's Wort on CYP2C19 activity with respect to genotype.

Induction of cytochrome p450 isozymes is the major cause for clinical drug interactions of St. John's wort. The relationships of St. John's wort to cytochrome p450 isoforms have been fully investigated, but its effect on CYP2C19 is lacking. Thus, the aim of the present study was to observe the effect of St. John's wort on CYP2C19 activity using CYP1A2 as a control. Twelve healthy adult men-6 extensive metabolizers of CYP2C19 (2C19(*)1/2C19(*)1) and 6 poor metabolizers (4 2C19(*)2/2C19(*)2 and 2 2C19(*)2/2C19(*)3)-were enrolled in a two-phase, randomized, crossover manner. All subjects took a 300-mg St. John's wort tablet or placebo three times daily for 14 days, and then the activities of CYP2C19 and CYP1A2 were measured using mephenytoin and caffeine. It was found that St. John's wort treatment significantly increased CYP2C19 activity in CYP2C19 wild-genotype subjects, with urinary 4'-hydroxymephenytoin excretion raised by 151.5% +/- 91.9% (p = 0.0156), whereas no significant alteration was observed for CYP2C19 poor metabolizers. Repeated St. John's wort administration did not affect the CYP1A2 phenotypic ratio for both CYP2C19 genotype subjects. In conclusion, St. John's wort is an inducer to the human CYP2C19, and clinicians should pay great attention when St. John's wort is added to or withdrawn from an existing drug regimen containing substrates for such enzymes.

Arg257Cys polymorphism of CYP2A13 in a Chinese population.

BACKGROUND: Human cytochrome P450 2A13 (CYP2A13) is involved in the activation of numerous toxicants and carcinogens, especially in the metabolic activation of 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a major tobacco-specific carcinogen. A functionally significant coding single nucleotide polymorphism (C3375T) in exon 5 of CYP2A13, which results in an amino acid substitution of Arg 257 to Cys, has been recently reported to exist in White, Black, Hispanic, and Asian individuals, with the variant 3375T allele frequencies being 1.9%, 14.4%, 5.8% and 7.7%, respectively. Since genetic background differs between ethnic groups, our present study aims to characterize the CYP2A13 Arg257Cys polymorphism in Chinese. METHODS: 258 healthy Chinese Han volunteers were involved in this study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was employed to genotype for the Arg257Cys polymorphism. RESULTS: Of all the 258 subjects, 27 (10.5%) heterozygotes and 1 (0.4%) homozygote for the 257Cys allele were detected. The frequency of the variant 257Cys allele in this Chinese population was 5.6% (95%CI: 4.2-7.0%). CONCLUSION: The CYP2A13 Arg257Cys variant represents a common polymorphism in Chinese, with the 257Cys allele frequency being similar to the Hispanic and Asian groups, but significantly lower than the Black.

Evidence for the involvement of human liver microsomes CYP1A2 in the mono-hydroxylation of daidzein.

BACKGROUND: In vitro studies with rats and human liver microsomes (HLM) demonstrated that daidzein is readily metabolized to mono-hydroxylated compounds. In this study, daidzein mono-hydroxylated metabolites was investigated using human liver microsomes to identify the cytochrome P450 (CYP) isoform(s) involved in this metabolic pathway. METHODS: Kinetic analysis for the formation rates of mono-hydroxylated metabolites of daidzein, including 7,8,4'-trihydroxyisoflavone (7,8,4'-THI), 7,3,4'-trihydroxyisoflavone (7,3,4'-THI) and 6,7,4'-trihydroxyisoflavone (6,7,4'-THI), were performed using human liver microsomes (HLM) and recombinant enzymes at substrate concentrations ranging from 0.5 to 400 micromol/l. Nine selective inhibitors or substrate probes specific for different CYP isoforms were applied for screening the isoform(s) responsible for mono-hydroxylated metabolism of daidzein. RESULTS: Michaelis-Menten kinetic parameters were best fitted to a one-component enzyme kinetic model. The mean K(m) (micromol/l) and V(max) (micromol/g min) values (+/-S.D.) were 26.86 (10.45) and 4.76 (2.07), 53.83 (22.25) and 2.29 (1.04), 51.48 (29.32) and 2.21(0.82), for the formation rates of 7,8,4'-THI, 7,3',4'-THI and 6,7,4'-THI, respectively. Furafylline, the CYP1A2-specific inhibitor, estrogen and monoclonal antibody raised against human CYP1A2 (MAB-1A2) substantially inhibited the formation rates of mono-hydroxylated metabolites. The IC(50) of Fur for the formation of 7,3',4'-THI, 6,7,4'-THI and 7,8,4'-THI was 1.0, 0.9 and 0.8 micromol/l, respectively. The IC(50) of estrogen for the formation of 7,3',4'-THI, 6,7,4'-THI and 7,8,4'-THI was 51, 60 and 64 micromol/l, respectively. The IC(50) of MAB-1A2 for the formation of the mono-hydroxylated products was 1 micromol/l, but neither other selective inhibitor nor substrate probes, including coumarin (CYP2D6), sulphaphenzole (CYP2C9/10), omeprazole (CYP2C19), quinidine (CYP2D6), diethyldithiocarbamate (CYP2E1), troleandomycin (CYP3A4) and keteconazole (CYP3A4), did so with human liver microsomes. CONCLUSION: Daidzein mono-hydroxylated products are principally metabolized by CYP1A2 in human.

Genotype-phenotype correlation for histamine N-methyltransferase in a Chinese Han population.

BACKGROUND: Two potential single-nucleotide polymorphisms (SNP) (C314T and A595G) exist in the gene for human histamine N-methyltransferase (HNMT). METHODS: A radiochemical microassay was used to measure the erythrocyte HNMT activities, whereas the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was employed to perform the genetic analysis in 247 unrelated Chinese Han subjects. RESULTS: All subjects had detectable HNMT activity. The activity of HNMT was gender related (males>females, p<0.0001), with a 5.5-fold individual variation. The distribution of HNMT activity was compatible with a normal distribution. There were 28 heterozygotes for the variant T314 allele among the 247 subjects, whereas no A595G transition was observed. All heterozygotes for the T314 allele displayed an intermediate or low HNMT activity, with an average HNMT activity being 34.0% lower than those with wild-type genotype (623.1+/-136.0 vs. 944.8+/-249.3 U/ml red blood cells [RBC], p<0.0001). CONCLUSION: The C314T polymorphism was functionally important and contributes in part to phenotypic variance of HNMT activity in Chinese Han population. Additional unknown genetic or epigenetic factors should also play important roles in the regulation of HNMT activity.

Analysis of the C314T and A595G mutations in histamine N-methyltransferase gene in a Chinese population.

BACKGROUND: Histamine N-methyltransferase (HNMT) plays an important role in the metabolism of histamine, a biogenic amine that has many physiologic and pathological roles in human tissues. A missense mutation C314T (Thr105Ile) in the HNMT gene has been identified to represent a common functional polymorphism in Caucasians, whereas an A595G (Ile199Val) variant has been reported in one HNMT cDNA from a Japanese subject. METHODS: By using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, the point mutations C314T and A595G within HNMT were both detected in 352 unrelated Chinese Han subjects. RESULTS: None of the 352 subjects contained the A595G mutation, whereas 40 (11.6%) heterozygotes and 1 (0.3%) homozygote for the variant T314 allele were detected. The frequency of the variant T314 allele in this Chinese population was 0.060 (95% CI: 0.042-0.078), not different from Japanese but significantly lower than American Caucasians. CONCLUSIONS: The C314T mutation represents a common functional genetic polymorphism in the Chinese Han population with a variant T314 allele frequency similar to Japanese but lower than American Caucasians, whereas the A595G mutation does not appear to exist in this population.