Common genetic variation in the 3'-untranslated region of gonadotropin-releasing hormone receptor regulates gene expression in cella and is associated with thyroid function, insulin secretion as well as insulin sensitivity in polycystic ovary syndrome patients.

Gonadotropin-releasing hormone receptor (GNRHR) is a member of the G protein-coupled Ca(2+)-dependent family of receptors. It interacts with GnRH, whose signaling plays an important role in thyroid-stimulating hormone (TSH) secretion and insulin activity. There has been no study on the genetic effect of GNRHR on TSH secretion and insulin action in polycystic ovary syndrome (PCOS). We decided to investigate whether naturally occurring genetic variation at the human GNRHR locus is associated with thyroid function, insulin secretion and insulin sensitivity in PCOS. We undertook a systematic search for polymorphisms in GNRHR by resequencing the gene and then genotyped common single-nucleotide polymorphisms across the locus in 261 PCOS patients well-phenotyped for several metabolic traits to determine associations. A test for association of common genetic variants with susceptibility to PCOS was carried out in a large cohort of 948 subjects. Finally, we experimentally validated the marker-on-trait associations using GNRHR 3'-UTR region/reporter analysis in 293T cells. The 3'-UTR variant rs1038426 was associated with serum thyroid concentration (P = 0.007), change of insulin levels during oral glucose tolerance test (P = 0.004) and insulin sensitivity index (P = 0.014). In a functional study, 3'-UTR variant T allele increased reporter expression by a transfected luciferase reporter/GNRHR 3'-UTR expression plasmid. In conclusion, our results strongly suggest that common genetic variant in GNRHR contributes to the phenotypic expression of PCOS. The findings suggest novel pathophysiological links between the GNRHR locus and thyroid function and insulin secretion in PCOS.

A common polymorphism in the human aromatase gene alters the risk for polycystic ovary syndrome and modifies aromatase activity in vitro.

Aromatase is a key enzyme involved in estradiol and estrone biosynthesis. Given that polymorphisms of the CYP19A1 gene encoding aromatase have been correlated with plasma testosterone levels, CYP19A1 may therefore act as a genetic modifier of the hyperandrogenic phenotype of polycystic ovary syndrome (PCOS). However, no functional CYP19A1 polymorphisms that predict the risk of PCOS have been identified. We explored the role of CYP19A1 genetic variation in a large case-control study involving 1078 samples, in which five common genetic polymorphisms were scored. Human embryonic kidney 293 cells were transiently transfected with a vector encoding either the CYP19A1 wild-type (WT) allele or an Arg(264)Cys variant to evaluate aromatase activity. Cells were cultured with androstenedione and estrone levels were measured using a specific ELISA. The Arg(264)Cys variant of CYP19A1 (rs700519) is associated with PCOS (P= 0.004, corrected P = 0.02). In this functional study, when cells were cultured in varying concentrations of androstenedione (100, 400 and 500 nM), transfection with the Arg(264)Cys variant resulted in increased conversion of androstenedione to estrogen when compared with transfection with the WT construct (P< 0.001). Our data suggest that the common missense polymorphism rs710059 is associated with susceptibility to PCOS and that the Arg(264)Cys variant may increase aromatase enzymatic activity. Overall, these findings imply that aromatase plays an important role in PCOS.

Side-chain effect on the structural evolution and properties of poly(9,9-dihexylfluorene-alt-2,5-dialkoxybenzene) copolymers.

The structural evolution and properties of poly(9,9-dihexylfluorene-alt-2,5-dialkoxybenzene) with different lengths of alkoxy side chains on phenylene have been systematically investigated by means of thermogravimetric analysis (TGA), X-ray diffraction (XRD), differential scanning calorimetry (DSC), polarizing light microscopy (PLM), atomic force microscopy (AFM), and cyclic voltammetry (CV) techniques. The polymer self-organizes into a lamellar structure consisting of both two- and one-layer packing, and the two-layer packing style is the dominant structure. In addition, the two-layer and one-layer packing structures also accompany the presence of planar stacking and/or crystalline and noncrystalline structures, thus maintaining the stability of the packing. PF6OC6 shows three ordered phases (two crystalline phases and one nematic phase) during the heating process. With further increase of the length of alkoxy side chains, only two ordered phases (one crystalline phase and one nematic phase) are observed and the polymers show a melting-recrystallization phenomenon, which is steadily inhibited as the length of the alkoxy side chains increases. The optical and electrochemical properties of the polymers do not exhibit noticeable dependence on the length of the alkoxy side chains. However, the thermal stability, the vibronic structures, and the full width at half-maximum (fwhm) in photoluminescence spectra of the films gradually decrease, and the oxidation onset potentials and the corresponding HOMO energy levels slightly increase with increasing length of alkoxy side chains on phenylene. These results indicate that the length variation of alkoxy side chains does not change the electronic structure of the polymer backbones, but remarkably affects the microphase separation between the flexible side chains and the conjugated backbones.

Inhibitory effect of ginsenoside Rb1 on cardiac hypertrophy induced by monocrotaline in rat.

Ginseng, the root of Panax ginseng, has been used as folk medicine in the treatment of various diseases for thousands of years in China. Ginsenoside Rb1 (Rb1), one of the effective components of ginseng, has been reported to release nitric oxide and decrease intracellular free Ca2+ in cardiac myocytes, both of which play important roles in antihypertrophic effect. This study was to investigate the potential effect of Rb1 on right ventricular hypertrophy (RVH) induced by monocrotaline (MCT) and its possible influence on calcineurin (CaN) signal trasnsduction pathway. MCT-treated animals were administered with Rb1 (10 and 40 mg /kg) from day 1 to day 14 (preventive administration) or from day 15 to day 28 (therapeutic administration), or with vehicle as corresponding controls. After 2 weeks, significantly hypertrophic reactions, including RVH index and the expressions of atrial natriuretic peptide mRNA, appeared in right ventricle of all MCT-treated animals (p < 0.05), which were significantly decreased with some improvements of myocardial pathomorphology in both Rb1 prevention- and therapy-groups (p < 0.05). Similarly, MCT-treatment caused the high expressions of mRNA and/or proteins of CaN, NFAT3 and GATA4 from cardiocytes (p < 0.05) and Rb1 could alleviate the expressions of these factors above (p < 0.05). These results suggest that Rb1 treatment can inhibit the RVH induced by MCT, which may be involved in its inhibitory effects on CaN signal transduction pathway.

Cardiac hypertrophy induced by prostaglandin F(2alpha) may be mediated by calcineurin signal transduction pathway in rats.

In this paper, we studied the relationship between the prostaglandin F(2alpha) (PGF(2alpha))-induced cardiac hypertrophy and calcineurin (CaN) signal transduction pathway in vivo and in vitro. Male Sprague-Dawley rats were given a single i.p. injection with monocrotaline (MCT) (60 mg/kg) and then given orally with celecoxib (20 mg/kg) or vehicle once a day for 14 d before (from d 1 to d 14) or after (from d 15 to d 28) right ventricular hypertrophy (RVH) was formed. Body weight (BW), right ventricular weight (RV), left ventricular with septum weight (LV), as well as lung weight were determined. RVH index (RVHI=RV/LV), RV/BW, and lung weight/BW were calculated and histological changes were observed with transmission electron microscope. PGF(2alpha) level, atrial natriuretic peptide (ANP) and CaN mRNA expressions, expression of CaN and its downstream effectors, NFAT(3) and GATA(4) protein were assayed by EIA kit, RT-PCR, and Western blotting, respectively. The cardiomyocyte hypertrophy in primary culture induced by PGF(2alpha) (0.1 micromol/L) was evaluated by measuring the cell diameter, protein content, and ANP mRNA as well as CaN mRNA expressions. It was found that 14 d or 28 d after MCT was given, the RVHI, RV/BW, and lung weight/BW were significantly increased by 47%, 53% and 118%, and by 64%, 94% and 156%, respectively; at the same time PGF(2alpha) levels in RV tissue were increased by 44% and by 51% with increasing RVHI, and elevated expressions of ANP and CaN mRNA, as well as CaN, NFAT(3) and GATA(4) proteins in a positive correlation manner. Furthermore, some histological injuries were found in RV tissue. Celecoxib, a cyclooxygenase inhibitor, obviously blunted the elevation of RVHI, RV/BW, and lung weight/BW no matter it was given before or after RVH. In vitro experiments showed that 0.1 micromol/L PGF(2alpha) significantly increased the cardiomyocyte diameter and protein content, and promoted ANP and CaN mRNA expressions, which was blocked by cyclosporin A, a CaN inhibitor. Our results indicate that PGF(2alpha) may be involved in cardiac hypertrophy induced by MCT in rats through CaN signal transduction pathway.

[RNA interference-mediated silencing of cytochrome P450 3A4 gene in CHL-3A4 cells].

OBJECTIVE: To investigate the effect of small hairpin interfering RNA (shRNA) in suppressing cytochrome P450 3A4 (CYP3A4) gene expression in CHL-3A4 cells. METHODS: Three shRNA expression vectors targeting CYP3A4 gene (CYP3A4 I, C YP3A4 II, and CYP3A4 III, respectively) were designed, synthesized and transfected into CHL-3A4 cells via liposomes. The inhibitory effect of shRNA on CYP 3A4 gene expression was detected by Western blotting and RT-PCR, and the effect of shRNA transfection in suppressing cyclophosphamide-induced cytotoxicity was measured using MTT assay. RESULTS: The vector carrying CYP3A4 III shRNA significantly reduced the expression of CYP3A4 gene at both the mRNA (75%) and protein levels (80%) in CHL3A4 cells. The cytotoxicity of cyclophosphamide was markedly inhibited by CYP3A4 III-mediated suppression of CYP3A4 gene expression by 75% in CHL-3A4 cells. CONCLUSION: The vector-mediated RNA interference can suppress CYP3A4 gene expression in CHL-3A4 cells, and RNA interference technique provides a new means for studying cytochrome P450 gene function in mammalian cells.

[Effect of ketoconazole on the activity of CYP4503A4 and CYP450 1A2 of hepatic microsomes in healthy adults].

OBJECTIVE: To observe the effect of ketoconazole on the activity of cytochrome P450 (CYP) 1A2 and 3A4 in hepatic microsomes of healthy adults. METHODS: Human hepatic microsomes obtained from healthy adults were randomly divided into control group and ketoconazole-treatment groups at different concentrations. After 15 min of culture, the substrates (testosterone for CYP3A4 and phenacetin for CYP1A2) were added and incubated for another 20 min. The metabolites (6-testosterone and acetaminophen) were then measured with high-performance liquid chromatography (HPLC) to assess the activities of CYP3A4 and 1A2. RESULTS: Significant difference was found between the groups in the quantity of 6-testosterone and the relative activity of CYP3A4 (P<0.05). The IC(50) of ketoconazole for CYP3A4 was 0. 16 mg/L. Both the quantity of 6-testosterone and the relative activity of CYP3A4 were reduced gradually with the increment of ketoconazole concentration. Significant differences were found between the ketoconazole groups and the control group in both the quantity of acetaminophen and the relative activity of CYP1A2 (P<0.05). Ketoconazole at low doses reduced CYP1A2 activity and but increased the activities at high doses (P<0.05). CONCLUSION: In the range of maximum clinical blood concentration, ketoconazole can inhibit the activity of CYP3A4, but not that of CYP1A2, in the hepatic microsomes in healthy adults.

Inhibitory effect of ginsenoside Rb1 on calcineurin signal pathway in cardiomyocyte hypertrophy induced by prostaglandin F2alpha.

AIM: To examine the antihypertrophic effect of ginsenoside Rb1 (Rb1) induced by prostaglandin F2alpha(PGF2alpha) in vitro and to investigate the possible mechanisms involved in the calcineurin (CaN) signal transduction pathway. METHODS: The cardiomyocyte hypertrophy induced by PGF2alpha and the antihypertrophic effect of Rb1 were evaluated in primary culture by measuring the cell diameter, protein content, and atrial natriuretic peptide (ANP) mRNA expression. ANP and CaN mRNA expressions, CaN and its downstream effectors NFAT3 and GATA4 protein expressions, and the intracellular free Ca2+ concentration ([Ca2+]i) were assayed by RT-PCR, Western blot, and fluorescent determination using Fura 2/AM, respectively. RESULTS: PGF2alpha (100 nmol/L) significantly increased the cardiomyocyte diameter, protein content and [Ca2+]i, and promoted ANP, CaN mRNA, and CaN/NFAT3/GATA4 protein expressions, which were inhibited by either Rb1 in a concentration-dependent manner (50, 100, and 200 microg/mL) or L-arginine (1 mmol/L). NG-nitro-L-arginine-methyl ester, a nitric oxide synthase inhibitor, could abolish the effects of L-arginine, but failed to change the effects of Rb1 in the experiments above. CONCLUSION: The present data implicate that Rb1 attenuates cardiac hypertrophy, the underlying mechanism may be involved in the inhibition of the Ca2+-CaN signal transduction pathway.