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Sample records for 7alpha 17alpha-dimethyl-19-nortestosterone mibolerone

  1. Use of radioactive 7alpha, 17alpha-dimethyl-19-nortestosterone (mibolerone) in the assay of androgen receptors

    SciTech Connect

    Schilling, K.; Liao, S.

    1984-01-01

    Tritiated 7alpha, 17alpha-dimethyl-19-nortestosterone (DMNT; mibolerone), a synthetic androgen stable to metabolic conversion in the rat ventral prostate, is an excellent radioactive ligand for the quantitation and characterization of androgen receptors in prostate, liver, and cultured cells. DMNT is more receptor-selective than 17alpha-methyl-17beta-hydroxy-estra-4,9,11-trien-3-one (R1881); DMNT interacts with glucocorticoid and progestin receptors much less strongly than R1881. Unlike 5alpha-dihydrotestosterone, DMNT does not bind tightly to testosterone-estradiol binding globulin of human serum. The hydroxylapatite-filter assay employed clearly distinguished between DMNT binding to androgen receptors of rat ventral prostate and interaction of DMNT with androgen binding protein of epididymides. The prostate cytosol (/sup 3/H)DMNT-receptor complex sediments in two forms (4 and 8 S) in a low salt medium. In 0.4 M KCl, both the prostate cytosol and nuclear (/sup 3/H)DMNT-receptor complexes migrated as 3-4 S components. The formation of both the cytosol and nuclear DMNT-receptor complexes is inhibited by antiandrogens and 17beta-estradiol.

  2. 21 CFR 558.348 - Mibolerone.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS NEW ANIMAL DRUGS FOR USE IN ANIMAL FEEDS Specific New Animal Drugs for Use in Animal Feeds § 558.348 Mibolerone. (a) Approvals. To No. 000009 in § 510.600(c) of this chapter for a...

  3. 21 CFR 558.348 - Mibolerone.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS NEW ANIMAL DRUGS FOR USE IN ANIMAL FEEDS Specific New Animal Drugs for Use in Animal Feeds § 558.348 Mibolerone. (a) Approvals. To No. 000009 in § 510.600(c) of this chapter for a...

  4. 21 CFR 558.348 - Mibolerone.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS NEW ANIMAL DRUGS FOR USE IN ANIMAL FEEDS Specific New Animal Drugs for Use in Animal Feeds § 558.348 Mibolerone. (a) Approvals. To No. 000009 in § 510.600(c) of this chapter for a...

  5. 21 CFR 558.348 - Mibolerone.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS NEW ANIMAL DRUGS FOR USE IN ANIMAL FEEDS Specific New Animal Drugs for Use in Animal Feeds § 558.348 Mibolerone. (a) Approvals. To No. 000009 in § 510.600(c) of this chapter for a...

  6. Androgen regulation of the androgen receptor of the quail uropygial gland: application of a [3H]mibolerone exchange assay.

    PubMed

    Amet, Y; Abalain, J H; di Stefano, S; Daniel, J Y; Tea, K; Floch, H H; Robel, P

    1986-06-01

    An exchange assay for androgen receptors in the quail uropygial gland using [3H]mibolerone was established. The most efficient exchange conditions were 3 days of incubation at 15 degrees C. Under these conditions, androgen receptors were stable in the presence of sodium molybdate, and the exchange of [3H]mibolerone with endogenous testosterone bound to cytosolic or nuclear androgen receptors was maximal. Less than 5% of [3H]mibolerone-binding sites occurred in the extracted nuclear pellets. Using this exchange technique, it was shown that androgen receptors in the uropygial gland of photostimulated male quail or castrated quail treated with testosterone were activated and that their concentrations in both cytosolic and nuclear fractions were increased. These results confirm the androgen dependency of the quail uropygial gland, and show that it is an organ which can be used as a model for the study of androgen action in sebaceous glands.

  7. Characterization of urinary metabolites of testosterone, methyltestosterone, mibolerone and boldebone in greyhound dogs.

    PubMed

    Williams, T M; Kind, A J; Hyde, W G; Hill, D W

    2000-06-01

    Androgenic steroids are used in female greyhound dogs to prevent the onset of estrus; moreover, these steroids also have potent anabolic activity. As anabolic steroids increase muscle mass and aggression in animals, the excessive use of these agents in racing greyhounds gives an unfair performance advantage to treated dogs. The biotransformation of most anabolic steroids has not been determined in greyhound dogs. The objective of the present study was to identify the urinary metabolites of testosterone, methyltestosterone, mibolerone, and boldenone in greyhound dogs. These steroids were administered orally (1 mg/kg) to either male or female greyhound dogs and urine samples were collected pre-administration and at 2, 4, 8, 12, 24, 72, and 96 h post-administration. Urine extracts were analyzed by high-performance liquid chromatography/mass spectrometry (HPLC/MS) to identify major metabolites and to determine their urinary excretion profiles. Major urinary metabolites, primarily glucuronide, conjugated and free, were detected for the selected steroids. Sulfate conjugation did not appear to be a major pathway for steroid metabolism and excretion in the greyhound dog. Phase I biotransformation was also evaluated using greyhound dog liver microsomes from untreated dogs. The identification of several in vivo steroid metabolites generated in this study will be useful in detecting these steroids in urine samples submitted for drug screening.

  8. Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites.

    PubMed

    Siddiqui, Mahwish; Ahmad, Malik Shoaib; Wahab, Atia-Tul-; Yousuf, Sammer; Fatima, Narjis; Naveed Shaikh, Nimra; Rahman, Atta-Ur-; Choudhary, M Iqbal

    2017-01-01

    Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone (1) with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina. Their structures were determined as 10β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6β,10β,17β-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11β,17β-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11β,17β-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of β-glucuronidase enzyme (IC50 = 42.98 ± 1.24 μM) during random biological screening, while its metabolites 2-4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 μM). Its transformed products 3 (IC50 = 79.09 ± 0.06 μM), and 8 (IC50 = 70.09 ± 0.05 μM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 μM), and its metabolite 8 (IC50 = 34.16 ± 5.3 μM) exhibited potent cytotoxicity against HeLa cancer cell line (human cervical carcinoma). Metabolite 2 (IC50 = 46.5 ± 5.4 μM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 μM) and 4 (IC50 = 152.5 ± 2.15 μM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 μM), and its transformed products 2 (IC50 = 43.3 ± 7.7 μM), 3 (IC50 = 65.6 ± 2.5 μM), and 4 (IC50 = 89.4 ± 2.7

  9. Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites

    PubMed Central

    Siddiqui, Mahwish; Ahmad, Malik Shoaib; Wahab, Atia-tul-; Yousuf, Sammer; Fatima, Narjis; Naveed Shaikh, Nimra; Rahman, Atta-ur-; Choudhary, M. Iqbal

    2017-01-01

    Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone (1) with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina. Their structures were determined as 10β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6β,10β,17β-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11β,17β-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11β,17β-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of β-glucuronidase enzyme (IC50 = 42.98 ± 1.24 μM) during random biological screening, while its metabolites 2–4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 μM). Its transformed products 3 (IC50 = 79.09 ± 0.06 μM), and 8 (IC50 = 70.09 ± 0.05 μM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 μM), and its metabolite 8 (IC50 = 34.16 ± 5.3 μM) exhibited potent cytotoxicity against HeLa cancer cell line (human cervical carcinoma). Metabolite 2 (IC50 = 46.5 ± 5.4 μM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 μM) and 4 (IC50 = 152.5 ± 2.15 μM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 μM), and its transformed products 2 (IC50 = 43.3 ± 7.7 μM), 3 (IC50 = 65.6 ± 2.5 μM), and 4 (IC50 = 89.4 ± 2

  10. Seasonal changes in the synthesis of the neurosteroid 7alpha-hydroxypregnenolone stimulating locomotor activity in newts.

    PubMed

    Haraguchi, Shogo; Matsunaga, Masahiro; Koyama, Teppei; Do Rego, Jean-Luc; Tsutsui, Kazuyoshi

    2009-04-01

    We recently found that the newt brain actively produces 7alpha-hydroxypregnenolone, a novel amphibian neurosteroid stimulating locomotor activity. It is well known that locomotor activity of male newts increases during the breeding period. To understand the physiological role of 7alpha-hydroxypregnenolone, we investigated seasonal changes in 7alpha-hydroxypregnenolone synthesis in the brain of male newts. Interestingly, 7alpha-hydroxypregnenolone synthesis in the brain showed marked changes during the annual breeding cycle, with a maximal level in the breeding period when locomotor activity of male newts increases. These results suggest that 7alpha-hydroxypregnenolone induces seasonal locomotor changes in male newts.

  11. Regulation of hepatic 7 alpha-hydroxylase expression by dietary psyllium in the hamster.

    PubMed Central

    Horton, J D; Cuthbert, J A; Spady, D K

    1994-01-01

    Soluble fiber consistently lowers plasma total and low density lipoprotein (LDL)-cholesterol concentrations in humans and various animal models including the hamster; however, the mechanism of this effect remains incompletely defined. We performed studies to determine the activity of dietary psyllium on hepatic 7 alpha-hydroxylase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and LDL receptor expression in the hamster. In animals fed a cholesterol-free semisynthetic diet containing 7.5% cellulose (avicel) as a fiber source, substitution of psyllium for avicel increased hepatic 7 alpha-hydroxylase activity and mRNA levels by 3-4-fold. Comparable effects on 7 alpha-hydroxylase expression were observed with 1% cholestyramine. Psyllium also increased hepatic 7 alpha-hydroxylase activity and mRNA in animals fed a diet enriched with cholesterol and triglyceride. Activation of 7 alpha-hydroxylase was associated with an increase in hepatic cholesterol synthesis that was apparently not fully compensatory since the cholesterol content of the liver declined. Although dietary psyllium did not increase hepatic LDL receptor expression in animals fed the cholesterol-free, very-low-fat diet, it did increase (or at least restore) receptor expression that had been downregulated by dietary cholesterol and triglyceride. Thus, 7.5% dietary psyllium produced effects on hepatic 7 alpha-hydroxylase and LDL metabolism that were similar to those of 1% cholestyramine. Induction of hepatic 7 alpha-hydroxylase activity by dietary psyllium may account, in large part, for the hypocholesterolemic effect of this soluble fiber. Images PMID:8182140

  12. Different in vitro metabolism of 7 alpha-methyl-19-nortestosterone by human and equine aromatases.

    PubMed

    Moslemi, S; Dintinger, T; Dehennin, L; Silberzahn, P; Gaillard, J L

    1993-06-01

    The ability of human and equine placental microsomes to aromatize 7 alpha-methyl-19-nortestosterone (MNT) was studied. Kinetic analysis indicates that MNT shares the androgen-binding site of human and equine placental microsomal aromatases. Human placental microsomal estrogen synthetase had about a 2.5-fold higher relative affinity for MNT than the equine placental enzyme (KiMNT/Km androstenedione of 32 versus 87). However, MNT was not metabolized by human placental microsomes, whereas it was very actively metabolized by equine placental microsomes. Further studies using purified equine cytochrome P-450arom indicated that the presence of a 7 alpha-methyl group and the absence of a C19 methyl group did not impair its conversion by the purified enzyme. The product of this reaction was separated and identified as 7 alpha-methylestradiol by gas chromatography coupled to mass spectrometry.

  13. [7alpha-hydroxylation of steroid 5-olefins by mold fungi].

    PubMed

    Andriushina, V G; Druzhinina, A V; Iaderets, V V; Stytsenko, T S; Voĭshvillo, N E

    2010-01-01

    Hydroxylation activity of the mold fungi belonging to the orders Dothideales, Hypocreales, and Mucorales towards delta(5)-3beta-hydroxysteroids was studied. The fungi Bipolaris sorokiniana, Fusarium sp., and Rhizopus nigricans were able to introduce hydroxy group at position 7alpha; however, this ability was detected only at a low substrate load and with a low yield. A 7alpha-hydroxylase activity of the Curvularia lunata VKPM F-981 culture was shown for the first time. It was demonstrated that the studied strain was capable of stereo- and regioselective transformations of androstane 5-olefins at a load not less than 2 g/l. Conversion of pregnane steroids by this culture yielded both 7alpha and 11beta-hydroxy derivatives. The introduction of 7alpha-hydroxy group by this strain occurred concurrently with enzymatic hydrolysis of ester groups, which proceeded under mild conditions to give the corresponding alcohols in the cases of both 3-acetate of delta(5)-androstenes and mono- and triacetates of delta(5)-pregnenes.

  14. Diurnal changes in the synthesis of the neurosteroid 7alpha-hydroxypregnenolone stimulating locomotor activity in newts.

    PubMed

    Koyama, Teppei; Haraguchi, Shogo; Vaudry, Hubert; Tsutsui, Kazuyoshi

    2009-04-01

    We recently identified 7alpha-hydroxypregnenolone as a novel amphibian neurosteroid stimulating locomotor activity in newts. Because male newts show marked diurnal changes in locomotor activity, we hypothesized that 7alpha-hydroxypregnenolone may be a key factor for the induction of diurnal changes in locomotor activity in male newts. In this study, we found diurnal changes in 7alpha-hydroxypregnenolone synthesis in the brain of male newts, which paralleled locomotor activity. Interestingly, the production of 7alpha-hydroxypregnenolone in the male newt brain increased during the dark phase when locomotor activity of males was high.

  15. Biotransformation of 7alpha-hydroxy- and 7-oxo-ent-atis-16-ene derivatives by the fungus Gibberella fujikuroi.

    PubMed

    Fraga, Braulio M; Gonzalez, Pedro; Gonzalez-Vallejo, Victoria; Guillermo, Ricardo; Diaz, Luz N

    2010-08-01

    The microbiological transformation of 7alpha,19-dihydroxy-ent-atis-16-ene by the fungus Gibberella fujikuroi gave 19-hydroxy-7-oxo-ent-atis-16-ene, 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene, 7alpha,11beta,19-trihydroxy-ent-atis-16-ene and 7alpha,16beta,19-trihydroxy-ent-atis-16-ene, while the incubation of 19-hydroxy-7-oxo-ent-atis-16-ene afforded 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene and 16beta,17-dihydroxy-7-oxo-ent-atisan-19-al. The biotransformation of 7-oxo-ent-atis-16-en-19-oic acid gave 6beta-hydroxy-7-oxo-ent-atis-16-en-19-oic acid, 6beta,16beta,17-trihydroxy-7-oxo-19-nor-ent-atis-4(18)-ene and 3beta,7alpha-dihydroxy-6-oxo-ent-atis-16-en-19-oic acid.

  16. Dietary fatty acids regulate cholesterol induction of liver CYP7alpha1 expression and bile acid production.

    PubMed

    Li, Yan; Hou, Meng Jun; Ma, Jing; Tang, Zhi Hong; Zhu, Hui Lian; Ling, Wen Hua

    2005-05-01

    In the present study we investigated the effects of dietary fats containing predominantly PUFA, monounsaturated FA (MUFA), or saturated FA (SFA) on lipid profile and liver cholesterol 7alpha-hydroxylase (CYP7alpha1) mRNA expression and bile acid production in C57BL/6J mice. The animals (n = 75) were randomly divided into five groups and fed a basic chow diet (AIN-93G) (BC diet), a chow diet with 1 g/100 g of cholesterol (Chol diet), a chow diet with 1 g/100 g of cholesterol and 14 g/100 g of safflower oil (Chol + PUFA diet), a chow diet with 1 g/100 g of cholesterol and olive oil (Chol + MUFA diet), or a chow diet with 1 g/100 g of cholesterol and myristic acid (Chol + SFA diet) for 6 wk. The results showed that the Chol + SFA diet decreased CYP7alpha1 gene expression and bile acid pool size, resulting in increased blood and liver cholesterol levels. Addition of PUFA and MUFA to a 1% cholesterol diet increased the bile acid pool production or bile acid excretion and simultaneously decreased liver cholesterol accumulation despite decreased CYP7alpha1 mRNA expression. The results indicate that the decreased bile acid pool size induced by the SFA diet is related to inhibition of the liver CYP7alpha1 gene expression, but an increased bile acid pool size and improved cholesterol homeostasis are disassociated from the liver CYP7alpha1 gene expression.

  17. Radioimmunoassay of 7 alpha-methyl-19-nortestosterone and investigation of its pharmacokinetics in animals.

    PubMed

    Kumar, N; Didolkar, A K; Ladd, A; Thau, R; Monder, C; Bardin, C W; Sundaram, K

    1990-11-30

    A method for the measurement of 7 alpha-methyl-19-nortestosterone (7MENT) in serum/plasma by radioimmunoassay (RIA) is described. The antiserum, raised against 7 alpha-methyl-19-nortestosterone-3-O-oxime-bovine serum albumin, had a low titer (final dilution = 1:4500) and low affinity (Ka = 1.17 x 10(9) l/mol) but showed little or no cross-reactivity with several of the steroids tested. The sensitivity of the RIA was 28.2 pg/ml and the mean recovery of added cold steroid was 86 to 100%. Intra- and inter-assay coefficients of variation ranged from 4.3 to 7.3% and 7.3 to 8.4%, respectively. This RIA was used to follow plasma 7MENT levels after a single i.v. injection of the steroid in rats and rabbits. The metabolic clearance rates (MCR) of 7MENT as determined from the plasma disappearance curve for rats and rabbits were 50 l/day and 336 l/day, respectively. The MCR of 7MENT in rats and rabbits lies in the same range as for testosterone. When compared to other nortestosterone derivatives such as norethisterone, 7MENT is metabolized relatively faster.

  18. A convenient synthesis of 7 alpha-hydroxycholest-4-en-3-one by the hydroxypropyl-beta-cyclodextrin-facilitated cholesterol oxidase oxidation of 3 beta,7 alpha-cholest-5-ene-3,7-diol.

    PubMed

    Alexander, D L; Fisher, J F

    1995-03-01

    The initial biosynthetic conversions of cholesterol to the bile acids involve sequential 7 alpha-hydroxylation (catalyzed by cholesterol 7 alpha-hydroxylase) followed by C-3 oxidation and concomitant double bond migration (to a delta 4-configuration, catalyzed by 3 beta-delta 5-C27-steroid oxidoreductase) to provide 7 alpha-hydroxycholest-4-en-3-one. A straightforward, and economical, preparation (on a 0.1 g scale) of this pivotal biosynthetic intermediate has been devised. Reduction of 3 beta-(benzoyloxy)-cholest-5-en-7-one with LiB(sec-butyl)3H provided a 4:1 mixture, respectively, of the 7 alpha- and 7 beta-hydroxy diastereomers, which were separated chromatographically. Solvolytic removal of the C-3 benzoyl group gave 3 beta,7 alpha-cholest-5-ene-3,7-diol. A suspension of the 1:1 (v/v) complex (formed by mutual dissolution in MeOH, followed by evaporation of the solvent) of this diol with hydroxypropyl-beta-cyclodextrin, at a concentration of 1 mg mL-1 (in neutral phosphate buffer), was converted by Brevibacterium sp cholesterol oxidase (0.25 U mg-1 of substrate) and catalase (70 U mg-1 of substrate, to recover O2 from the H2O2 produced by the enzymatic oxidation) to a suspension of 7 alpha-hydroxycholest-4-en-3-one and the hydroxypropyl-beta-cyclodextrin. The yield for the enzymatic conversion was in excess of 90%. A much poorer and less reproducible yield (< 20%) was seen in the absence of the hydroxypropyl-beta-cyclodextrin. Routine extraction of this aqueous suspension, and chromatographic purification (85:15 CHCl3/acetone v/v on silica) of the residue, gave pure 7 alpha-hydroxycholest-4-en-3-one in 68% isolated yield. This route is a significant improvement, in terms of reaction scale and convenience, over the previous procedures for the preparation of this steroid.

  19. Quantitative determination of cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides in rat skin.

    PubMed

    Yamazaki, S; Ozawa, N; Hiratsuka, A; Watanabe, T

    1999-07-01

    An assay method for determination of cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides (ChOOHs) in rat skin using high-performance liquid chromatography (HPLC) with a chemiluminescence detector has been developed. In the assay method, free form and free plus ester forms of ChOOHs could be separately determined by HPLC in combination with the treatment of a tissue extract by cholesterol esterase. Lower limits of quantitation for cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides were 0.2, 0.1, and 0.5 nmol/g skin, respectively. This assay method showed that (i) good absolute recoveries of ChOOHs from rat skin (80-90% of radiolabeled ChOOHs added to rat skin); (ii) negligible autoxidation of cholesterol caused by the assay procedure (<9.4x10(-5)% of radiolabeled cholesterol added to rat skin); and (iii) good correlation between ChOOHs added to rat skin and ChOOHs determined, indicating this assay method is applicable to quantify ChOOHs in rat skin. By using this assay method, we observed that (i) cholesterol 5alpha-hydroperoxide was detected in skin of rats pretreated with oral doses of pheophorbide a and subsequent visible irradiation; (ii) concentrations of cholesterol 7-hydroperoxides in skin of rats in an ambient light room were not significantly different from those in a dark room for 12 weeks; and (iii) ultraviolet light B irradiation markedly enhanced the concentrations of cholesterol 7-hydroperoxides in the skin of rats.

  20. Aromatization of 7 alpha-methyl-19-nortestosterone by human placental microsomes in vitro.

    PubMed

    LaMorte, A; Kumar, N; Bardin, C W; Sundaram, K

    1994-02-01

    Part of the biological effects of testosterone (T) are mediated by its enzymatic reduction to 5 alpha-dihydrotestosterone (DHT) or aromatization to estradiol (E2). 7 alpha-Methyl-19-nortestosterone (MENT) is a synthetic androgen that is considerably more potent than T. Previous studies have shown that MENT is not 5 alpha-reduced. The studies reported here were undertaken to determine whether MENT undergoes enzymatic aromatization in vitro. Human placental microsomes were used as the source of the aromatase. Radioactive or nonradioactive T or MENT was incubated with the microsomes in the presence of NADPH and the metabolites extracted out with ethyl ether. Following evaporation of ether, the residue was dissolved in benzene-petroleum ether and extracted with 0.4 N NaOH which selectively removes phenolic metabolites of the androgens. When either radioactive T or MENT was incubated with the aromatase in the presence of NADPH, there was a 20-fold increase in the amount of radioactivity extracted with NaOH. In contrast, if the incubation was carried out in the absence of NADPH or in the presence of R76713, an aromatase inhibitor, most of the radioactivity remained in the benzene-petroleum ether phase. To further identify the enzymatic reaction products, thin layer chromatography (TLC) was performed. The Rf value for MENT was 0.22 while that of the major reaction product was 0.34, which corresponded with the RF value of the estrogen, 7 alpha-methyl-estradiol (MeE2). This was further verified by using a second solvent system for the chromatographic separation. In an effort to ascertain whether the metabolites bind to estrogen receptors (ER), rat uterine cytosol was used. NaOH extracts of medium following incubation of nonradioactive MENT with microsomes showed competitive inhibition of [3H]E2 binding to rat uterine ER. Furthermore, after [3H]MENT was incubated with microsomes, the radioactive metabolite extracted in NaOH showed specific binding to the ER which could

  1. Pharmacokinetics of 7 alpha-methyl-19-nortestosterone (MENT) delivery using subdermal implants in healthy men.

    PubMed

    Suvisaari, J; Moo-Young, A; Juhakoski, A; Elomaa, K; Saleh, S I; Lähteenmäki, P

    1999-11-01

    We studied the pharmacokinetics of 7 alpha-methyl-19-nortestosterone (MENT), a potent synthetic androgen, administered by subdermal implants. The implants contained 112 +/- 4 mg of MENT acetate in a polyethylene vinyl acetate copolymer. MENT acetate released from the implants is rapidly hydrolyzed to MENT in vivo. Fifteen healthy Finnish men were randomized to have either one, two, or four implants inserted in the medial aspect of the upper arm. The implants remained in place for 4 weeks. Blood samples were obtained before implant insertion, 1, 2, 3, and 4 weeks after insertion, and 1 and 2 weeks after removal. Serum MENT concentrations were determined by gas chromatography with mass selective detection. The MENT levels attained in each implant group remained at a steady level during the 4 weeks of implant use. The mean steady state MENT concentrations in the one, two, and four implant groups were 0.6, 1.4, and 2.3 nmol/L, respectively. Serum MENT concentrations during implant use were clearly dose dependent; the between-subject effect of implants as well as the differences between each pair of groups were all statistically significant. The release rate of MENT from one, two, and four implants was calculated to be approximately 0.3, 0.8, and 1.3 mg/day, respectively. This study suggests that MENT acetate implants are a promising method for long-term androgen administration in hypogonadism and male contraception.

  2. 7 alpha-methyl-nortestosterone (MENT): the optimal androgen for male contraception.

    PubMed

    Sundaram, K; Kumar, N; Bardin, C W

    1993-04-01

    Androgens are needed in male fertility control methods to impede gonadotropin secretion. Large and frequent doses of testosterone esters are used to induce this effect, but these large and frequent doses are linked to wide fluctuations of plasma testosterone levels. Thus, men need a contraceptive that supplies effective, appropriate, continuous replacement doses over long periods. The synthetic androgen 7alpha-methyl-19-nortestosterone (MENT) can likely address this problem. Studies in primates, rats, and adult men show that it is much more potent than testosterone and can be administered via subdermal implants in effective amounts, which mimic physiologic doses and effects of testosterone, for 12 months. There will most likely be 2 subdermal implants, 1 releasing MENT and the other releasing a luteinizing hormone releasing hormone. Unlike testosterone, MENT is not reduced (5alpha-reduction) to a 5alpha-dihydrosteroid in the prostate. If MENT is administered in a dose sufficient to not disturb normal muscle mass and gonadotropin secretion, it will not hyperstimulate the prostate. Thus, it is less apt to cause benign prostatic hypertrophy and, possibly, prostate cancer than is testosterone. MENT is the first androgen to promote health (i.e., reduction of the incidence of prostate disease). Clinicians may also be able to use MENT to treat hypogonadism, prostatic hyperplasia, and muscle wasting.

  3. Metabolism of the bile acid analogues 7 beta-methyl-cholic acid and 7 alpha-methyl-ursocholic acid

    SciTech Connect

    Kuroki, S.; Mosbach, E.H.; Cohen, B.I.; McSherry, C.K.

    1987-04-01

    The metabolism of two new bile acid analogues, 7 beta-methyl-cholate and 7 alpha-methyl-ursocholate, was compared with that of cholate in the hamster. After intraduodenal administration of /sup 14/C-labeled compounds into bile fistula hamsters, radioactivity was exclusively recovered in bile; the more hydrophobic bile acid was absorbed more rapidly. Hepatic extraction of intravenously infused compounds was efficient and administered analogues became major biliary bile acids. Amidation of cholate was essentially complete, whereas 39% of 7 beta-methyl-cholate and 65% of 7 alpha-methyl-ursocholate were secreted in unconjugated form. After intragastric administration of the compounds, radioactivity was quantitatively recovered in feces. Cholate was 7-dehydroxylated to deoxycholate, whereas 31% of 7 beta-methyl-cholate and 78% of 7 alpha-methyl-ursocholate were recovered unchanged. Fifty percent of 7 beta-methyl-cholate and 15% of 7 alpha-methyl-ursocholate were transformed into ketonic derivatives, without loss of the 7-hydroxyl group. It is concluded that the introduction of the 7-methyl group did not interfere with intestinal absorption, hepatic extraction, and biliary secretion but did affect enzymatic amidation and bacterial 7-dehydroxylation of the analogues.

  4. 7alpha,11beta-Dimethyl-19-nortestosterone: a potent and selective androgen response modulator with prostate-sparing properties.

    PubMed

    Cook, C Edgar; Kepler, John A

    2005-02-15

    7alpha,11beta-Dimethyl-19-nortestosterone, made by 1,6-methyl addition to 17beta-acetoxy-11beta-methylestra-4,6-dien-3-one, was a highly potent and selective androgen response modulator, with enhanced androgen receptor binding, androgenic activity and anabolic:androgenic ratio over its two monomethyl homologs.

  5. Neonatal dietary cholesterol and alleles of cholesterol 7-alpha hydroxylase affect piglet cerebrum weight, cholesterol concentration, and behavior

    USDA-ARS?s Scientific Manuscript database

    This experiment was designed to test the effect of polymorphism in the cholesterol 7-alpha hydroxylase (CYP7) gene locus, and dietary cholesterol (C) on cerebrum C in neonatal pigs fed sow's milk formulas. Thirty-six pigs (18 male and 18 female) genetically selected for high (HG), or low (LG) plasma...

  6. Unexpected inhibition of cholesterol 7 alpha-hydroxylase by cholesterol in New Zealand white and Watanabe heritable hyperlipidemic rabbits.

    PubMed Central

    Xu, G; Salen, G; Shefer, S; Ness, G C; Nguyen, L B; Parker, T S; Chen, T S; Zhao, Z; Donnelly, T M; Tint, G S

    1995-01-01

    We investigated the effect of cholesterol feeding on plasma cholesterol concentrations, hepatic activities and mRNA levels of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase and hepatic LDL receptor function and mRNA levels in 23 New Zealand White (NZW) and 17 Watanabe heritable hyperlipidemic (WHHL) rabbits. Plasma cholesterol concentrations were 9.9 times greater in WHHL than NZW rabbits and rose significantly in both groups when cholesterol was fed. Baseline liver cholesterol levels were 50% higher but rose only 26% in WHHL as compared with 3.6-fold increase with the cholesterol diet in NZW rabbits. In both rabbit groups, hepatic total HMG-CoA reductase activity was similar and declined > 60% without changing enzyme mRNA levels after cholesterol was fed. In NZW rabbits, cholesterol feeding inhibited LDL receptor function but not mRNA levels. As expected, receptor-mediated LDL binding was reduced in WHHL rabbits. Hepatic cholesterol 7 alpha-hydroxylase activity and mRNA levels were 2.8 and 10.4 times greater in NZW than WHHL rabbits. Unexpectedly, cholesterol 7 alpha-hydroxylase activity was reduced 53% and mRNA levels were reduced 79% in NZW rabbits with 2% cholesterol feeding. These results demonstrate that WHHL as compared with NZW rabbits have markedly elevated plasma and higher liver cholesterol concentrations, less hepatic LDL receptor function, and very low hepatic cholesterol 7 alpha-hydroxylase activity and mRNA levels. Feeding cholesterol to NZW rabbits increased plasma and hepatic concentrations greatly, inhibited LDL receptor-mediated binding, and unexpectedly suppressed cholesterol 7 alpha-hydroxylase activity and mRNA to minimum levels similar to WHHL rabbits. Dietary cholesterol accumulates in the plasma of NZW rabbits, and WHHL rabbits are hypercholesterolemic because reduced LDL receptor function is combined with decreased catabolism of cholesterol to bile acids. Images PMID:7706454

  7. Pharmacokinetics and pharmacodynamics of 7alpha-methyl-19-nortestosterone after intramuscular administration in healthy men.

    PubMed

    Suvisaari, J; Sundaram, K; Noé, G; Kumar, N; Aguillaume, C; Tsong, Y Y; Lähteenmäki, P; Bardin, C W

    1997-05-01

    7alpha-Methyl-19-nortestosterone (MENT) is a potent synthetic androgen that is resistant to 5alpha-reductases and therefore less prone to over-stimulate the prostate. It is a good candidate for implant administration in long-term androgen replacement therapy for hypogonadal men or as part of a male contraceptive system. To investigate the pharmacokinetics of MENT after i.m. administration, single i.m. injections of 2, 4 or 8 mg of micronized MENT were given in aqueous suspension to 18 healthy men in two clinics. Blood was sampled frequently for 8 h and 1, 2, 3, 4 and 9 days after the injections. Serum MENT concentrations were determined by radioimmunoassay. Peak MENT concentrations were dose-dependent and were reached about 1-2 h after the injections. Doubling the dose of MENT resulted in an increase of 60% in peak serum MENT concentrations. The mean +/- SE clearance rate was 1790 +/- 140 l/day. The antigonadotrophic activity of MENT was investigated by giving six consecutive daily i.m. injections of 1, 2 or 4 mg of MENT to 24 healthy men in two clinics. Blood was sampled before each injection and up to 24 days after the last injection. Serum testosterone and gonadotrophin concentrations (determined by radioimmunoassay and fluoroimmunoassay respectively) decreased in a dose-dependent and statistically significant manner. The highest dose caused a 74% fall in testosterone, a 70% fall in luteinizing hormone, and a 57% fall in follicle stimulating hormone concentrations. MENT injections did not cause any side-effects. The results show that MENT is a potent antigonadotrophic agent in men.

  8. Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

    SciTech Connect

    Sun, Zhichao; Yu, Xuemei; Wu, Weibin; Jia, Dongwei; Chen, Yinle; Ji, Lingling; Liu, Xijun; Peng, Xiaomin; Li, Yintao; Yang, Lili; Ruan, Yuanyuan; Gu, Jianxin; Ren, Shifang; Zhang, Songwen

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated that the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.

  9. Prolactin increases the synthesis of 7alpha-hydroxypregnenolone, a key factor for induction of locomotor activity, in breeding male Newts.

    PubMed

    Haraguchi, Shogo; Koyama, Teppei; Hasunuma, Itaru; Vaudry, Hubert; Tsutsui, Kazuyoshi

    2010-05-01

    We recently found that the Japanese red-bellied newt, Cynops pyrrhogaster, actively produces 7alpha-hydroxypregnenolone, a previously undescribed amphibian neurosteroid. 7alpha-Hydroxypregnenolone stimulates locomotor activity of male newts. Locomotor activity of male newts increases during the breeding period as in other wild animals, but the molecular mechanism for such a change in locomotor activity is poorly understood. Here we show that the adenohypophyseal hormone prolactin (PRL) stimulates 7alpha-hydroxypregnenolone synthesis in the brain, thus increasing locomotor activity of breeding male newts. In this study, cytochrome P450(7alpha) (CYP7B), a steroidogenic enzyme catalyzing the formation of 7alpha-hydroxypregnenolone, was first identified to analyze seasonal changes in 7alpha-hydroxypregnenolone synthesis. Only males exhibited marked seasonal changes in 7alpha-hydroxypregnenolone synthesis and CYP7B expression in the brain, with a maximum level in the spring breeding period when locomotor activity of males increases. Subsequently we identified PRL as a key component of the mechanism regulating 7alpha-hydroxypregnenolone synthesis. Hypophysectomy decreased 7alpha-hydroxypregnenolone synthesis in the male brain, whereas administration of PRL but not gonadotropins to hypophysectomized males caused a dose-dependent increase in 7alpha-hydroxypregnenolone synthesis. To analyze the mode of PRL action, CYP7B and the receptor for PRL were localized in the male brain. PRL receptor was expressed in the neurons expressing CYP7B in the magnocellular preoptic nucleus. Thus, PRL appears to act directly on neurosteroidogenic magnocellular preoptic nucleus neurons to regulate 7alpha-hydroxypregnenolone synthesis, thus inducing seasonal locomotor changes in male newts. This is the first report describing the regulation of neurosteroidogenesis in the brain by an adenohypophyseal hormone in any vertebrate.

  10. Tibolone is not converted by human aromatase to 7alpha-methyl-17alpha-ethynylestradiol (7alpha-MEE): analyses with sensitive bioassays for estrogens and androgens and with LC-MSMS.

    PubMed

    de Gooyer, Marcel E; Oppers-Tiemissen, Hendrika M; Leysen, Dirk; Verheul, Herman A M; Kloosterboer, Helenius J

    2003-03-01

    To exclude that aromatization plays a role in the estrogenic activity of tibolone, we studied the effect tibolone and metabolites on the aromatization of androstenedione and the aromatization of tibolone and its metabolites to 7alpha-methyl-17alpha-ethynylestradiol (7alpha-MEE) by human recombinant aromatase. Testosterone (T), 17alpha-methyltestosterone (MT), 19-nortestosterone (Nan), 7alpha-methyl-19-nortestosterone (MENT) and norethisterone (NET) were used as reference compounds. Sensitive in vitro bioassays with steroid receptors were used to monitor the generation of product and the reduction of substrate. LC-MSMS without derivatization was used for structural confirmation. A 10 times excess of tibolone and its metabolites did not inhibit the conversion of androstenedione to estrone by human recombinant aromatase as determined by estradiol receptor assay whereas T, MT, Nan, and MENT inhibited the conversion for 75, 53, 85 and 67%, respectively. Tibolone, 3alpha- and 3beta-hydroxytibolone were not converted by human aromatase whereas the estrogenic activity formed with the Delta4-isomer suggests a conversion rate of 0.2% after 120 min incubation. In contrast T, MT, Nan, and MENT were completely converted to their A-ring aromates within 15 min while NET could not be aromatized. Aromatization of T, MT, Nan and MENT was confirmed with LC-MSMS. Structure/function analysis indicated that the 17alpha-ethynyl-group prevents aromatization of (19-nor)steroids while 7alpha-methyl substitution had no effect. Our results with the sensitive estradiol receptor assays show that in contrast to reference compounds tibolone and its metabolites are not aromatized.

  11. Formation of varanic acid, 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid from 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid in Bombina orientalis.

    PubMed

    Une, M; Inoue, A; Hoshita, T

    1996-11-01

    Varanic acid (3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid; 24-OH-THCA) is almost the sole component of bile acids in the bile of Bombina orientalis. To examine in the mechanism of the formation of 24-OH-THCA, radiolabeled (25R)- and (25S)-3 alpha, 7 alpha, 12 alpha-trihdroxy-5 beta-cholestanoic acids [(25R)- and (25S)-THCA] and (24E)-3 alpha, 7 alpha, 12 alpha-trihdroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) were administered intraperitoneally to B. orientalis, gallbladder bile was collected after 24 h, and bile acids were subsequently extracted. Then the bile acids were analyzed by means of radio thin-layer chromatography and radio high-performance liquid chromatography after conversion to p-bromophenacyl ester derivatives. Although delta 24-THCA was not converted to 24-OH-THCA, (25R)-THCA and (25S)-THCA were transformed to (24R,25R)-24-OH-THCA and (24R,25S)-24-OH-THCA, respectively. These results strongly suggest that 24-OH-THCA was transformed via direct hydroxylation of the saturated side chain of THCA, not via hydration to an alpha, beta-unsaturated acid, delta 24-THCA, in B. orientalis.

  12. Estrogenic and progestational activity of 7alpha-methyl-19-nortestosterone, a synthetic androgen.

    PubMed

    Beri, R; Kumar, N; Savage, T; Benalcazar, L; Sundaram, K

    1998-11-01

    Synthetic androgens exhibit estrogenic/antiestrogenic and progestational activities in addition to their androgenic effects. To investigate the pharmacological action of the synthetic androgen, 7alpha-methyl-19-nortestosterone (MENT), we examined its action in female rodents. The criteria employed for estrogenic/antiestrogenic effects were, uterine weight increase, vaginal cornification, induction of progesterone receptors (PR) synthesis and stimulation of peroxidase activity in the uteri of ovariectomized rats and mice. MENT increased uterine weight in a dose dependent manner, but did not cause vaginal cornification or stimulate PR synthesis in the uterus. The uterotropic activity of MENT was 200-fold lower than that of estradiol. Estrogen receptor (ER) bound [3H]-E2 was displaced by E2 and MENT with ED50 values of 70 pg and 250 ng, respectively, a 3,500 fold difference in their binding affinity. The low binding of MENT to ER, in contrast to its relatively high uterotropic action, suggested that receptors other than ER may be involved in its action on the uterus. The progestational activity of MENT in immature rabbits using the McPhail index assay was comparable to that of progesterone. Binding affinities of MENT and progesterone to PR were also comparable. However, the action of MENT on the uterus does not seem to be a progestational effect since mifepristone, an antiprogestin, had no effect on MENT-induced uterine growth. Specific androgen receptors (AR) in uterine cytosol were demonstrated. The involvement of AR in MENT action was confirmed by using an antiandrogen (flutamide) and an antiestrogen (ICI-182) in ovariectomized mice. Although MENT did not block the uterotropic effect of E2, it inhibited the E2-induced cornification of vaginal epithelium, induction of uterine PR synthesis and increase in uterine peroxidase activity in ovariectomized rats. The antiestrogenic effect of MENT was also blocked by flutamide. These results suggest that the uterotropic and

  13. Pharmacokinetics of 7 alpha-methyl-19-nortestosterone in men and cynomolgus monkeys.

    PubMed

    Kumar, N; Suvisaari, J; Tsong, Y Y; Aguillaume, C; Bardin, C W; Lähteenmaki, P; Sundaram, K

    1997-01-01

    Testosterone and its esters are widely used for androgen replacement therapy. In the prostate, testosterone ins 5 alpha-reduced to dihydrotestosterone (DHT), which leads to an amplification of its stimulatory activity in this and other tissues that have significant 5 alpha-reductase activity. While this amplification is essential during fetal development, it has potentially undesirable consequences during adult life. 7 alpha-Methyl-19-nortestosterone (MENT) is a potent synthetic androgen that does not undergo 5 alpha reduction and is therefore being investigated for long-term clinical use because it is expected to be less stimulatory to the prostate. Since we anticipate using MENT acetate (MENT Ac) rather than MENT as the form of this androgen in humans, the bioavailability of MENT following the administration of MENT and MENT Ac was investigated in cynomolgus monkeys. Equimolar concentrations of MENT or MENT Ac were administered as a continuous subcutaneous infusion via Alzet osmotic pumps. Serum MENT levels were measured by radioimmunoassay (RIA) in blood samples collected daily for 4 days during steady state. The serum MENT levels were not significantly different in the two groups (11.3 +/- 1.6 vs. 13.1 +/- 1.2 nmol/L). This suggested that MENT Ac was rapidly converted to MENT in circulation. The hydrolysis of MENT Ac to MENT was confirmed by the in vitro incubation of MENT Ac with blood or plasma and the demonstration of MENT in products following separation by high-performance liquid chromatography (HPLC). Following the demonstration of the safety of MENT Ac in subchronic toxicity studies in rats and rabbits, a pharmacokinetic study was performed in men. In normal men, a single intravenous bolus of 500 micrograms of MENT led to peak serum MENT levels at 3 minutes after dosing (when the first samples were collected), followed by an exponential decline, reaching undetectable levels by 180 minutes. The average terminal half-life and the metabolic clearance rate

  14. [Optimization of hydroxylating DHEA to 7alpha,15alpha-diOH-DHEA by compound mutation and fermentation optimization].

    PubMed

    Li, Chuanpeng; Li, Hui; Wu, Yan; Li, Heng; Zhang, Rujin; Zhang, Zhengbin; Shi, Jinsong; Xu, Zhenghong

    2014-01-01

    Combined with method of ketoconazole resistance screening, a 7alpha,15alpha-diOH-DHEA high-producing mutant Colletotrichum lini ST-1 was obtained by compound mutation of NTG and low energy N+ ion beam implantation. With the substrate concentration of 10 g/L DHEA, the molar yield of 7alpha,15alpha-diOH-DHEA reached 34.2%, increased by 46.2% than that of the original strain. Then we optimized the medium. First, Plackett-Burman design was used to evaluate the effects of medium components on molar yield of the product. Results show that glucose, yeast extract and MgSO4 x 7H2O were the important parameters for the biotransformation process. Subsequently, the path of steepest ascent was used to approach the optimal levels. To obtain the optimal levels, central composite design and response surface analysis were carried out. The optimal medium was as follows (g/L): glucose 26.34, yeast extract 12.15, corn flour 3.00, FeSO4 x 7H2O 0.015, MgSO4 x 7H2O 0.14, KH2PO4 0.90. Under the optimal conditions, the molar yield of 7alpha,15alpha-diOH-DHEA reached 49.3%, which was 44.2% higher than that of using the medium before optimization.

  15. 7Alpha-methyl-19-nortestosterone maintains sexual behavior and mood in hypogonadal men.

    PubMed

    Anderson, R A; Martin, C W; Kung, A W; Everington, D; Pun, T C; Tan, K C; Bancroft, J; Sundaram, K; Moo-Young, A J; Baird, D T

    1999-10-01

    The synthetic steroid 7alpha-methyl-19-nortestosterone (MENT) is a potent androgen that is resistant to 5alpha-reductase. It thus has decreased activity at the prostate and may have advantages over testosterone-based regimens in long term treatment or as part of a male contraceptive. Administration to eugonadal men results in suppression of gonadotropins, but its ability to support androgen-dependent behavior has not been investigated. For sustained release administration, MENT acetate was used, because its diffusion characteristics were more suitable for use in implants. However, upon release the acetate is rapidly hydrolyzed, and MENT is the biologically active moiety in circulation. We studied the effects of MENT on sexual interest and activity, spontaneous erection, and mood states in comparison with testosterone enanthate (TE) in 20 Caucasian and Chinese hypogonadal men recruited in Edinburgh and Hong Kong (n = 10 in each center). Outcomes were measured using a combination of daily diaries, semistructured interviews, and questionnaires. Nocturnal penile tumescence (NPT) was also recorded in the Edinburgh group. After withdrawal of androgen replacement treatment (wash-out phase) for a minimum of 6 weeks, subjects were randomized to two groups in a cross-over design. Drug treatment regimens were of 6-week duration and consisted of two implants, each containing 115 mg MENT acetate, inserted s.c. into the upper arm and removed after 6 weeks and two injections of TE (200 mg, i.m.) 3 weeks apart. MENT treatment resulted in stable plasma MENT concentrations of 1.4 +/- 0.1 nmol/L after 3 weeks and 1.3 +/- 0.1 nmol/L after 6 weeks (mean +/- SEM; all men). Nadir testosterone concentrations were 3.6 +/- 0.6 nmol/L at the end of the wash-out phase and 9.4 +/- 0.6 nmol/L 3 weeks after each injection. There were no differences in hormone concentrations between centers. There were no adverse toxicological effects. There were only minor differences between the two treatments

  16. Adenovirus-mediated transfer of a gene encoding cholesterol 7 alpha-hydroxylase into hamsters increases hepatic enzyme activity and reduces plasma total and low density lipoprotein cholesterol.

    PubMed Central

    Spady, D K; Cuthbert, J A; Willard, M N; Meidell, R S

    1995-01-01

    Clinical interventions that accelerate conversion of cholesterol to bile acids reduce circulating low density lipoprotein (LDL) cholesterol concentrations. The initial and rate-limiting step in the bile acid biosynthetic pathway is catalyzed by hepatic cholesterol 7 alpha-hydroxylase. To examine the effects of transient primary overexpression of this enzyme on sterol metabolism and lipoprotein transport, we constructed a recombinant adenovirus in which a cDNA encoding rat 7 alpha-hydroxylase is expressed from the human cytomegalovirus immediate-early promoter (AdCMV7 alpha). Syrian hamsters administered AdCMV7 alpha intravenously accumulated transgene-specific mRNA in the liver and demonstrated a dose-dependent increase in hepatic microsomal 7 alpha-hydroxylase activity. The increased conversion of cholesterol to bile acids resulted in a compensatory increase in hepatic cholesterol synthesis. In addition, overexpression of 7 alpha-hydroxylase reduced the rate of LDL cholesterol entry into the plasma space and, in animals maintained on a Western-type diet, restored hepatic LDL receptor expression. As a consequence, plasma LDL concentrations fell by approximately 60% in animals maintained on control diet and by approximately 75% in animals consuming a Western-type diet. Plasma high density lipoprotein cholesterol levels were reduced to a lesser degree. These results demonstrate that transient upregulation of bile acid synthesis by direct transfer of a 7 alpha-hydroxylase gene favorably alters circulating lipoprotein profiles and suggest one potential molecular target for genetic strategies aimed at reducing cardiovascular risk. Images PMID:7635963

  17. [Synthesis of 4-substituted 17 beta-hydroxy-7 alpha-methyl-4-estren-3-one and their 17-acetates as antifertility compounds].

    PubMed

    Ma, J B; Li, Z S

    1990-01-01

    Guided by antiprogestational screening results, 4-substituted(4-OH, 4-Cl, 4-Br, and 4-OMe) 17 beta-hydroxy-7 alpha-methyl-4-estren-3-one and their 17-acetate were synthesized via 4 beta, 5 beta-epoxy-17 beta-hydroxy-7 alpha-methylestran-3-one from 17 beta-hydroxy-7 alpha-methyl-4-estren-3-one. Some of these compounds possess strong affinity to human decidual progesterone receptor, inhibit in vitro the growth of decidual cells, and prevent the implantation in rats at the dose of 1 mumol/kg.

  18. Synthesis and biochemical studies of 7 alpha-substituted androsta-1,4-diene-3,17-diones as enzyme-activated irreversible inhibitors of aromatase.

    PubMed

    Ebrahimian, S; Chen, H H; Brueggemeier, R W

    1993-09-01

    Several 7 alpha-thiosubstituted derivatives of androstenedione have demonstrated effective inhibition of aromatase, the cytochrome P450 enzyme complex responsible for the biosynthesis of estrogens. Introduction of an additional double bond in the A ring resulted in 7 alpha-(4'-amino)phenylthioandrosta-1,4-diene-3,17-dione (7 alpha-APTADD), a potent inhibitor that inactivated aromatase by an enzyme-catalyzed process. Additional 7 alpha-thiosubstituted androsta-1,4-diene-3,17-dione derivatives were designed to further examine enzyme-catalyzed inactivation. Two halogenated and one unsubstituted 7 alpha-phenylthioandrosta-1,4-diene-3,17-diones were synthesized via an acid-catalyzed conjugate Michael addition of substituted thiophenols with androsta-1,4,6-triene-3,17-dione. Two 7 alpha-naphthylthioandrosta-1,4-diene-3,17-diones were synthesized via either acid-catalyzed or based-catalyzed conjugate Michael addition of substituted thionaphthols with androsta-1,4,6-triene-3,17-dione. These agents were evaluated for aromatase inhibitory activity in the human placental microsomal preparation. Under initial velocity assay conditions of low product formation, the inhibitors demonstrated potent inhibition of aromatase, with apparent Ki's ranging from 12 to 27 nM. Furthermore, these compounds produced time-dependent, first-order inactivation of aromatase in the presence of NADPH, whereas no aromatase inactivation was observed in the absence of NADPH. This enzyme-activated irreversible inhibition, also referred to as mechanism-based inhibition, can be prevented by the substrate androstenedione. Thus, the apparent Ki values for these inhibitors are consistent with earlier studies on 7 alpha-substituted competitive inhibitors that indicate bulky substituents can be accommodated at the 7 alpha-position.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Regulation of diurnal variation of cholesterol 7alpha-hydroxylase (CYP7A1) activity in healthy subjects.

    PubMed

    Kovár, J; Lenícek, M; Zimolová, M; Vítek, L; Jirsa, M; Pitha, J

    2010-01-01

    Cholesterol 7alpha-hydroxylase (CYP7A1), the key regulatory enzyme of bile acid synthesis, displays a pronounced diurnal variation. To better understand the regulation of CYP7A1 activity, three day-long examinations were carried out in 12 healthy men. The concentrations of 7alpha-hydroxycholest-4-en-3-one (C4), a surrogate marker of CYP7A1 activity, bile acids (BA), insulin, glucose, nonesterified fatty acids, triglycerides, and cholesterol were measured in serum in 90-min intervals from 7 AM till 10 PM. To lower and to increase BA concentration during the study, the subjects received cholestyramine and chenodeoxycholic acid (CDCA), respectively, in two examinations. No drug was used in the control examination. There was a pronounced diurnal variation of C4 concentration with a peak around 1 PM in most of the subjects. The area under the curve (AUC) of C4 concentration was five times higher and three times lower when subjects were treated with cholestyramine and CDCA, respectively. No relationship was found between AUC of C4 and AUC of BA concentration, but AUC of C4 correlated positively with that of insulin. Moreover, short-term treatment with cholestyramine resulted in about 10 % suppression of glycemia throughout the day. Our results suggest that insulin is involved in the regulation of diurnal variation of CYP7A1 activity in humans.

  20. Dietary cholesterol fails to stimulate the human cholesterol 7alpha-hydroxylase gene (CYP7A1) in transgenic mice.

    PubMed

    Agellon, Luis B; Drover, Victor A B; Cheema, Sukhinder K; Gbaguidi, G Franck; Walsh, Annemarie

    2002-06-07

    Dietary cholesterol has been shown to have a stimulatory effect on the murine cholesterol 7alpha-hydroxylase gene (Cyp7a1), but its effect on human cholesterol 7alpha-hydroxylase gene (CYP7A1) expression in vivo is not known. A transgenic mouse strain harboring the human CYP7A1 gene and homozygous for the disrupted murine Cyp7a1 gene was created. Cholesterol feeding increased the expression of the endogenous modified Cyp7a1 allele but failed to stimulate the human CYP7A1 transgene. In transfected hepatoma cells, 25-hydroxycholesterol increased murine Cyp7a1 gene promoter activity, whereas the human CYP7A1 gene promoter was unresponsive. Electrophoretic mobility shift assays demonstrated the interaction of the liver X receptor alpha (LXRalpha): retinoid X receptor (RXR) heterodimer, a transcription factor complex that is activated by oxysterols, with the murine Cyp7a1 gene promoter, whereas no binding to the human CYP7A1 gene promoter was detected. The results demonstrate that the human CYP7A1 gene is not stimulated by dietary cholesterol in the intact animal, and this is attributable to the inability of the CYP7A1 gene promoter to interact with LXRalpha:RXR.

  1. Distribution, metabolism and excretion of a synthetic androgen 7alpha-methyl-19-nortestosterone, a potential male-contraceptive.

    PubMed

    Prasad, Pramod Vishwanath; Arumugam, Ramamani; Willman, Mark; Ge, Ren-Shan; Sitruk-Ware, Regine; Kumar, Narender

    2009-01-01

    A synthetic androgen 7alpha-Methyl-19-nortestosterone (MENT) has a potential for therapeutic use in 'androgen replacement therapy' for hypogonadal men or as a hormonal male-contraceptive in normal men. Its tissue distribution, excretion and metabolic enzyme(s) have not been reported. Therefore, the present study tested the distribution and excretion of MENT in Sprague-Dawley rats castrated 24h prior to the injection of tritium-labeled MENT ((3)H-MENT). Rats were euthanized at different time intervals after dosing, and the amount of radioactivity in various tissues/organs was measured following combustion in a Packard oxidizer. The radioactivity (% injected dose) was highest in the duodenal contents in the first 30min of injection. Specific uptake of the steroid was observed in target tissues such as ventral prostate and seminal vesicles at 6h, while in other tissues radioactivity equilibrated with blood. Liver and duodenum maintained high radioactivity throughout, as these organs were actively involved in the metabolism and excretion of most drugs. The excretion of (3)H-MENT was investigated after subcutaneous injection of (3)H-MENT into male rats housed in metabolic cages. Urine and feces were collected at different time intervals (up to 72h) following injection. Results showed that the radioactivity was excreted via feces and urine in equal amounts by 30h. Aiming to identify enzyme(s) involved in the MENT metabolism, we performed in vitro metabolism of (3)H-MENT using rat and human liver microsomes, cytosol and recombinant cytochrome P(450) (CYP) isozymes. The metabolites were separated by thin-layer chromatography (TLC). Three putative metabolites (in accordance with the report of Agarwal and Monder [Agarwal AK, Monder C. In vitro metabolism of 7alpha-methyl-19-nortestosterone by rat liver, prostate, and epididymis. Endocrinology 1988;123:2187-93]), [i] 3-hydroxylated MENT by both rat and human liver cytosol; [ii] 16alpha-hydroxylated MENT (a polar metabolite

  2. 7alpha-Hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts by means of the dopaminergic system.

    PubMed

    Matsunaga, Masahiro; Ukena, Kazuyoshi; Baulieu, Etienne-Emile; Tsutsui, Kazuyoshi

    2004-12-07

    It is becoming clear that steroids can be synthesized de novo by the brain and other nervous systems. Such steroids are called neurosteroids, and de novo neurosteroidogenesis from cholesterol is a conserved property of vertebrate brains. In this study, we show that the newt brain actively produces 7alpha-hydroxypregnenolone, a previously undescribed amphibian neurosteroid that stimulates locomotor activity. 7alpha-hydroxypregnenolone was identified as a most abundant amphibian neurosteroid in the newt brain by using biochemical techniques combined with HPLC, TLC, and GC-MS analyses. The production of 7alpha-hydroxypregnenolone in the diencephalon and rhombencephalon was higher than that in the telencephalon and peripheral steroidogenic glands. In addition, 7alpha-hydroxypregnenolone synthesis in the brain showed marked changes during the annual breeding cycle, with a maximal level in the spring breeding period when locomotor activity of the newt increases. Behavioral analysis of newts in the nonbreeding period demonstrated that administration of this previously undescribed amphibian neurosteroid acutely increased locomotor activity. In vitro analysis further revealed that 7alpha-hydroxypregnenolone treatment resulted in a dose-dependent increase in the release of dopamine from cultured brain tissue of nonbreeding newts. The effect of this neurosteroid on locomotion also was abolished by dopamine D(2)-like receptor antagonists. These results indicate that 7alpha-hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts through the dopaminergic system. This study demonstrates a physiological function of 7alpha-hydroxypregnenolone that has not been described previously in any vertebrate class. This study also provides findings on the regulatory mechanism of locomotor activity from a unique standpoint.

  3. Cholesterol 7alpha-hydrolase (CYP7A1) c.-278A>C promoter polymorphism in gallstone disease patients.

    PubMed

    Juzyszyn, Zygmunt; Kurzawski, Mateusz; Lener, Agnieszka; Modrzejewski, Andrzej; Pawlik, Andrzej; Droździk, Marek

    2008-03-01

    There is growing evidence that gallstone formation may be genetically determined. Cholesterol 7alpha-hydrolase (CYP7A1) is an enzyme that catalyzes the first, rate-limiting reaction of cholesterol catabolic pathway. Recently, a common c.-278A>C polymorphism (rs3808607:G>T) has been described in CYP7A1 gene, associated with altered plasma lipid levels. The aim of this study was to verify the finding that CYP7A1 polymorphism may be associated with gallstone disease. Frequency and distribution of the studied alleles did not differ significantly between the patients (-278C; minor allele frequency: 0.45) and the controls (0.48). No significant gender-related differences of allele frequencies or distribution were noted. We conclude that CYP7A1 promoter polymorphism is not a valuable marker of gallstone disease susceptibility in a Polish population.

  4. Cloning of a Streptomyces clavuligerus DNA fragment encoding the cephalosporin 7 alpha-hydroxylase and its expression in Streptomyces lividans.

    PubMed Central

    Xiao, X; Hintermann, G; Häusler, A; Barker, P J; Foor, F; Demain, A L; Piret, J

    1993-01-01

    A 26-mer DNA probe was designed from N-terminal sequence data for the cephalosporin 7 alpha-hydroxylase (CH) of Streptomyces clavuligerus NRRL 3585 and used to screen a DNA library from this organism. The library was constructed in the lambda GEM-11 phage system. After plaque purification and reprobing, positive recombinant phages were chosen for further analysis. Characterization of the cloned DNA by restriction mapping and Southern hybridization showed that a 1.5-kb SalI fragment hybridized to the probe. Polymerase chain reaction assays using this fragment as a template and the probe as a primer indicated that the fragment carries the entire putative CH gene (cmcI). This was confirmed through the expression of CH enzymatic activity when the fragment was introduced into Streptomyces lividans. A putative beta-lactamase activity was detected in S. lividans. Images PMID:8431021

  5. Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous (4- sup 14 C)cholesterol as substrate

    SciTech Connect

    Hylemon, P.B.; Studer, E.J.; Pandak, W.M.; Heuman, D.M.; Vlahcevic, Z.R.; Chiang, J.Y. )

    1989-11-01

    The HPLC-spectrophotometric method for measuring cholesterol 7 alpha-hydroxylase activity was modified by using a C-18 reverse-phase column to separate 7 alpha-hydroxy-4-cholesten-3-one and 4-cholesten-3-one and by adding 7 beta-hydroxycholesterol to each reaction mixture as an internal recovery standard. With this method, we were able to simultaneously measure cholesterol 7 alpha-hydroxylase activity using endogenous cholesterol and exogenous (4-{sup 14}C)cholesterol as substrate. Rat liver cytosol differentially stimulated (286%) the 7 alpha-hydroxylation of exogenous (4-{sup 14}C)-cholesterol. In contrast, total cholesterol 7 alpha-hydroxylase activity was stimulated only 35% by cytosol. This method should prove useful for studying mechanisms of cholesterol delivery to cholesterol 7 alpha-hydroxylase.

  6. 7alpha-methyl-19-nortestosterone, a synthetic androgen with high potency: structure-activity comparisons with other androgens.

    PubMed

    Kumar, N; Crozat, A; Li, F; Catterall, J F; Bardin, C W; Sundaram, K

    1999-12-31

    CNNT. There was a good correlation between bioactivity and binding affinity to AR for the 7alpha-substituted androgens compared to T. In contrast, relative to their binding affinity to AR, the androgenic potency of DHT and 19-NT was lower compared to T. The reason for the lower in vivo androgenic activity of 19-NT is attributable to its enzymatic conversion to 5alpha-reduced-19-NT in the prostate. In the case of DHT, the lower bioactivity could be attributed to its faster metabolic clearance rate relative to T. The correlation was further investigated in vitro by co-transfection of rat ARcDNA expression plasmid and a reporter plasmid encoding the chloramphenicol acetyl transferase (CAT) gene driven by an androgen inducible promoter into CV-1 cells. All the androgens led to a dose-dependent increase in the CAT activity. MENT was found to be the most potent followed by DHT, 19-NT, T, and CNNT. The specificity of the androgenic response was confirmed by its inhibition with hydroxyflutamide, an antiandrogen. Thus, there was a good correlation between binding affinity and in vitro bioactivity in the transient transfection assay for the androgens. This suggests that the in vivo bioactivity of androgens could be influenced not only by binding affinity to receptors but also by factors such as absorption, binding to serum proteins and metabolism. However, the high potency of MENT is primarily related to its higher affinity to AR.

  7. Role of cholesterol 7alpha-hydroxylase (CYP7A1) in nutrigenetics and pharmacogenetics of cholesterol lowering.

    PubMed

    Hubacek, Jaroslav A; Bobkova, Dagmar

    2006-01-01

    The relationship between dietary composition/cholesterol-lowering therapy and final plasma lipid levels is to some extent genetically determined. It is clear that these responses are under polygenic control, with multiple variants in many genes participating in the total effect (and with each gene contributing a relatively small effect). Using different experimental approaches, several candidate genes have been analyzed to date.Interesting and consistent results have been published recently regarding the A-204C promoter variant in the cholesterol 7alpha-hydroxylase (CYP7A1) gene. CYP7A1 is a rate-limiting enzyme in bile acid synthesis and therefore plays an important role in maintaining cholesterol homeostasis. CYP7A1-204CC homozygotes have the greatest decrease in total cholesterol level in response to dietary changes in different types of dietary intervention studies. In contrast, one study has reported that the effect of statins in lowering low-density lipoprotein (LDL)-cholesterol levels was slightly greater in -204AA homozygotes. The CYP7A1 A-204C variant accounts for a significant proportion of the genetic predisposition of the response of plasma cholesterol levels.

  8. Species-specific mechanisms for cholesterol 7alpha-hydroxylase (CYP7A1) regulation by drugs and bile acids.

    PubMed

    Handschin, Christoph; Gnerre, Carmela; Fraser, David J; Martinez-Jimenez, Celia; Jover, Ramiro; Meyer, Urs A

    2005-02-01

    The gene encoding cholesterol 7alpha-hydroxylase (CYP7A1) is tightly regulated in order to control intrahepatic cholesterol and bile acid levels. Ligands of the xenobiotic-sensing pregnane X receptor inhibit CYP7A1 expression. To retrace the evolution of the molecular mechanisms underlying CYP7A1 inhibition, we used a chicken hepatoma cell system that retains the ability to be induced by phenobarbital and other drugs. Whereas bile acids regulate CYP7A1 via small heterodimer partner and liver receptor homolog-1, mRNA expression of these nuclear receptors is unchanged by xenobiotics. Instead, drugs repress chicken hepatic nuclear factor 4alpha (HNF4alpha) transcript levels concomitant with a reduction in CYP7A1 expression. Importantly, no reduction of HNF4alpha levels is found in mouse liver in vivo and in human primary hepatocyte cultures, respectively. Thus, besides the importance of HNF4alpha in CYP7A1 regulation in all species, birds and mammals use different signaling pathways to adjust CYP7A1 levels after exposure to xenobiotics.

  9. Determination of 7alpha-OH cholesterol by LC-MS/MS: Application in assessing the activity of CYP7A1 in cholestatic minipigs

    USDA-ARS?s Scientific Manuscript database

    An LC-MS/MS method was developed and validated to determine 7alpha-OH cholesterol in liver microsome. This method was convenient and fast with high specificity and sensitivity. Briefly, a gradient elution was performed on a Synergi polar-C18 column (50 x 4.6mm i.d., 3microm). The mobile phase (cons...

  10. Bile alcohol metabolism in man. Conversion of 5beta-cholestane-3alpha, 7alpha,12alpha, 25-tetrol to cholic acid.

    PubMed Central

    Salen, G; Shefer, S; Setoguchi, T; Mosbach, E H

    1975-01-01

    To study the role of C25-HYDROXY BILE ALCOHOLS AS PRECURSORS OF CHOlic acid, [G-3-H]5beta-cholestane-3alpha,7alpha12alpha,25-tetrol was administered intravenously to two subjects with cerebrotendinous xanthomatosis (CTX) and two normal individuals. One day after pulse labeling, radioactivity was present in the cholic acid isolated from the bile and feces of the subjects with CTX and the bile of the normal individuals. In the two normal subjects, the sp act decay curves of [G-3-H]-cholic acid were exponential, and no traces of [G-3-H]-5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol were detected. In contrast, appreciable quantities of labeled 5beta-cholestane-3alpha,-7aopha,12alpha,25-tetrol were present in the bile and feces of the CTX subjects. The sp act vs. time curves of fecal [G-3-H]5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol and [G-3-H]-cholic acid showed a precursor-product relationship. Although these results suggest that 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol may be a precursor of cholic acid in man, the possibility that C26-hydroxy intermediates represent the normal pathway can not be excluded. PMID:1141434

  11. Synthesis of 17 beta-hydroxyandrost-4-en-3-one-7 alpha-(biotinyl-6-N-hexylamide), a conjugate useful for affinity chromatography and for testosterone immunoassays.

    PubMed

    Luppa, P; Hauck, S; Schwab, I; Birkmayer, C; Hauptmann, H

    1996-01-01

    We describe the synthesis of 17 beta-hydroxyandrost-4-en-3-one-7 alpha-(biotinyl-6-N-hexylamide) from 17 beta-hydroxyandrost-4-en-3-one (testosterone) via copper-catalyzed 1,6 Michael addition of a 6-(tertbutyldimethylsilyloxyhexyl) chain to 6-dehydrotestosterone 17 beta-acetate. After chromatographic separation of the 7 alpha-isomer from the alpha / beta mixture and cleavage of the silyl ether, the alcohol was oxidized to the 6-hexanal side chain and then subjected to reductive amination. The resulting primary amine is easily biotinylated using biotinyl-N-hydroxysuccinimide ester. The overall yield for the epimeric 7 alpha-end product was 30%. The absolute configurations of the epimers were investigated by 1H NMR studies by the nuclear Overhauser effect. We introduced a biotin label to the testosterone molecule at ring position 7 in compliance with Landsteiner's principle, which states that antibody specificity is directed primarily at that portion of the hapten furthest from the functional group linking it to the carrier protein. Thus, this negligible alteration in comparison to the structure of the respective testosterone hapten used to elicit antibodies offers the feasibility of applying the testosterone derivative as an optimal immunoadsorbent in affinity chromatography. The 7 alpha-biotinylated testosterone was used to obtain active antitestosterone antibodies from a specific antiserum by affinity chromatography. This was achieved by attaching the biotinylated testosterone to agarose-coupled streptavidin beads. Accordingly, a 3H-testosterone-binding test demonstrated a 20-fold increase in affinity of the purified antibody to the steroid compared to the original antiserum, and a recovery of > 80% could be obtained. The antitestosterone antibody, obtained by that method, is an effective component for use in a competitive immunoassay for testosterone in human sera. An assay configuration is conceivable with the same 7 alpha-biotinylated testosterone employed as

  12. The biological activity of 7 alpha-methyl-19-nortestosterone is not amplified in male reproductive tract as is that of testosterone.

    PubMed

    Kumar, N; Didolkar, A K; Monder, C; Bardin, C W; Sundaram, K

    1992-06-01

    Based on the premise that testosterone, but not 7 alpha-methyl-androgens, is reduced at the 5 alpha-position in the prostate and seminal vesicles, the differential bioactivities of these androgens were investigated in castrated rats. The ability of 7 alpha-methyl-19-nortestosterone acetate (MENT) to increase the weights of ventral prostate and seminal vesicles of castrated rats was four times higher than that of testosterone, while its effect on the weights of bulbocavernosus plus levator ani muscles (muscle), was 10 times that of testosterone. MENT was also approximately 12 times more potent than testosterone in the suppression of serum gonadotropin levels. A dose of testosterone that maintains serum gonadotropin levels and muscle mass also maintains prostate and seminal vesicle weights in castrated rats. By contrast, a dose of MENT that maintains muscle and gonadotropins does not maintain prostate and seminal vesicles. The action of other 7 alpha-methylated androgens were similar to that of MENT. The importance of 5 alpha reductase in the differential action of testosterone and MENT on prostate was confirmed by using a 5 alpha-reductase inhibitor. The activity of testosterone was significantly suppressed in the ventral prostate and seminal vesicles but not on muscle by the 5 alpha-reductase inhibitor (N,N-diethyl-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta-carboxamide). The enzyme inhibitor, however, had no influence on the activity of MENT on either tissue. In contrast, cyproterone acetate, an antiandrogen that competitively binds to the androgen receptors, inhibited the action of MENT and of testosterone on the prostate as well as on the muscle. In conclusion, these observations show that 7 alpha-methylated androgens can maintain muscle mass and normal gonadotropin levels in androgen deficient rats without hyperstimulating the prostate. These findings suggest that 7 alpha-methylated androgens may offer some health benefits to men who require androgen treatment.

  13. Neonatal dietary cholesterol and alleles of cholesterol 7-alpha hydroxylase affect piglet cerebrum weight, cholesterol concentration, and behavior.

    PubMed

    Pond, Wilson G; Mersmann, Harry J; Su, Dairong; McGlone, John J; Wheeler, Matthew B; Smith, E O'Brian

    2008-02-01

    This experiment was designed to test the effect of polymorphism in the cholesterol 7-alpha hydroxylase (CYP7) gene locus and dietary cholesterol (C) on cerebrum C in neonatal pigs fed sow's milk formulas. Thirty-six pigs (18 male and 18 female) genetically selected for high (HG) or low (LG) plasma total C were weaned at 24-36 h after birth and assigned in a 2 x 2 x 2 factorial arrangement of treatments with 2 diets (0 or 0.5% C), 2 sexes, and 2 genotypes (HG and LG). Individually housed pigs consumed diets ad libitum for 42 d. Open-field behavior was tested at wk 2 and 4. All pigs were killed at 42 d of age, the cerebrum was weighed, and C content and concentration measured. All data were analyzed by general linear model ANOVA. Cerebrum weight was greater in HG than LG pigs (P < 0.03) but was not affected by diet or sex. Pigs fed C tended to have a higher cerebrum C concentration than those deprived (P = 0.12). At 2 wk, LG pigs explored a novel open-field environment less often (P < 0.001) than did HG pigs. At 4 wk, some LG pigs explored the open field but fewer (P < 0.001) vs. HG pigs retreated back to the safe area. There were no genotype x diet, genotype x sex, or diet x sex interactions affecting cerebrum weight, or C content or concentration. Polymorphism in the CYP7 gene locus affected cerebrum weight and behavior and dietary C tended to increase cerebrum C concentration in neonatal pigs. These findings in neonatal pigs have considerable potential importance in human infant nutrition and behavioral development.

  14. 7alpha-methyl-19-nortestosterone (MENT) vs testosterone in combination with etonogestrel implants for spermatogenic suppression in healthy men.

    PubMed

    Walton, Melanie J; Kumar, Narender; Baird, David T; Ludlow, Helen; Anderson, Richard A

    2007-01-01

    Testosterone with a progestogen can suppress spermatogenesis for contraception. The synthetic androgen 7alpha-methyl-19-nortestosterone (MENT) may offer advantages because it is resistant to 5alpha-reduction and is therefore less active at the prostate. This study aimed to investigate MENT implants in combination with etonogestrel on spermatogenesis, gonadotropins, and androgen-dependent tissues in comparison with a testosterone/etonogestrel regimen. Healthy men (n = 29) were recruited and randomized to receive 2 etonogestrel implants with either 600-mg testosterone pellets repeated every 12 weeks or 2 MENT implants for up to 48 weeks. Testosterone concentrations in the testosterone group remained in the normal range. Subjects with 2 MENT implants showed peak MENT levels at 4 weeks with testosterone concentrations of 2 nmol/L. Sperm concentrations fell rapidly to less than 1 x 10(6)/mL at 12 weeks in 8 of 10 subjects in the MENT group and 13 of 16 subjects in the testosterone group with equally suppressed gonadotropins. Thereafter, suppression was not maintained in the MENT group, and 6 men noted loss of libido. Fourteen men completed 48 weeks of testosterone treatment, and all became azoospermic. Hemoglobin concentrations rose, and high density lipoprotein-cholesterol (HDL-C) fell in both groups. The MENT group showed a fall in prostate-specific antigen with no change in bone mass. MENT with a progestogen can achieve rapid suppression of spermatogenesis similar to testosterone, but this promising result was not sustained due to a decline in MENT release from the implants. This dose of testosterone, compared with previous studies using a lower dose with a higher dose of etonogestrel, had nonreproductive side effects without any increase in spermatogenic suppression. These data indicate the importance of the doses of progestogen and testosterone for optimum spermatogenic suppression while minimizing side effects.

  15. Gonadotrophin and testosterone suppression by 7alpha-methyl-19-nortestosterone acetate administered by subdermal implant to healthy men.

    PubMed

    Noé, G; Suvisaari, J; Martin, C; Moo-Young, A J; Sundaram, K; Saleh, S I; Quintero, E; Croxatto, H B; Lähteenmäki, P

    1999-09-01

    The synthetic androgen 7alpha-methyl-19-nortestosterone (MENT) is a potent suppressor of gonadotrophin that has several advantages for long term administration to normal or hypoandrogenic men. The aim of this study was to examine MENT serum concentrations following subdermal insertion of MENT acetate (MENT Ac) implants and their effects on gonadotrophins, testosterone, dihydrotestosterone (DHT), sex hormone-binding globulin, prostate specific antigen and insulin-like growth factor-1 serum concentrations in normal men. A total of 45 healthy men were recruited at three clinics. Each subject received one, two or four implants for 28 days. Serum samples were obtained before insertion and on days 8, 15, 22, 29, 36 and 43 after implant insertion. The average daily dose delivered in vivo by one implant was approximately 500 microg. One, two or four MENT Ac implants produced dose dependent and sustained serum MENT concentrations for the entire duration of treatment of 0.7 +/- 0.1, 1.2 +/- 0.1 and 2.0 +/- 0.1 nmol/l respectively. This treatment induced a dose dependent decrease in gonadotrophin and androgen serum levels. Two and four implants induced maximal suppression that was maintained throughout treatment and was completely reversed after removal of the implants. The mean decreases were 93 +/- 1% for testosterone, 80 +/- 3% for DHT, 97 +/- 1% for luteinizing hormone and 95 +/- 1% for follicle stimulating hormone. No serious adverse reactions were reported by the volunteers and no consistent changes in clinical chemistry and haematology were found. These results indicate that MENT Ac implants are an efficient way of MENT administration and confirm the potent gonadotrophin and androgen suppressive effect of this drug.

  16. Genetic polymorphism of cholesterol 7alpha-hydroxylase (CYP7A1) and colorectal adenomas: Self Defense Forces Health Study.

    PubMed

    Tabata, Shinji; Yin, Guang; Ogawa, Shinsaku; Yamaguchi, Keizo; Mineshita, Masamichi; Kono, Suminori

    2006-05-01

    Bile acids have long been implicated in colorectal carcinogenesis, but epidemiological evidence is limited. Cholesterol 7alpha-hydroxylase (CYP7A1) is the rate-limiting enzyme producing bile acids from cholesterol. A recent case-control study showed a decreased risk of proximal colon cancer associated with the CC genotype of the CYP7A1 A-203C polymorphism. The present study examined the relationship between the CYP7A1 A-203C polymorphism and colorectal adenoma, which is a well-established precursor lesion of colorectal cancer. The study subjects comprised 446 cases of colorectal adenomas and 914 controls of normal total colonoscopy among men receiving a preretirement health examination at two hospitals of the Self Defense Forces (SDF). The CYP7A1 genotype was determined by the polymerase chain reaction-restriction fragment length polymorphism method. Statistical adjustment was made for age, hospital, rank in the SDF, smoking, alcohol use, body mass index, physical activity and parental history of colorectal cancer. The CYP7A1 polymorphism was not measurably related to the overall risk of colorectal adenomas. However, the CC genotype was associated with a decreased risk of proximal colon adenomas, but not of distal colon and rectal adenomas. Adjusted odds ratios of proximal colon adenomas (95% confidence intervals) for the AC and CC genotype versus AA genotype were 0.82 (0.54-1.24) and 0.56 (0.34-0.95), respectively. The findings add to evidence for the role of bile acids in colorectal carcinogenesis. The CC genotype of the CYP7A1 A-203C polymorphism probably renders lower activity of the enzyme synthesizing bile acids.

  17. 21 CFR 558.348 - Mibolerone.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... breeding purposes. (3) Limitations. Administer daily at least 30 days before expected initiation of heat... being used primarily for breeding purposes. Use orally in adult female dogs only. Federal law...

  18. 21 CFR 520.1430 - Mibolerone.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... for use. For the prevention of estrus (heat) in adult female dogs not intended primarily for breeding... being used primarily for breeding purposes. Use orally in adult female dogs only. Federal law...

  19. 21 CFR 520.1430 - Mibolerone.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS... for use. For the prevention of estrus (heat) in adult female dogs not intended primarily for breeding... being used primarily for breeding purposes. Use orally in adult female dogs only. Federal law...

  20. Mechanism of rifampicin and pregnane X receptor inhibition of human cholesterol 7 alpha-hydroxylase gene transcription.

    PubMed

    Li, Tiangang; Chiang, John Y L

    2005-01-01

    Bile acids, steroids, and drugs activate steroid and xenobiotic receptor pregnane X receptor (PXR; NR1I2), which induces human cytochrome P4503A4 (CYP3A4) in drug metabolism and cholesterol 7 alpha-hydroxylase (CYP7A1) in bile acid synthesis in the liver. Rifampicin, a human PXR agonist, inhibits bile acid synthesis and has been used to treat cholestatic diseases. The objective of this study is to elucidate the mechanism by which PXR inhibits CYP7A1 gene transcription. The mRNA expression levels of CYP7A1 and several nuclear receptors known to regulate the CYP7A1 gene were assayed in human primary hepatocytes by quantitative real-time PCR (Q-PCR). Rifampicin reduced CYP7A1 and small heterodimer partner (SHP; NR02B) mRNA expression suggesting that SHP was not involved in PXR inhibition of CYP7A1. Rifampicin inhibited CYP7A1 reporter activity and a PXR binding site was localized to the bile acid response element-I. Mammalian two-hybrid assays revealed that PXR interacted with hepatic nuclear factor 4 alpha (HNF4 alpha, NR2A1) and rifampicin was required. Coimmunoprecipitation assay confirmed PXR interaction with HNF4 alpha. PXR also interacted with peroxisome proliferator-activated receptor gamma coactivator (PGC-1 alpha), which interacted with HNF4 alpha and induced CYP7A1 gene transcription. Rifampicin enhanced PXR interaction with HNF4 alpha and reduced PGC-1 alpha interaction with HNF4 alpha. Chromatin immunoprecipitation assay showed that PXR, HNF4 alpha, and PGC-1 alpha bound to CYP7A1 chromatin, and rifampicin dissociated PGC-1 alpha from chromatin. These results suggest that activation of PXR by rifampicin promotes PXR interaction with HNF4 alpha and blocks PGC-1 alpha activation with HNF4 alpha and results in inhibition of CYP7A1 gene transcription. Rifampicin inhibition of bile acid synthesis may be a protective mechanism against drug and bile acid-induced cholestasis.

  1. Chenodeoxycholic acid synthesis in the hamster: a metabolic pathway via 3 beta, 7 alpha-dihydroxy-5-cholen-24-oic acid

    SciTech Connect

    Kulkarni, B.; Javitt, N.B.

    1982-11-01

    The quantitative significance of the metabolism of 3 beta, 7 alpha-dihydroxy-5-cholen-24-oic acid to chenodeoxycholic acid was evaluated in the hamster. A precursor-product relationship was established in this species by the finding that intravenous administration to an animal previously given cholesterol-4-14C caused a significant reduction in the specific activity of chenodeoxycholic acid. Administration of 12.9 mumole of the precursor was followed by a 10-fold increase in chenodeoxycholic acid excretion although the predominant excretory pathway was via biliary excretion as a monosulfate. The data indicate that synthesis of bile acid from cholesterol via the intermediate 3 beta, 7 alpha-dihydroxy-5-cholen-24-oic acid can be a quantitatively important pathway.

  2. Nicotine inhibits Fc epsilon RI-induced cysteinyl leukotrienes and cytokine production without affecting mast cell degranulation through alpha 7/alpha 9/alpha 10-nicotinic receptors.

    PubMed

    Mishra, Neerad C; Rir-sima-ah, Jules; Boyd, R Thomas; Singh, Shashi P; Gundavarapu, Sravanthi; Langley, Raymond J; Razani-Boroujerdi, Seddigheh; Sopori, Mohan L

    2010-07-01

    Smokers are less likely to develop some inflammatory and allergic diseases. In Brown-Norway rats, nicotine inhibits several parameters of allergic asthma, including the production of Th2 cytokines and the cysteinyl leukotriene LTC(4). Cysteinyl leukotrienes are primarily produced by mast cells, and these cells play a central role in allergic asthma. Mast cells express a high-affinity receptor for IgE (FcepsilonRI). Following its cross-linking, cells degranulate and release preformed inflammatory mediators (early phase) and synthesize and secrete cytokines/chemokines and leukotrienes (late phase). The mechanism by which nicotine modulates mast cell activation is unclear. Using alpha-bungarotoxin binding and quantitative PCR and PCR product sequencing, we showed that the rat mast/basophil cell line RBL-2H3 expresses nicotinic acetylcholine receptors (nAChRs) alpha7, alpha9, and alpha10; exposure to exceedingly low concentrations of nicotine (nanomolar), but not the biologically inactive metabolite cotinine, for > or = 8 h suppressed the late phase (leukotriene/cytokine production) but not degranulation (histamine and hexosaminidase release). These effects were unrelated to those of nicotine on intracellular free calcium concentration but were causally associated with the inhibition of cytosolic phospholipase A(2) activity and the PI3K/ERK/NF-kappaB pathway, including phosphorylation of Akt and ERK and nuclear translocation of NF-kappaB. The suppressive effect of nicotine on the late-phase response was blocked by the alpha7/alpha9-nAChR antagonists methyllycaconitine and alpha-bungarotoxin, as well as by small interfering RNA knockdown of alpha7-, alpha9-, or alpha10-nAChRs, suggesting a functional interaction between alpha7-, alpha9-, and alpha10-nAChRs that might explain the response of RBL cells to nanomolar concentrations of nicotine. This "hybrid" receptor might serve as a target for novel antiallergic/antiasthmatic therapies.

  3. Role of integrin alpha 4 beta 7/alpha 4 beta P in lymphocyte adherence to fibronectin and VCAM-1 and in homotypic cell clustering

    PubMed Central

    1992-01-01

    Integrins are heterodimeric cell surface proteins that mediate both cell-cell and cell-extracellular matrix interactions. We and others recently identified cDNAs encoding a novel integrin beta subunit, beta 7, in lymphocytes. We have now detected beta 7 mRNA in mouse TK-1 T lymphoma cells, which are known to express the putative Peyer's patch homing receptor alpha 4 beta P. We used an anti-peptide antiserum and a novel mAb against the beta 7 subunit to show that TK-1 cells express beta 7 as the only subunit associated with alpha 4. We conclude that beta 7 and beta P are identical. We also show that activated peripheral blood T cells express alpha 4 beta 7. We studied the function of alpha 4 beta 7/alpha 4 beta P in TK-1 cells, which do not express very late antigen (VLA)-4 (alpha 4 beta 1). Cells adhered to intact fibronectin and to a fibronectin fragment containing the CS-1 region, but not to a fragment containing the RGD sequence. Adhesion to fibronectin was inhibited by antibodies to alpha 4, suggesting that alpha 4 beta 7 is a fibronectin receptor. We confirmed that alpha 4 beta 7 binds to the CS- 1 region of fibronectin using affinity chromatography. TK-1 cell adhesion to the vascular cell adhesion molecule VCAM-1 was also inhibited by antibodies to alpha 4, implying that alpha 4 beta 7 also plays a role in the adherence of lymphocytes to endothelial cells. TK-1 cell binding to fibronectin and VCAM-1 is markedly increased by brief PMA stimulation. We also found that mAbs against alpha 4 and beta 7 induce homotypic clustering of TK-1 cells. Taken together these results suggest that alpha 4 beta 7/alpha 4 beta P recognizes some or all of the same widely distributed ligands recognized by VLA-4 (alpha 4 beta 1) and that the role of alpha 4 beta 7/alpha 4 beta P may not be restricted to lymphocyte homing. PMID:1372909

  4. A two-protein component 7 alpha-cephem-methoxylase encoded by two genes of the cephamycin C cluster converts cephalosporin C to 7-methoxycephalosporin C.

    PubMed Central

    Coque, J J; Enguita, F J; Martín, J F; Liras, P

    1995-01-01

    Two genes, cmcI and cmcJ, corresponding to open reading frames 7 and 8 (ORF7 and ORF8) of the cephamycin C cluster of Nocardia lactamdurans encode enzymes that convert cephalosporin C to 7-methoxycephalosporin C. Proteins P7 and P8 (the products of ORF7 and ORF8 expressed in Streptomyces lividans) introduce the methoxyl group at C-7 of the cephem nucleus. Efficient hydroxylation at C-7 and transfer of the methyl group from S-adenosylmethionine require both proteins P7 and P8, although P7 alone shows weak C-7 hydroxylase activity and strong cephalosporin-dependent NADH oxidase activity. Both P7 and P8 appear to be synthesized in a coordinated form by translational coupling of cmcI and cmcJ. Protein P7 contains domains that correspond to conserved sequences in cholesterol 7 alpha-monooxygenases and to the active center of O-methyltransferases by comparison with the crystal structure of catechol-O-methyltransferase. Protein P8 may act as a coupling protein for efficient hydroxylation at C-7 in a form similar to that of the two-component system of Pseudomonas putida p-hydroxyphenylacetate-3-hydroxylase. PMID:7721717

  5. Effect of chronic administration of 7alpha-methyl-19-nortestosterone on serum testosterone, number of spermatozoa and fertility in adult male bonnet monkeys (Macaca radiata).

    PubMed

    Ramachandra, S G; Ramesh, V; Krishnamurthy, H N; Kumar, N; Sundaram, K; Hardy, M P; Rao, A Jagannadha

    2002-08-01

    Hormonal approaches to male contraception that are based on the suppression of LH secretion require androgen replacement treatment to maintain sexual behaviour and secondary sexual characteristics. Androgen supplementation not only involves large and frequent doses of testosterone esters but also results in undesirable effects on the prostate gland. In an attempt to avoid such problems, a synthetic androgen, 7alpha-methyl-19-nortestosterone (MENT), which is much more potent than testosterone, has been developed. In the present study, MENT was administered at different doses (25, 50, 100, 300 and 1000 microg day(-1)) either alone or in combination with oestradiol via Silastic implants for a specified period to adult male bonnet monkeys (Macaca radiata). Blood and semen samples were collected at specific intervals and analysed for serum testosterone and seminal parameters, respectively. The results of the present study clearly indicate that administration of MENT at all doses tested results in suppression of the nocturnal surge of testosterone (by day 3), as well as a decrease in the number of spermatozoa (by day 45). Co-administration of oestradiol resulted in a reduction in the dose of MENT required to suppress the nocturnal surge. None of the male bonnet monkeys treated with MENT were able to impregnate females, clearly demonstrating the efficacy of MENT in blocking fertility in male bonnet monkeys.

  6. Prostate-sparing effects in primates of the potent androgen 7alpha-methyl-19-nortestosterone: a potential alternative to testosterone for androgen replacement and male contraception.

    PubMed

    Cummings, D E; Kumar, N; Bardin, C W; Sundaram, K; Bremner, W J

    1998-12-01

    7alpha-Methyl-19-nortestosterone (MENT) is a potent synthetic androgen that cannot be converted to dihydrotestosterone. In this study we determined the relative androgenic, antigonadotropic, and anabolic potencies of testosterone vs. MENT in the nonhuman primate M. fascicularis. In castrated monkeys, dose-response relationships were generated for the effects of testosterone and MENT on gonadotropin levels, prostate growth, body weight, and lipid metabolism. In a pilot study, four monkeys were castrated, and magnetic resonance imaging (MRI) was used to document a 50% loss of prostate volume within 8 weeks, verifying that MRI is a reliable means to measure prostate size in this species. Two additional groups of six monkeys each were then castrated and serially administered four graded dosages of testosterone or MENT via osmotic minipumps over 20 weeks. Complete suppression of LH was achieved with a minimum of 0.3 mg/day MENT, compared to 3.0 mg/day testosterone. MENT supported body weight 10 times more potently than did testosterone. Baseline prostate volumes were maintained with 0.1-0.2 mg/day MENT vs. 0.3 mg/day testosterone. Thus, in monkeys, MENT is 10 times more potent than testosterone with regard to the clinically desirable end points of gonadotropin suppression and anabolism, but only twice as potent at stimulating prostate growth. These results suggest that MENT may have a wider therapeutic index than testosterone for human androgen replacement and male contraception.

  7. Induction of male sexual behavior in the rat by 7 alpha-methyl-19-nortestosterone, an androgen that does not undergo 5 alpha-reduction.

    PubMed

    Moralí, G; Lemus, A E; Munguía, R; Arteaga, M; Pérez-Palacios, G; Sundaram, K; Kumar, N; Bardin, C W

    1993-09-01

    The synthetic steroid 7 alpha-methyl-19-nortestosterone (MENT) binds with high affinity to the androgen receptor and exerts biological effects at some peripheral target tissues with a potency greater than that of naturally occurring androgens. In vivo, MENT does not undergo enzymatic 5 alpha-reduction and as a consequence, its biologic action on prostate and other organs of the male reproductive tract is not amplified as is that of testosterone (T). Thus, in castrated rats, a dose of MENT that will maintain normal muscle mass and gonadotropin levels will not maintain normal prostate and seminal vesicle weights. To investigate the ability of MENT to restore male sexual behavior in castrated rats, varying doses of MENT acetate were administered for 4 wk by use of s.c. mini-osmotic pumps. Animals treated with T acetate (200 micrograms/day) and nontreated intact animals served as positive controls, while a group of animals receiving vehicle alone were the negative controls. Steroid acetates are rapidly converted to T and MENT in blood. Appropriate steroid delivery was assessed by measurement of serum androgen concentrations. Male behavioral parameters were recorded twice per week. At the end of treatment, the weights of sex accessory organs were also recorded. The administration of MENT acetate at daily doses of 100 micrograms and 10 micrograms induced full copulatory behavior in a manner similar to that observed with doses of 200 micrograms T acetate.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. An LC/MS method for the quantitative determination of 7alpha-OH DHEA and 7beta-OH DHEA: an application for the study of the metabolism of DHEA in rat brain.

    PubMed

    Li, Aiqun; May, Matthew P; Bigelow, James C

    2010-08-01

    Dehydroepiandrosterone (DHEA) is an important neurosteroid with neuronal protection and memory enhancement functions. 7alpha-OH DHEA and 7beta-OH DHEA are the two important metabolites of DHEA in the brain. We have developed an LC/MS method to quantitatively analyze 7alpha-OH DHEA and 7beta-OH DHEA. Chromatographic separation was carried out on a C18 column with gradient elution using mobile phases of formic acid in acetonitrile and in water formic acid. Mass spectral detection was performed with a ThermoFinnigan LCQ advantage quadruple ion trap mass spectrometer with electrospray ionization. Positive ion chromatograms were acquired using single ion monitoring. The protonated molecule was 305 m/z, but the most abundant ion (269 m/z) was used for quantification. This method was validated and applied to investigate the 7-hydroxylation of DHEA. When incubating DHEA with rat brain microsomes, both 7alpha-OH DHEA and 7beta-OH DHEA were observed, but 7alpha-OH DHEA was the major metabolite.

  9. An evaluation of the use of serum 7-alpha-hydroxycholestenone as a diagnostic test of bile acid malabsorption causing watery diarrhea

    PubMed Central

    Brydon, W Gordon; Culbert, Pearl; Kingstone, Kathleen; Jarvie, Ann; Iacucci, Marietta; Tenhage, Merel; Ghosh, Subrata

    2011-01-01

    BACKGROUND: Bile acid malabsorption (BAM) is a recognized cause of watery diarrhea, often diagnosed empirically based on clinical response to cholestyramine. The radionuclide selenium-labelled homocholic acid-taurine whole body retention test is expensive, labour intensive and of limited availability. OBJECTIVE: To report on the clinical performance of serum 7-alpha-hydroxy-4-cholesten-3-one (7HCO) as a test of BAM in adult patients with unexplained diarrhea. METHODS: Patients with unexplained diarrhea were investigated over a three-year period. Final diagnosis was determined based on medical history and investigations, serum levels of 7HCO and response to cholestyramine. ROC analysis was used to determine the ideal upper reference range cut-off value to optimize sensitivity/specificity for BAM. Time of blood specimen collection was recorded to investigate possible variation in results throughout the working day. RESULTS: ROC analysis yielded a sensitivity/specificity of 90%/77% for type 1 BAM (ileal disease/resection) and 97%/74% for type 2 BAM (idiopathic) using 30 ng/mL as the upper limit of normal for serum 7HCO when compared with all other patients. Of 813 patients, 196 tested positive. Serum 7HCO levels were significantly higher in blood specimens that were collected between 12:00 and 13:00 (median 24 ng/mL) than in specimens collected between 09:00 and 10:00 (median 17 ng/mL) (P<0.05). CONCLUSION: Serum 7HCO testing is a simple, sensitive, noninvasive, inexpensive alternative to other more commonly used tests for BAM. Time of specimen collection, however, resulted in small but significant result variations and, although unlikely to have much impact on test value, it should ideally be standardized. PMID:21766092

  10. Glucagon and cAMP inhibit cholesterol 7alpha-hydroxylase (CYP7A1) gene expression in human hepatocytes: discordant regulation of bile acid synthesis and gluconeogenesis.

    PubMed

    Song, Kwang-Hoon; Chiang, John Y L

    2006-01-01

    The gene encoding cholesterol 7alpha-hydroxylase (CYP7A1) is tightly regulated to control bile acid synthesis and maintain lipid homeostasis. Recent studies in mice suggest that bile acid synthesis is regulated by the fasted-to-fed cycle, and fasting induces CYP7A1 gene expression in parallel to the induction of peroxisome proliferators-activated receptor gamma co-activator 1alpha (PGC-1alpha) and phosphoenolpyruvate carboxykinase (PEPCK). How glucagon regulates CYP7A1 gene expression in the human liver is not clear. Here we show that glucagon and cyclic adenosine monophosphate (cAMP) strongly repressed CYP7A1 mRNA expression in human primary hepatocytes. Reporter assays confirmed that cAMP and protein kinase A (PKA) inhibited human CYP7A1 gene transcription, in contrast to their stimulation of the PEPCK gene. Mutagenesis analysis identified a PKA-responsive region located within the previously identified HNF4alpha binding site in the human CYP7A1 promoter. Glucagon and cAMP increased HNF4alpha phosphorylation and reduced the amount of HNF4alpha present in CYP7A1 chromatin. Our findings suggest that glucagon inhibited CYP7A1 gene expression via PKA phosphorylation of HNF4alpha, which lost its ability to bind the CYP7A1 gene and resulted in inhibition of human CYP7A1 gene transcription. In conclusion, this study unveils a species difference in nutrient regulation of the human and mouse CYP7A1 gene and suggests a discordant regulation of bile acid synthesis and gluconeogenesis by glucagon in human livers during fasting.

  11. Differential hepatocellular zonation pattern of cholesterol 7alpha-hydroxylase (Cyp7a1) and sterol 12alpha-hydroxylase (Cyp8b1) in the mouse.

    PubMed

    Wang, Jin; Olin, Maria; Rozell, Björn; Björkhem, Ingemar; Einarsson, Curt; Eggertsen, Gösta; Gåfvels, Mats

    2007-03-01

    The synthesis of primary bile acids is confined to the hepatocytes. This study aimed to evaluate the expression pattern within the liver architecture of the rate-limiting enzyme of the neutral pathway, cholesterol 7alpha-hydroxylase (Cyp7a1), and sterol 12alpha-hydroxylase (Cyp8b1), the enzyme necessary for the synthesis of cholic acid. Specific Cyp8b1 and Cyp7a1 peptide antiserums were used for immunohistochemical staining of livers from wild type and Cyp8b1 null mice, the latter instead expressing beta-galactosidase (beta-Gal) as a replacement reporter gene. Cyp8b1 was mainly expressed in the hepatocytes in a zonal pattern surrounding the central vein while the areas surrounding the portal zones showed much lower levels. The zonation was maintained in cholic acid-depleted mice using beta-Gal as a reporter protein. Cyp7a1 expression in wild type mice also showed a zonal distribution pattern, although less distinct, with a maximal expression within a 1-2 cell thick layer of hepatocytes surrounding the central vein. In Cyp8b1 null mice, a more intense staining was obtained, in accordance with the higher expression level of Cyp7a1, although the overall expression pattern was maintained. Our results in mice indicate possible differences in the regulation of the cellular zonation of Cyp7a1 and Cyp8b1. Also, cholic acid affects the set-point of Cyp7a1 expression but not its zonal distribution.

  12. Role of genetic variant A-204C of cholesterol 7alpha-hydroxylase (CYP7A1) in susceptibility to gallbladder cancer.

    PubMed

    Srivastava, Anvesha; Pandey, Sachchida Nand; Choudhuri, Gourdas; Mittal, Balraj

    2008-05-01

    Gallbladder carcinoma (GBC) usually arises in the background of gallstone disease. Cholesterol 7alpha-hydroxylase (CYP7A1) is a rate-limiting enzyme for cholesterol catabolism and bile acid synthesis. A-204C genetic polymorphism in CYP7A1 may influence gene expression and thus affect the risk of gallstone disease and GBC. We aimed to study the association of A-204C variation of CYP7A1 gene promoter polymorphism in GBC patients, gallstone patients and healthy subjects. The study included 141 histopathologically proven GBC patients, ultrasonographically proven 185 symptomatic gallstone patients and 200 gallstone-free healthy subjects. Genotyping was done by PCR-RFLP method. CYP7A1 A-204C genotypes in control population were in Hardy-Weinberg equilibrium. The CC genotype conferred marginally significant risk for gallstone disease (p=0.051; OR=1.54; 95% CI=0.9-3.4). In GBC patients, the CYP7A1 A-204C polymorphism conferred high risk for GBC at genotype (p=0.005; OR=2.78; 95% CI: 1.3-5.6) as well as allele levels (p=0.008; OR=1.58 and 95% CI: 1.1-2.2). After stratification of GBC patients on the basis of presence or absence of gallstones, CC genotype imparted higher risk for GBC without stones (p=0.002; OR=4.44: 95% CI=1.7-11.3). The association of the polymorphism with GBC was more pronounced in female GBC patients, and also in cancer patients who developed GBC at advanced age. The CC genotype of CYP7A1 is an independent genetic risk factor for GBC but plays a modest role in susceptibility to gallstone disease. The GBC pathogenesis by CYP7A1 polymorphism appears to be independent of gallstone pathway and probably involves genotoxicity due to lipid peroxidation mechanisms.

  13. Hypocholesterolemic mechanism of Chlorella: Chlorella and its indigestible fraction enhance hepatic cholesterol catabolism through up-regulation of cholesterol 7alpha-hydroxylase in rats.

    PubMed

    Shibata, Shinya; Hayakawa, Kazuhito; Egashira, Yukari; Sanada, Hiroo

    2007-04-01

    Chlorella powder (CP) has a hypocholesterolemic effect and high bile acid-binding capacity; however, its effects on hepatic cholesterol metabolism are still unclear. In the present study, male Wistar rats were divided into four groups and fed a high sucrose + 10% lard diet (H), an H + 10% CP diet (H+CP), an H + 0.5% cholesterol + 0.25% sodium cholate diet (C), or a C + 10% CP diet (C+CP) for 2 weeks. CP decreased serum and liver cholesterol levels significantly in rats fed C-based diets, but did not affect these parameters in rats fed H-based diets. CP increased the hepatic mRNA level and activity of cholesterol 7alpha-hydroxylase (CYP7A1). CP increased hepatic HMG-CoA reductase (HMGR) activity in the rats fed H-based diets, but not in rats fed C-based diets. CP did not affect hepatic mRNA levels of sterol 27-hydroxylase, HMGR, low-density lipoprotein (LDL) receptor, scavenger receptor class B1, ATP-binding cassette (ABC) A1, ABCG5, or ABCB11. Furthermore, the effect of a 3.08% Chlorella indigestible fraction (CIF, corresponding to 10% CP) on hepatic cholesterol metabolism was determined using the same animal models. CIF also decreased serum and liver cholesterol levels significantly in rats fed C-based diets. CIF increased hepatic CYP7A1 mRNA levels. These results suggest that the hypocholesterolemic effect of CP involves enhancement of cholesterol catabolism through up-regulation of hepatic CYP7A1 expression and that CIF contributes to the hypocholesterolemic effect.

  14. Bone and muscle protective potential of the prostate-sparing synthetic androgen 7alpha-methyl-19-nortestosterone: evidence from the aged orchidectomized male rat model.

    PubMed

    Venken, Katrien; Boonen, Steven; Van Herck, Erik; Vandenput, Liesbeth; Kumar, Narender; Sitruk-Ware, Regine; Sundaram, Kalyan; Bouillon, Roger; Vanderschueren, Dirk

    2005-04-01

    This study reports the preclinical evaluation of the bone and muscle protective potential of the synthetic androgen 7alpha-methyl-19-nortestosterone (MENTtrade mark), as assessed in the aged orchidectomized rat model. Aged (13-month-old) orchidectomized Wistar rats were treated with different doses of MENT (4, 12 or 36 microg/day) subcutaneously for 16 weeks via mini-osmotic pumps. Analysis of the effects of androgen deficiency versus MENT replacement was performed using quantitative computed tomography (pQCT), dual energy X-ray absorptiometry (DEXA) and biochemical markers of bone turnover. At the end of the study period, prostate weight in orchidectomized rats treated with low- (4 microg/day) or mid-dose (12 mug/day) MENT remained significantly lower compared to the sham-operated animals (-47% and -25%, respectively). High-dose MENT (36 microg/day), on the other hand, induced prostate hypertrophy (+21% versus sham). Low-, mid- and high-dose MENT were found to be effective in suppressing the acceleration of bone remodeling following orchidectomy, as assessed by osteocalcin and deoxypyridinoline. In addition, low-, mid- and high-dose were able to prevent the orchidectomy-induced bone loss, as evaluated by DEXA at the femur and total-body and by pQCT at the femur. Compared to sham-operated animals, the low- and mid-dose MENT groups showed no decline in lean body mass and no muscle atrophy (as measured by m. quadriceps weight) at 16 weeks, whereas high-dose MENT was associated with a significant decline in lean body mass (-8.5% versus sham) and quadriceps weight (-10.6%). We conclude that, in the aged orchidectomized rat model, low- and mid-doses of the synthetic androgen MENT have bone and muscle protective effects and do not induce prostate hypertrophy. The bone protective action of high-dose MENT, however, occurs at the expense of muscle wasting and prostate hypertrophy. Our findings support the need for human studies to explore the potential of MENT as an option

  15. Bile acids and cytokines inhibit the human cholesterol 7 alpha-hydroxylase gene via the JNK/c-jun pathway in human liver cells.

    PubMed

    Li, Tiangang; Jahan, Asmeen; Chiang, John Y L

    2006-06-01

    Cholesterol 7 alpha-hydroxylase (CYP7A1) of the bile acid biosynthesis pathway is suppressed by bile acids and inflammatory cytokines. Bile acids are known to induce inflammatory cytokines to activate the mitogen-activated protein kinase/c-Jun N-terminal kinase (JNK) signaling pathway that inhibits CYP7A1 gene transcription. c-Jun has been postulated to mediate bile acid inhibition of CYP7A1. However, the c-Jun target involved in the regulation of CYP7A1 is unknown. Human primary hepatocytes and HepG2 cells were used as models to study chenodeoxycholic acid (CDCA) and interleukin-1 beta (IL-1 beta) regulation of human CYP7A1 gene expression via real-time polymerase chain reaction, reporter assays, co-immunoprecipitation and chromatin immunocipitation (ChIP) assays. IL-1 beta and CDCA reduced CYP7A1 but induced c-Jun messenger RNA expression in human primary hepatocytes. IL-1beta inhibited human CYP7A1 reporter activity via the HNF4 alpha binding site. A JNK-specific inhibitor blocked the inhibitory effect of IL-1 beta on HNF4 alpha expression and CYP7A1 reporter activity. c-Jun inhibited HNF4 alpha and PPARgamma coactivator-1 alpha (PGC-1 alpha) coactivation of CYP7A1 reporter activity, whereas a dominant negative c-Jun did not. Co-immunoprecipitation and ChIP assays revealed that IL-1 beta and CDCA reduced HNF4 alpha bound to the CYP7A1 chromatin, and that c-Jun interacted with HNF4 alpha and blocked HNF4 alpha recruitment of PGC-1 alpha to the CYP7A1 chromatin. In conclusion, IL-1 beta and CDCA inhibit HNF4 alpha but induce c-Jun, which in turn blocks HNF 4 alpha recruitment of PGC-1 alpha to the CYP7A1 chromatin and results in inhibition of CYP7A1 gene transcription. The JNK/c-Jun signaling pathway inhibits bile acid synthesis and protects hepatocytes against the toxic effect of inflammatory agents.

  16. A new, major C27 biliary bile acid in the red-winged tinamou (Rhynchotus rufescens):25R-1beta, 3alpha,7alpha-trihydroxy-5beta-cholestan-27-oic acid.

    PubMed

    Hagey, Lee R; Kakiyama, Genta; Muto, Akina; Iida, Takashi; Mushiake, Kumiko; Goto, Takaaki; Mano, Nariyasu; Goto, Junichi; Oliveira, Cleida A; Hofmann, Alan F

    2009-04-01

    The chemical structures of the three major bile acids present in the gallbladder bile of the Red-winged tinamou (Rhynchotus rufescens), an early evolving, ground-living bird related to ratites, were determined. Bile acids were isolated by preparative reversed-phase HPLC. Two of the compounds were identified as the taurine N-acylamidates of 25R-3alpha,7alpha-dihydroxy-5beta-cholestan-27-oic acid (constituting 22% of biliary bile acids) and 25R-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-27-oic acid (constituting 51%). The remaining compound, constituting 21% of biliary bile acids, was an unknown C27 bile acid. Its structure was elucidated by LC/ESI-MS/MS and NMR and shown to be the taurine conjugate of 25R-1beta, 3alpha, 7alpha-trihydroxy-5beta-cholestan-27-oic acid, a C27 trihydroxy bile acid not previously reported. Although C27 bile acids with a 1beta-hydroxyl group have been identified as trace bile acids in the alligator, this is the first report of a major biliary C27 bile acid possessing a 1beta-hydroxyl group.

  17. Substrate specificity of THCA-CoA oxidases from rat liver light mitochondrial fractions on dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid CoA thioester.

    PubMed

    Ikegawa, S; Goto, T; Mano, N; Goto, J

    1998-11-01

    The substrate specificity of rat liver peroxisomal 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) oxidases, which catalyze the dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) CoA thioester, having an asymmetric center at C-25, to form (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) CoA thioester, was studied. The stable isotope labeled substrates, [3,7,12-18O3]-(25R)- and (25S)-THCA CoA thioesters were synthesized by an exchange reaction of carbonyl oxygens on a steroid nucleus of 3,7,12-trioxo-5 beta-cholestanoic acid, followed by metal hydride reduction and condensation reaction with CoA. After incubation of a mixture of unlabeled (25R)- and 18O-labeled (25S)-THCA CoA thioester, or vice versa, with hepatic peroxisomal THCA-CoA oxidases, biotransformed delta 24-THCA was determined by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. The delta 24-THCA was derived only from (25S)-THCA CoA thioester, indicating that the 25S epimer of THCA is a preferential substrate on dehydrogenation by THCA-CoA oxidases.

  18. Superiority of dietary safflower oil over olive oil in lowering serum cholesterol and increasing hepatic mRnas for the LDL receptor and cholesterol 7alpha-hydroxylase in exogenously hypercholesterolemic (exHC) rats.

    PubMed

    Sato, M; Yoshida, S; Nagao, K; Imaizumi, K

    2000-06-01

    The exogenously hypercholesterolemic (ExHC) rat is a strain segregated from SD rats with a high response to dietary cholesterol. To understand the underlying mechanism(s) for this hypercholesterolemia, the interactive effects of dietary fatty acid and the susceptibility of rats to dietary cholesterol on the serum cholesterol concentration and hepatic mRNA abundance of the low-density lipoprotein (LDL) receptor, cholesterol 7alpha-hydroxylase (7alpha-hydroxylase) and 3-hydroxyl-3methylglutaryl (HMG) CoA reductase were examined. Both strains were fed on a diet supplemented with 10% each of olive, safflower or coconut oil with or without the addition of 1% cholesterol for one week. The ExHC rats fed on olive, safflower and coconut oil in combination with cholesterol respectively resulted in a 3.5-, 2.0- and 2.1-fold higher serum cholesterol concentration than that in the animals fed on the corresponding dietary fats without any supplementation of cholesterol (p < 0.01 by dietary cholesterol or type of fat). The dietary cholesterol dependent-elevation of serum cholesterol in the SD rats was less than 1.5-fold (p<0.01) and there was no dietary fat effect. The ExHC rats fed on the safflower oil-containing diet supplemented with cholesterol resulted in a higher mRNA abundance of the LDL receptor and 7alpha-hydroxylase than in the corresponding fat-fed rats without cholesterol (p<0.05). There was no dietary cholesterol-dependent change of mRNA abundance in either strain fed on olive or coconut oil, except for a decreased abundance of HMG CoA reductase mRNA in the olive oil-fed ExHC rats and coconut oil-fed Sprague-Dawley (SD) rats (p<0.05). These results indicate that the hepatic mRNA abundance of the LDL receptor and of 7alpha-hydroxylase depended on the dietary combination of cholesterol and a fatty acid and suggest that a linoleic acid-rich diet may alleviate exogenous hypercholesterolemia by activating the process involved in the hepatic uptake and biliary excretion of

  19. Configuration at C-25 in 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol excreted by patients with cerebrotendinous xanthomatosis: circular dichroism and 13C-NMR studies.

    PubMed

    Dayal, B; Salen, G; Toome, V; Tint, G S

    1986-12-01

    The configuration at C-25 in 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,25,26-pentol isolated from the bile and feces of patients with cerebrotendinous xanthomtosis (CTX) was determined from the lanthanide-induced circular dichroism (CD) Cotton effects and 13C-NMR measurements. Under anhydrous conditions, CD spectra of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,25,26-pentol in the presence of Eu(fod)3 exhibited a large induced negative Cotton effect at 320 nm. On the basis of the empirical rule (primary-tertiary-alpha-diols) in which R compounds have positive Cotton effects and S compounds have negative Cotton effects at 320 nm, it was concluded that 25,26-pentol has the 1,2,glycol structure with C-25 having the S-configuration. This assignment was based upon comparison with model compounds, 25(R and S),26-dihydroxy cholesterols and 25(R and S),26-dihydroxy cholecalciferols whose single-crystal X-ray structure and 13C-NMR studies have been performed. It is suggested that these data may be helpful to clarify the stereospecificity of the hydroxylation of the terminal methyl group of the cholesterol side chain in CTX.

  20. The metabolism of 3alpha, 7alpha, 12alpha-trihydorxy-5beta-cholestan-26-oic acid in two siblings with cholestasis due to intrahepatic bile duct anomalies. An apparent inborn error of cholic acid synthesis.

    PubMed Central

    Hanson, R F; Isenberg, J N; Williams, G C; Hachey, D; Szczepanik, P; Klein, P D; Sharp, H L

    1975-01-01

    Studies were carried out in a family in which two children with cholestasis due to intrahepatic bile duct anomalies were shown to have increased amounts of the cholic acid precursor, 3alpha, 7alpha, 12alpha-trihydorxy-5beta-cholestan-26-oic acid (THCA). The metabolism of THCA was studied in one of these patients after an intravenous injection of (3H)THCA, and the cause of the increased amounts of THCA in this condition was found to be due to a metabolic defect in the conversion of this compound into cholic acid. A small amount of (3H)cholic acid was also identified after (3H)THCA administration, confirming that this metabolic defect was incomplete. Varanic acid (3alpha, 7alpha, 12alpha, 24xi-tetrahydorxy-5beta-cholestan-26-oic acid), a metabolite of THCA, could not be identified in either of these patients. By assuming that this compound would be conjugated and excreted if the metabolic block occurred after the formation of varanic acid, the defect in these patients appears to be due to a deficiency of a 24-hydroxylating enzyme system required to convert THCA into varanic acid. This condition appears to be transmitted in an autosomal recessive fashion, because the two affected patients were of opposite sex, and neither a normal sibling nor the two parents have increased amount of THCA in their bile. Images PMID:1159074

  1. Stereospecific dehydrogenation of (25R)- and (25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acids by acyl-CoA oxidase in rat liver light mitochondrial fraction.

    PubMed

    Ikegawa, S; Watanabe, H; Goto, T; Mano, N; Goto, J; Nambara, T

    1995-08-01

    From a stereochemical point of view, the dehydrogenation mechanism of the biotransformation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) has been studied with capillary gas chromatography (GC)/negative ion chemical ionization (NICI)-mass spectrometry. After incubation of (24R,25R)- or (24S,25S)-[24,25-2H2]THCA, synthesized from (24E)-delta 24-THCA by a deuterated diimide reduction, with a rat liver light mitochondrial fraction, 5 beta-cholestanoic acids were extracted and derivatized into a pentafluorobenzyl (PFB) ester-dimethylethylsilyl (DMES) ether. Subsequent resolution into THCA and delta 24-THCA was attained by GC on a cross-linked 5% phenylmethyl silicone fused-silica capillary column monitored with a corresponding characteristic carboxylate anion [M-PFB]- in the NICI mode. The stereospecific elimination of a pro-R hydrogen at C-24 in both (25R)- and (25S)-THCA indicated syn-elimination for the former, whereas anti-elimination for the latter was observed.

  2. 3{alpha}-6{alpha}-Dihydroxy-7{alpha}-fluoro-5{beta}-cholanoate (UPF-680), physicochemical and physiological properties of a new fluorinated bile acid that prevents 17{alpha}-ethynyl-estradiol-induced cholestasis in rats

    SciTech Connect

    Clerici, Carlo . E-mail: clerici@unipg.it; Castellani, Danilo; Asciutti, Stefania; Pellicciari, Roberto; Setchell, Kenneth D.R. |; O'Connell, Nancy C. |; Sadeghpour, Bahman; Camaioni, Emidio; Fiorucci, Stefano; Renga, Barbara; Nardi, Elisabetta; Sabatino, Giuseppe; Clementi, Mattia; Giuliano, Vittorio; Baldoni, Monia; Orlandi, Stefano; Mazzocchi, Alessandro; Morelli, Antonio; Morelli, Olivia

    2006-07-15

    3{alpha}-6{alpha}-Dihydroxy-7{alpha}-fluoro-5{beta}-cholanoate (UPF-680), the 7{alpha}-fluorine analog of hyodeoxycholic acid (HDCA), was synthesized to improve bioavailability and stability of ursodeoxycholic acid (UDCA). Acute rat biliary fistula and chronic cholestasis induced by 17{alpha}-ethynyl-estradiol (17EE) models were used to study and compare the effects of UPF-680 (dose range 0.6-6.0 {mu}mol/kg min) with UDCA on bile flow, biliary bicarbonate (HCO{sub 3} {sup -}), lipid output, biliary bile acid composition, hepatic enzymes and organic anion pumps. In acute infusion, UPF-680 increased bile flow in a dose-related manner, by up to 40.9%. Biliary HCO{sub 3} {sup -} output was similarly increased. Changes were observed in phospholipid secretion only at the highest doses. Treatment with UDCA and UPF-680 reversed chronic cholestasis induced by 17EE; in this model, UDCA had no effect on bile flow in contrast to UPF-680, which significantly increased bile flow. With acute administration of UPF-680, the biliary bile acid pool became enriched with unconjugated and conjugated UPF-680 (71.7%) at the expense of endogenous cholic acid and muricholic isomers. With chronic administration of UPF-680 or UDCA, the main biliary bile acids were tauro conjugates, but modification of biliary bile acid pool was greater with UPF-680. UPF-680 increased the mRNA for cytochrome P450 7A1 (CYP7A1) and cytochrome P450 8B (CYP8B). Both UDCA and UPF-680 increased the mRNA for Na{sup +} taurocholate co-transporting polypeptide (NCTP). In conclusion, UPF-680 prevented 17EE-induced cholestasis and enriched the biliary bile acid pool with less detergent and cytotoxic bile acids. This novel fluorinated bile acid may have potential in the treatment of cholestatic liver disease.

  3. A rapid single-tube multiplex polymerase chain reaction assay for the seven most prevalent alpha-thalassemia deletions and alphaalphaalpha(anti 3.7) alpha-globin gene triplication.

    PubMed

    de Mare, Arjan; Groeneger, Antoinette Heijs-Oude; Schuurman, Sander; van den Bergh, Frank A T J M; Slomp, Jennichjen

    2010-01-01

    alpha-Globin gene triplications may exacerbate the alpha chain and beta chain imbalance in beta-thalassemia (beta-thal) and may compensate for the effect of alpha-globin gene deletion in alpha-thal. Identification of an alpha-globin gene triplication is, therefore, valuable in predicting the clinical phenotype of the thalassemias. To be able to detect alpha-globin gene triplications, we have modified an existing multiplex polymerase chain reaction (PCR) assay for the seven most prevalent alpha-globin gene deletions by incorporating two triplication-specific primers and concurrently substituting one of the original primers by a newly designed primer. This modified multiplex PCR assay was evaluated by performing the assay on archival DNA samples and on peripheral blood samples from 163 suspected thalassemia cases. It was found to function properly. Our assay thereby represents the first multiplex PCR assay that can detect both the seven most prevalent alpha-globin gene deletions and the alphaalphaalpha(anti 3.7) alpha-globin gene triplication in a single-tube reaction.

  4. Comparison of the growth-promoting effects of testosterone and 7-alpha-methyl-19-nor-testosterone (MENT) on the prostate and levator ani muscle of LPB-tag transgenic mice.

    PubMed

    Shao, T C; Li, H L; Kasper, S; Matusik, R; Ittmann, M; Cunningham, G R

    2006-03-01

    7-alpha-methyl-19-nortestosterone (MENT) is being considered for androgen replacement in testosterone deficient men and as a male contraceptive. Because androgenic effects on the prostate are a major concern, we have evaluated MENT in a transgenic model of prostate cancer. LPB-Tag mice were castrated and infused with testosterone (T; 5 or 30 microg/day) or MENT (5 or 30 microg/day) for 4 weeks. Prostate, seminal vesicle, and levator ani muscle (LAM) weights were compared. At an equivalent dose, MENT maintained or stimulated the mean weights of these organs more than T. However, the dorsolateral prostate/LAM ratio of weights did not favor MENT, but DNA/mg tissue and Ki 67 immunostaining suggested that MENT may increase DNA less than T. MENT is more potent than T in maintaining or stimulating prostate, seminal vesicle, and LAM. Using doses that resulted in comparable stimulation of the levator ani muscle, MENT had similar effect on prostate weight, but increased DNA/mg prostate less than T in this transgenic mouse model of prostate cancer. (c) 2005 Wiley-Liss, Inc.

  5. Dimethandrolone (7alpha,11beta-dimethyl-19-nortestosterone) and 11beta-methyl-19-nortestosterone are not converted to aromatic A-ring products in the presence of recombinant human aromatase.

    PubMed

    Attardi, Barbara J; Pham, Trung C; Radler, Lisa C; Burgenson, Janet; Hild, Sheri A; Reel, Jerry R

    2008-06-01

    Dimethandrolone undecanoate (DMAU: 7alpha,11beta-dimethyl-19-nortestosterone 17beta-undecanoate) is a potent orally active androgen in development for hormonal therapy in men. Cleavage of the 17beta-ester bond by esterases in vivo leads to liberation of the biologically active androgen, dimethandrolone (DMA), a 19-norandrogen. For hormone replacement in men, administration of C19 androgens such as testosterone (T) may lead to elevations in circulating levels of estrogens due to aromatization. As several reports have suggested that certain 19-norandrogens may serve as substrates for the aromatase enzyme and are converted to the corresponding aromatic A-ring products, it was important to investigate whether DMA, the related compound, 11beta-methyl-19-nortestosterone (11beta-MNT), also being tested for hormonal therapy in men, and other 19-norandrogens can be converted to aromatic A-ring products by human aromatase. The hypothetical aromatic A-ring product corresponding to each substrate was obtained by chemical synthesis. These estrogens bound with high affinity to purified recombinant human estrogen receptors (ER) alpha and beta in competitive binding assays (IC50's: 5-12 x 10(-9) M) and stimulated transcription of 3XERE-luciferase in T47Dco human breast cancer cells with a potency equal to or greater than that of estradiol (E2) (EC50's: 10(-12) to 10(-11) M). C19 androgens (T, 17alpha-methyltestosterone (17alpha-MT), androstenedione (AD), and 16alpha-hydroxyandrostenedione (16alpha-OHAD)), 19-norandrogens (DMA, 11beta-MNT, 19-nortestosterone (19-NT), and 7alpha-methyl-19-nortestosterone (MENT)) or the structurally similar 19-norprogestin, norethindrone (NET) were incubated at 50 microM with recombinant human aromatase for 10-180 min at 37 degrees C. The reactions were terminated by extraction with acetonitrile and centrifugation, and substrate and potential product were separated by HPLC. Retention times were monitored by UV absorption, and UV peaks were quantified

  6. Formation of ursodeoxycholic acid from chenodeoxycholic acid by a 7 beta-hydroxysteroid dehydrogenase-elaborating Eubacterium aerofaciens strain cocultured with 7 alpha-hydroxysteroid dehydrogenase-elaborating organisms.

    PubMed Central

    MacDonald, I A; Rochon, Y P; Hutchison, D M; Holdeman, L V

    1982-01-01

    A gram-positive, anaerobic, chain-forming, rod-shaped anaerobe (isolate G20-7) was isolated from normal human feces. This organism was identified by cellular morphology as well as fermentative and biochemical data as Eubacterium aerofaciens. When isolate G20-7 was grown in the presence of Bacteroides fragilis or Escherichia coli (or another 7 alpha-hydroxysteroid dehydrogenase producer) and chenodeoxycholic acid, ursodeoxycholic acid produced. Time course curves revealed that 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid produced by B. fragilis or E. coli or introduced into the medium as a pure substance was reduced by G20-7 specifically to ursodeoxycholic acid. The addition of glycine- and taurine-conjugated primary bile acids (chenodeoxycholic and cholic acids) and other bile acids to binary cultures of B. fragilis and G20-7 revealed that (i) both conjugates were hydrolyzed to give free bile acids, (ii) ursocholic acid (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acid) was produced when conjugated (or free) cholic acid was the substrate, and (iii) the epimerization reaction was at least partially reversible. Corroborating these observations, an NADP-dependent 7 beta-hydroxysteroid dehydrogenase (reacting specifically with 7 beta-OH-groups) was demonstrated in cell-free preparations of isolate G20-7; production of the enzyme was optimal at between 12 and 18 h of growth. This enzyme, when measured in the oxidative direction, was active with ursodeoxycholic acid, ursocholic acid, and the taurine conjugate of ursodeoxycholic acid (but not with chenodeoxycholic, deoxycholic, or cholic acids) and displayed an optimal pH range of 9.8 to 10.2 Images PMID:6758698

  7. A validated LC-MS/MS method for the sensitive quantitation of serum 7alpha hydroxy-, 7beta hydroxy- and 7keto-dehydroepiandrosterone using a novel derivatization reagent.

    PubMed

    Ke, Yuyong; Gonthier, Renaud; Simard, Jean-Nicolas; Labrie, Fernand

    2016-04-01

    7alpha hydroxy-, 7beta hydroxy- and 7keto-dehydroepiandrosterone (7α OH-DHEA, 7β OH-DHEA and 7 oxo-DHEA) are oxidized metabolites of dehydroepiandrosterone (DHEA). Their concentrations are low in the circulation, especially in postmenopausal women, thus resulting in a considerable challenge for their reliable measurement. A sensitive and accurate LC-MS/MS method has been developed using a simple sample preparation procedure and a novel derivatization with 1-amino-4-methyl piperazine (MP). The derivatized metabolites are stable in high water content reagents. A 10 pg/mL (0.2 pg on column) for the low limit of quantitation (LLOQ) has been achieved for all three compounds. A proper choice of multiple reaction monitoring (MRM) transitions provides good specificity. The excess amount of reagent can be removed from the sample during the derivatization process. Within the calibration range of 10-2000 pg/mL, a good linearity was obtained with R>0.99 where the weighing factor is 1/X while the bias and coefficient of variance (CV) are within 8% for all levels of QCs and calibration curves. This method has been fully validated according to the FDA guidelines, where the results of the matrix effect meet the acceptance criteria while freeze-thaw stability, short and long term stability in matrix and solution as well as post-processed sample stability meet the requirements. With this method, the concentrations of 7α OH-DHEA, 7β OH-DHEA and 7 oxo-DHEA were measured in premenopausal and postmenopausal serum. The average concentration of 7α OH-DHEA is equivalent to that of 7β OH-DHEA in both types of sera. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. The antigonadotropic action of testosterone but not 7alpha-methyl-19-nortestosterone is attenuated through the 5alpha-reductase pathway in the castrated male rat pituitary gland.

    PubMed

    Bandivdekar, A H; Karp, R; Sundaram, K; Kumar, N

    2000-01-01

    The enzyme 5alpha-reductase plays a significant role in the prostate to amplify the action of testosterone (T) by converting it to a more potent androgen, dihydrotestosterone (DHT). The role of 5alpha-reductase in the testosterone feedback inhibition of gonadotropin secretion from the pituitary has not been elucidated. Therefore, we investigated the role of 5alpha-reductase on T action in in vitro and in vivo models. Castration has been reported to increase the 5alpha-reductase activity in pituitary glands. Hence, the effect of castration duration on the conversion of T to DHT by pituitary homogenates and the responsiveness of pituitary monolayer cell cultures to gonadotropin-releasing hormone (GnRH) challenge exposure were investigated. Incubation of [3H]-T with pituitary homogenates showed that the conversion of T to 5alpha-reduced metabolites was two- to threefold greater in pituitaries from rats who had been castrated for 14 days compared with those castrated for 1 day. In addition, the GnRH-stimulated release of LH from monolayer cell cultures of pituitaries from rats castrated for 1 day was twofold greater, whereas that from rats castrated for 2 weeks was six- to sevenfold greater compared with basal luteinizing hormone (LH) release. Hence we used rats castrated for 2 weeks to elucidate the role of 5alpha-reductase in T feedback inhibition. The inhibitory effects of the androgens T, 19-nortestosterone (19-NT), and 7alpha-methyl-19-nortestosterone (MENT) at 3 different concentrations (10(-9), 10(-7), and 10(-5) mol/L) on GnRH-stimulated LH release from monolayer cell cultures of pituitaries from rats castrated for 2 weeks were examined. All 3 androgens showed dose-dependent inhibition of LH release. MENT showed the greatest inhibition, followed by 19-NT and T. In the presence of finasteride (a 5alpha-reductase inhibitor), the inhibition of LH released by T and 19-NT were significantly greater. The inhibitory effect of MENT, which does not undergo 5alpha

  9. Chemical synthesis of (22E)-3alpha,6alpha,7alpha,12alpha-Tetrahydroxy-5beta-chol-22-en-24-oic acid and its N-acylamidated conjugates with glycine or taurine: precursors of the [22,23-(3)H] labelled tracers.

    PubMed

    Ogawa, Shoujiro; Adachi, Yuuki; Kakiyama, Genta; Shimada, Miki; Mano, Nariyasu; Goto, Junichi; Iida, Takashi

    2010-08-01

    (22E)-3alpha,6alpha,7alpha,12alpha-Tetrahydroxy-5beta-chol-22-en-24-oic acid and its N-acylamidated conjugates with glycine or taurine were synthesized from cholic acid. The key reactions employed are: 1) degradation of the side chain in intermediary C(24) 3alpha,6alpha,7alpha,12alpha-tetrahydroxylated bile acid to the corresponding C(22) 23,24-dinor-aldehyde, followed by Wittig reaction with methyl (triphenylphosphoranylidene)acetate and 2) N-acylamidation of the unconjugated tetrahydroxy-Delta(22)-5beta-cholenoic acid with glycine (or taurine) in the presence of diethylphosphorocyanide and triethylamine as coupling reagents.

  10. The negative effects of bile acids and tumor necrosis factor-alpha on the transcription of cholesterol 7alpha-hydroxylase gene (CYP7A1) converge to hepatic nuclear factor-4: a novel mechanism of feedback regulation of bile acid synthesis mediated by nuclear receptors.

    PubMed

    De Fabiani, E; Mitro, N; Anzulovich, A C; Pinelli, A; Galli, G; Crestani, M

    2001-08-17

    Bile acids regulate the cholesterol 7alpha-hydroxylase gene (CYP7A1), which encodes the rate-limiting enzyme in the classical pathway of bile acid synthesis. Here we report a novel mechanism whereby bile acid feedback regulates CYP7A1 transcription through the nuclear receptor hepatocyte nuclear factor-4 (HNF-4), which binds to the bile acid response element (BARE) at nt -149/-118 relative to the transcription start site. Using transient transfection assays of HepG2 cells with Gal4-HNF-4 fusion proteins, we show that chenodeoxycholic acid (CDCA) dampened the transactivation potential of HNF-4. Overexpression of a constitutive active form of MEKK1, an upstream mitogen-activated protein kinase (MAPK) module triggered by stress signals, strongly repressed the promoter activity of CYP7A1 via the consensus sequence for HNF-4 embedded in the BARE. Similarly, MEKK1 inhibited the activity of HNF-4 in the Gal4-based assay. The involvement of the MEKK1-dependent pathway in the bile acid-mediated repression of CYP7A1 was confirmed by co-transfecting a dominant negative form of the stress-activated protein kinase kinase, SEK, which abolished the effect of CDCA upon CYP7A1 transcription. Treatment of transfected HepG2 cells with tumor necrosis factor alpha (TNF-alpha), an activator of the MEKK1 pathway, led to the repression of CYP7A1 via the HNF-4 site in the BARE. TNF-alpha also inhibited the transactivation potential of HNF-4. Collectively, our results demonstrate for the first time that HNF-4, in combination with a MAPK signaling pathway, acts as a bile acid sensor in the liver. Furthermore, the effects of CDCA and TNF-alpha converge to HNF-4, which binds to the BARE of CYP7A1, suggesting a link between the cascades elicited by bile acids and pro-inflammatory stimuli in the liver.

  11. 7 alpha-Methyl-19-nortestosterone: an ideal androgen for replacement therapy.

    PubMed

    Sundaram, K; Kumar, N; Bardin, C W

    1994-01-01

    MENT is a synthetic androgen which cannot be 5 alpha-reduced. Therefore, relative to T, its stimulatory action on the prostate is lower than that on the muscle and pituitary. Like T, MENT undergoes enzymatic aromatization to an estrogen. We conclude that the use of MENT instead of T for androgen replacement therapy could have health-promoting effects by reducing the occurrence of prostate disease.

  12. 7 alpha-methyl-19-nortestosterone facilitates sexual behavior in the male Syrian hamster.

    PubMed

    Wood, R I; Bean, A R; Sundaram, K; Kumar, N; Bardin, C W

    1996-06-01

    Steroid hormones from the testes promote attraction to estrous females and facilitate copulation in the male Syrian hamster. We compared the ability of testosterone (T) and MENT, a potent synthetic androgen that does not undergo 5 alpha-reduction, to maintain sexual behavior in castrated males. Steroid treatment was initiated immediately after castration at three levels by means of Alzet osmotic pumps in sexually experienced adult male hamsters. Daily doses were 5, 25, or 100 micrograms T and 1, 5, or 25 micrograms MENT (n = 5/group). Additional castrated males (n = 5) remained untreated. Sexual behavior was recorded during two 10-min tests before, and at 2, 4, 6, and 8 weeks after orchidectomy. MENT and T maintained equivalent levels of behavior at each corresponding dose of androgen (high, medium, or low). The low dose of T or MENT failed to sustain mating behavior. Eight weeks after castration, males receiving the high and medium doses of androgens continued to express intromissions and ejaculations at gonadally intact levels. However, only males receiving the high dose showed anogenital investigation at the same level as intact males. From these data, we conclude that MENT sustains mating behavior in the male hamster, and that chemoinvestigatory behavior requires higher levels of androgens than those necessary for copulation.

  13. Thyroid hormone induction of human cholesterol 7 alpha-hydroxylase (Cyp7a1) in vitro.

    PubMed

    Lammel Lindemann, Jan A; Angajala, Anusha; Engler, David A; Webb, Paul; Ayers, Stephen D

    2014-05-05

    Thyroid hormone (TH) modulates serum cholesterol by acting on TH receptor β1 (TRβ1) in liver to regulate metabolic gene sets. In rodents, one important TH regulated step involves induction of Cyp7a1, an enzyme in the cytochrome P450 family, which enhances cholesterol to bile acid conversion and plays a crucial role in regulation of serum cholesterol levels. Current models suggest, however, that Cyp7a1 has lost the capacity to respond to THs in humans. We were prompted to re-examine TH effects on cholesterol metabolic genes in human liver cells by a recent study of a synthetic TH mimetic which showed that serum cholesterol reductions were accompanied by increases in a marker for bile acid synthesis in humans. Here, we show that TH effects upon cholesterol metabolic genes are almost identical in mouse liver, mouse and human liver primary cells and human hepatocyte cell lines. Moreover, Cyp7a1 is a direct TR target gene that responds to physiologic TR levels through a set of distinct response elements in its promoter. These findings suggest that THs regulate cholesterol to bile acid conversion in similar ways in humans and rodent experimental models and that manipulation of hormone signaling pathways could provide a strategy to enhance Cyp7a1 activity in human patients.

  14. Effects of early cholesterol intake on cholesterol 7 alpha hydroxylase (Cyp7a1) expression in piglets receiving sow's breast milk or infant formula until weaning

    USDA-ARS?s Scientific Manuscript database

    Unlike breast milk, infant formulas are not rich in cholesterol. To compensate for the dietary loss, hepatic cholesterol synthesis is increased in formula-fed infants. Observational studies have reported significant increases in serum cholesterol and triglycerides in adults that received formula dur...

  15. Genetic polymorphisms of cytochrome P450cl7alpha (CYP17) and progesterone receptor genes (PROGINS) in the assessment of endometriosis risk.

    PubMed

    De Carvalho, Cristina Valleta; Nogueira-De-Souza, Naiara Correa; Costa, Ana Maria Massad; Baracat, Edmund Chada; Girão, Manoel J B C; D'Amora, Paulo; Schor, Eduardo; da Silva, Ismael D C G

    2007-01-01

    We designed the present study in order to evaluate the eventual role of polymorphisms in the genes encoding cytochrome P450c17alpha (CYP17) and the progesterone receptor (PROGINS) as risk factors for endometriosis development. Eligible cases consisted of 121 women with surgically confirmed endometriosis who underwent treatment in a hospital in São Paulo, Brazil during the period from September 2003 to September 2005. The 281 controls were participants with normal gynecological as well as pelvic ultrasound evaluation, who did not have any gynecological conditions during their reproductive lives such as pelvic pain and/or dyspareunia nor infertility history. Genomic DNA was obtained from buccal cells and processed for DNA extraction using the GFX DNA extraction kit (GE Healthcare). The CYP17 (-34T-->C) polymerase chain reaction-restriction fragment length polymorphism assay has been described previously, as has the progesterone receptor polymorphism (PROGINS) detection assay. PROGINS heterozygosis genotype frequencies were shown to be statistically higher in endometriosis cases compared with controls. On the other hand, differences in the CYP17 polymorphism (-34T-->C) frequencies were not even close to significance (p = 0.278) according to our findings.

  16. An evaluation of the use of serum 7-alpha-hydroxycholestenone as a diagnostic test of bile acid malabsorption causing watery diarrhea.

    PubMed

    Brydon, W Gordon; Culbert, Pearl; Kingstone, Kathleen; Jarvie, Ann; Iacucci, Marietta; Tenhage, Merel; Ghosh, Subrata

    2011-06-01

    Bile acid malabsorption (BAM) is a recognized cause of watery diarrhea, often diagnosed empirically based on clinical response to cholestyramine. The radionuclide selenium-labelled homocholic acid-taurine whole body retention test is expensive, labour intensive and of limited availability. To report on the clinical performance of serum 7-alphahydroxy-4-cholesten-3-one (7HCO) as a test of BAM in adult patients with unexplained diarrhea. Patients with unexplained diarrhea were investigated over a three-year period. Final diagnosis was determined based on medical history and investigations, serum levels of 7HCO and response to cholestyramine. ROC analysis was used to determine the ideal upper reference range cut-off value to optimize sensitivity/specificity for BAM. Time of blood specimen collection was recorded to investigate possible variation in results throughout the working day. ROC analysis yielded a sensitivity/specificity of 90%/77% for type 1 BAM (ileal disease/resection) and 97%/74% for type 2 BAM (idiopathic) using 30 ng/mL as the upper limit of normal for serum 7HCO when compared with all other patients. Of 813 patients, 196 tested positive. Serum 7HCO levels were significantly higher in blood specimens that were collected between 12:00 and 13:00 (median 24 ng/mL) than in specimens collected between 09:00 and 10:00 (median 17 ng/mL) (P<0.05). Serum 7HCO testing is a simple, sensitive, noninvasive, inexpensive alternative to other more commonly used tests for BAM. Time of specimen collection, however, resulted in small but significant result variations and, although unlikely to have much impact on test value, it should ideally be standardized.

  17. 27-Hydroxycholesterol and 7alpha-hydroxycholesterol trigger a sequence of events leading to migration of CCR5-expressing Th1 lymphocytes

    SciTech Connect

    Kim, Sun-Mi; Kim, Bo-Young; Lee, Sae-A; Eo, Seong-Kug; Yun, Yungdae; Kim, Chi-Dae; Kim, Koanhoi

    2014-02-01

    Th1 lymphocytes are predominant in atherosclerotic lesions. However, mechanisms involved in the Th1 predominance are unknown. We have investigated the possibility of Th1 lymphocyte recruitment in a cholesterol-rich milieu. A high cholesterol diet resulted in enhanced expression of CCR5 ligands, including CCL3 and CCL4, but not of proatherogenic CXCR3 ligands, in atherosclerotic arteries of ApoE{sup −/−} mice. 27-Hydroxycholesterol and 7α-hydroxycholesterol, cholesterol oxides (oxysterols) detected in abundance in atherosclerotic lesions, greatly induced the transcription of CCL3 and CCL4 genes in addition to enhancing secretion of corresponding proteins by THP-1 monocytic cells. However, an identical or even higher concentration of cholesterol, 7β-hydroxycholesterol, and 7-ketocholsterol did not influence expression of these chemokines. Conditioned media containing the CCR5 ligands secreted from THP-1 cells induced migration of Jurkat T cells expressing CCR5, a characteristic chemokine receptor of Th1 cells, but not of Jurkat T cells that do not express CCR5. The migration of CCR5-expressing Jurkat T cells was abrogated in the presence of a CCR5-neutralizing antibody. 27-Hydroxycholesterol and 7α-hydroxycholesterol enhanced phosphorylation of Akt. Pharmacological inhibitors of phosphoinositide-3-kinase/Akt pathways blocked transcription as well as secretion of CCL3 and CCL4 in conjunction with attenuated migration of CCR5-expressing Jurkat T cells. This is the first report on the involvement of cholesterol oxides in migration of distinct subtype of T cells. We propose that 27-hydroxycholesterol and 7α-hydroxycholesterol can trigger a sequence of events that leads to recruitment of Th1 lymphocytes and phosphoinositide-3-kinase/Akt pathways play a major role in the process. - Graphical abstract: Th1 lymphocytes are predominant in atherosclerotic lesions. However, mechanisms involved in the Th1 predominance are unknown. We have investigated the possibility of Th1 lymphocyte recruitment in a cholesterol-rich milieu. We propose a model via which 27OHChol and 7αOHChol contribute to the predominance of Th1 cells in atherosclerotic lesions on the basis of our results and previous findings. Cholesterol deposited in the artery undergoes oxidative modification to oxysterols. Exposure of monocytic cells to 27OHChol or 7αOHChol results in increased transcription and secretion of CCR5 ligands, like CCL3 and CCL4, which leads to a concentration gradient of the chemokines. Among the lymphocytes attached to cell adhesion molecules expressed on endothelial cells, Th1 cells that express CCR5 recognize the gradient and follow the signal of increasing chemokine concentration towards the source of the chemokines, whereas other subtypes of T cells that do not express CCR5 (Tregs and Th2 cells) do not respond. The preferential infiltration of Th1 cells leads to predominance of Th1 cells. Since oxidized LDL (oxLDL) enhances the expression of cell adhesion molecules on endothelial cells, existence of oxLDL will accelerate the recruitment of Th1 lymphocytes into atherosclerotic lesions in response to the oxysterols. - Highlights: • High-cholesterol diet induces CCR5L expression, like CCL3 and CCL4, in ApoE{sup −/−} mice. • 27OHChol and 7αOHChol enhance secretion of CCL3 and CCL4 by monocytic cells. • The secreted CCR5 ligands promote migration of CCR5-expressing Th1 cells. • We report a mechanism underlying Th1 cell recruitment into atherosclerotic lesions.

  18. Screening study of lead compounds for natural product-based fungicides: antifungal activity and biotransformation of 6alpha,7alpha-dihydroxy-beta-himachalene by Botrytis cinerea.

    PubMed

    Daoubi, Mourad; Hernández-Galán, Rosario; Benharref, Ahmed; Collado, Isidro G

    2005-08-24

    Eleven beta-himachalene derivatives were tested, using the poisoning food technique, for their potential antifungal activity against the phytopathogen Botrytis cinerea. Compounds 1-11 displayed moderate activity, whereas the 6,7-diol derivative (12) produced an inhibition of 91% after 6 days. The microbial transformation of 12 was investigated and yielded four new compounds hydroxylated at positions C-5 (13), C-2 (14), C-4 (15), and C-12 (16). The structures were established on the basis of their spectroscopic data including two-dimensional NMR analysis (HMQC, HMBC, nOesy) and nOes. The results obtained from biotransformation experiments shed further light on the detoxification mechanism of the phytopathogenic fungus against this compound and give an indication of the structural modifications that may be necessary if substrates of this type are to be further developed as selective fungal control agents for B. cinerea.

  19. Elucidating the role of the TRPM7 alpha-kinase: TRPM7 kinase inactivation leads to magnesium deprivation resistance phenotype in mice

    PubMed Central

    Ryazanova, Lillia V.; Hu, Zhixian; Suzuki, Sayuri; Chubanov, Vladimir; Fleig, Andrea; Ryazanov, Alexey G.

    2014-01-01

    TRPM7 is an unusual bi-functional protein containing an ion channel covalently linked to a protein kinase domain. TRPM7 is implicated in regulating cellular and systemic magnesium homeostasis. While the biophysical properties of TRPM7 ion channel and its function are relatively well characterized, the function of the TRPM7 enzymatically active kinase domain is not understood yet. To investigate the physiological role of TRPM7 kinase activity, we constructed mice carrying an inactive TRPM7 kinase. We found that these mice were resistant to dietary magnesium deprivation, surviving three times longer than wild type mice; also they displayed decreased chemically induced allergic reaction. Interestingly, mutant mice have lower magnesium bone content compared to wild type mice when fed regular diet; unlike wild type mice, mutant mice placed on magnesium-depleted diet did not alter their bone magnesium content. Furthermore, mouse embryonic fibroblasts isolated from TRPM7 kinase-dead animals exhibited increased resistance to magnesium deprivation and oxidative stress. Finally, electrophysiological data revealed that the activity of the kinase-dead TRPM7 channel was not significantly altered. Together, our results suggest that TRPM7 kinase is a sensor of magnesium status and provides coordination of cellular and systemic responses to magnesium deprivation. PMID:25534891

  20. Synthesis of high affinity fluorine-substituted ligands for the androgen receptor. Potential agents for imaging prostatic cancer by positron emission tomography.

    PubMed

    Liu, A; Carlson, K E; Katzenellenbogen, J A

    1992-05-29

    We have prepared nine androgens substituted with fluorine at C-16 or C-20 to evaluate their potential, as positron emission tomographic (PET) imaging agents for prostatic cancer when labeled with the positron emitting radionuclide fluorine-18 (t1/2 = 110 min). These compounds represent members from the following classes of androgens: testosterone (T), 5 alpha-dihydrotestosterone (DHT), 7 alpha-methyl-19-nortestosterone (MNT), mibolerone (Mib), and metribolone (R1881). All of these compounds were prepared by functionalization of suitable androgen precursors, and the synthetic routes were developed to allow the introduction of fluorine by a fluoride ion displacement reaction late in the synthesis, as is required for the preparation of these compounds in fluorine-18 labeled form. We have also prepared four androgens in which the C-3 carbonyl or 17 beta-hydroxyl groups are replaced by fluorine. Most of the fluorine-substituted androgens show high affinity for the androgen receptor (AR), although fluorine substitution lowers their affinity by a small factor. None of the androgens where fluorine replaces oxygen functions at C-3 or C-17 have substantial affinity for AR. Derivatives of the natural androgens (T and DHT) as well as MNT have little affinity for other steroid hormone receptors (progesterone and mineralocorticoid receptors), whereas the Mib and R1881 derivatives have somewhat greater heterologous binding. With sex steroid binding protein, a human serum binding protein, the pattern of binding affinities is nearly the reverse, with derivatives of Mib, R1881 and MNT having low affinity, and DHT and T, high affinity. From these fluorine-substituted compounds, we can select several whose preparation in fluorine-18 labeled form for further tissue distribution studies is merited.

  1. The inhibition of the human cholesterol 7alpha-hydroxylase gene (CYP7A1) promoter by fibrates in cultured cells is mediated via the liver x receptor alpha and peroxisome proliferator-activated receptor alpha heterodimer.

    PubMed

    Gbaguidi, G Franck; Agellon, Luis B

    2004-01-01

    In previous work, we showed that the binding of the liver x receptor alpha:peroxisome proliferator-activated receptor alpha (LXRalpha:PPARalpha) heterodimer to the murine Cyp7a1 gene promoter antagonizes the stimulatory effect of their respective ligands. In this study, we determined if LXRalpha:PPARalpha can also regulate human CYP7A1 gene promoter activity. Co-expression of LXRalpha and PPARalpha in McArdle RH7777 hepatoma cells decreased the activity of the human CYP7A1 gene promoter in response to fibrates and 25-hydroxycholesterol. In vitro, the human CYP7A1 Site I bound LXRalpha:PPARalpha, although with substantially less affinity compared with the murine Cyp7a1 Site I. The binding of LXRalpha:PPARalpha to human CYP7A1 Site I was increased in the presence of either LXRalpha or PPARalpha ligands. In HepG2 hepatoblastoma cells, fibrates and 25-hydroxycholesterol inhibited the expression of the endogenous CYP7A1 gene as well as the human CYP7A1 gene promoter when co-transfected with plasmids encoding LXRalpha and PPARalpha. However, a derivative of the human CYP7A1 gene promoter that contains a mutant form of Site I that does not bind LXRalpha:PPARalpha was not inhibited by WY 14,643 or 25-hydroxycholesterol in both McArdle RH7777 and HepG2 cells. The ligand-dependent recruitment of LXRalpha:PPARalpha heterodimer onto the human CYP7A1 Site I can explain the inhibition of the human CYP7A1 gene promoter in response to fibrates and 25-hydroxycholesterol.

  2. Unusually high rates of metabolism of DHT in cytosols of the quail uropygial gland.

    PubMed

    Amet, Y; di Stefano, S; Quemener, E; Abalain, J H; Floch, H H; Daniel, J Y

    1990-05-01

    The metabolism of DHT in the cytosol of the quail uropygial gland was found to be so high that the steroid was almost completely inactivated within 2 hours of incubation at 0 C. In these conditions, DHT cannot be used for the characterization of androgen receptors. By contrast, R 1881 and mibolerone, which are not metabolized, can be used as alternative ligands. Moreover, the extremely high metabolism of DHT questions the physiologic role of this steroid in the quail uropygial gland.

  3. (Radiolabeled androgens and progestins as imaging agents for tumors of the prostate and breast)

    SciTech Connect

    Katzenellenbogen, J.A.

    1991-01-01

    The specific aims of the previous grant application can be summarized as follows: Synthesize fluorine-substituted progestins from the following high affinity classes: R5020 (promegestone), norgestrel, RU486, and retroprogestins; Synthesize fluorine-substituted androgens from the following high affinity classes: mibolerone, R1881 (metribolone) and 2-oxometribolone; Evaluate the receptor binding and non-specific binding of these fluorosteroids by in vitro binding assays; Develop and optimize fluoride ion substitution reactions suitable for the rapid, efficient and convenient preparation of these fluorosteroids in high specific activity, F-18 labeled form; and Evaluate the target tissue uptake of the F-18 labeled androgens and progestins in experimental animals.

  4. [Radiolabeled androgens and progestins as imaging agents for tumors of the prostate and breast]. Progress report

    SciTech Connect

    Katzenellenbogen, J.A.

    1991-12-31

    The specific aims of the previous grant application can be summarized as follows: Synthesize fluorine-substituted progestins from the following high affinity classes: R5020 (promegestone), norgestrel, RU486, and retroprogestins; Synthesize fluorine-substituted androgens from the following high affinity classes: mibolerone, R1881 (metribolone) and 2-oxometribolone; Evaluate the receptor binding and non-specific binding of these fluorosteroids by in vitro binding assays; Develop and optimize fluoride ion substitution reactions suitable for the rapid, efficient and convenient preparation of these fluorosteroids in high specific activity, F-18 labeled form; and Evaluate the target tissue uptake of the F-18 labeled androgens and progestins in experimental animals.

  5. Anti-Cancer Effect of Lambertianic Acid by Inhibiting the AR in LNCaP Cells

    PubMed Central

    Lee, Myoung-Sun; Lee, Seon-Ok; Kim, Sung-Hoon; Lee, Eun-Ok; Lee, Hyo-Jeong

    2016-01-01

    Lambertianic acid (LA) is known to have anti-allergic and antibacterial effects. However, the anticancer activities and mechanism of action of LA have not been investigated. Therefore, the anticancer effects and mechanism of LA are investigated in this study. LA decreased not only AR protein levels, but also cellular and secretory levels of PSA. Furthermore, LA inhibited nuclear translocation of the AR induced by mibolerone. LA suppressed cell proliferation by inducing G1 arrest, downregulating CDK4/6 and cyclin D1 and activating p53 and its downstream molecules, p21 and p27. LA induced apoptosis and the expression of related proteins, including cleaved caspase-9 and -3, c-PARP and BAX, and inhibited BCl-2. The role of AR in LA-induced apoptosis was assessed by using siRNA. Collectively, these findings suggest that LA exerts the anticancer effect by inhibiting AR and is a valuable therapeutic agent in prostate cancer treatment. PMID:27399684

  6. Lignan derivatives from the stem bark of Syzygium cumini (L.) Skeels.

    PubMed

    Mir, Qurat Y; Ali, M; Alam, Prawez

    2009-01-01

    Phytochemical investigation of the stem bark of Syzygium cumini (L.) Skeels (Myrtaceae) yielded four new lignan derivatives characterised as (7alpha,8alpha,2'alpha)-3,4,5-trimethoxy-7,3',1',9'-diepoxylignan (cuminiresinol), (7alpha,7'alpha,8alpha,8'alpha)-3,4-dioxymethylene-3',4'-dimethoxy-7,9',7',9-diepoxylignan-5'-ol (5'-hydroxy-methyl-piperitol), (7alpha,7'alpha,8alpha,8'alpha)-3'-methoxy-9-oxo-7,9',7',9-diepoxylignan-3,4,4'-triol or 3-demethyl-9-oxo-pinoresinol (syzygiresinol A), (7alpha,7'alpha,8alpha,8'alpha)-9-oxo-7,9',7',9-diepoxylignan-3,4,3',4',5'-pentaol or 3,3'-didemethyl-9-oxo-pinoresinol (syzygiresinol B) along with the known lignans di-demethyl-5-hydroxypinoresinol, dimethylpinoresinol, didemethoxypinoresinol, pinoresinol and 4'-methyl-5'-hydroxypinoresinol. The structures of these lignans were elucidated on the basis of structural data analysis and chemical reactions.

  7. Novel labdane diterpenes from the insecticidal plant hyptis spicigera1

    PubMed

    Fragoso-Serrano; Gonzalez-Chimeo; Pereda-Miranda

    1999-01-01

    Seven new labdane diterpenes with insecticidal properties were isolated from the aerial parts of Hyptis spicigera. Their structures were established on the basis of spectral (MS, 1H NMR, and 13C NMR) and chemical evidences as: 19-acetoxy-2alpha,7alpha, 15-trihydroxylabda-8(17),(13Z)-diene (1); 15,19-diacetoxy-2alpha, 7alpha-dihydroxylabda-8(17),(13Z)-diene (2); 7alpha,15, 19-triacetoxy-2alpha-hydroxylabda-8(17),(13Z)-diene (3); 19-acetoxy-2alpha,7alpha-dihydroxylabda-8(17),(13Z)-dien-15-al (4); 19-acetoxy-7alpha,15-dihydroxylabda-8(17),(13Z)-dien-2-one (5); 19-acetoxy-2alpha,7alpha-dihydroxylabda-14, 15-dinorlabd-8(17)-en-13-one (6); and 2alpha,7alpha,15, 19-tetrahydroxy-ent-labda-8(17),(13Z)-diene (7). Absolute configurations were established by application of Mosher's method. Compound 2 significantly inhibited larval growth of the European corn borer.

  8. Microbial transformation of the diterpene 7-epi-foliol by Fusarium fujikuroi.

    PubMed

    Fraga, Braulio M; Bressa, Carlo; González, Pedro; Guillermo, Ricardo

    2014-08-01

    The incubation of 3alpha,7alpha,18-trihydroxy-ent-kaur-16-ene (7-epi-foliol) with the fungus Fusarium fujikuroi gave 3alpha,7alpha,18-trihydroxy-ent-kaur-16-en-18-al as the sole product. The biotransformation of other 7alpha- or 7beta-hydroxy derivatives had led to the oxidation of C-19, which is a main step in the biosynthesis of gibberellins and kaurenolides. Now, the presence of the 3alpha-hydroxyl impedes that oxidation, which is directed to the adjacent C-18 hydroxymethyl forming the corresponding aldehyde.

  9. 21 CFR 1308.11 - Schedule I.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ..., levomethadyl acetate, or LAAM) 9603 (5) Alphameprodine 9604 (6) Alphamethadol 9605 (7) Alpha-methylfentanyl (N... acid; sodium oxybate; sodium oxybutyrate) 2010 (2) Mecloqualone 2572 (3) Methaqualone 2565 (f...

  10. 21 CFR 1308.11 - Schedule I.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ..., levomethadyl acetate, or LAAM) 9603 (5) Alphameprodine 9604 (6) Alphamethadol 9605 (7) Alpha-methylfentanyl (N... acid; sodium oxybate; sodium oxybutyrate) 2010 (2) Mecloqualone 2572 (3) Methaqualone 2565 (f...

  11. 21 CFR 1308.11 - Schedule I.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ..., levomethadyl acetate, or LAAM) 9603 (5) Alphameprodine 9604 (6) Alphamethadol 9605 (7) Alpha-methylfentanyl (N... acid; sodium oxybate; sodium oxybutyrate) 2010 (2) Mecloqualone 2572 (3) Methaqualone 2565 (f...

  12. 21 CFR 1308.11 - Schedule I.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ..., levomethadyl acetate, or LAAM) 9603 (5) Alphameprodine 9604 (6) Alphamethadol 9605 (7) Alpha-methylfentanyl (N...-hydroxybutyrate; 4-hydroxybutyrate; 4-hydroxybutanoic acid; sodium oxybate; sodium oxybutyrate) 2010 (2...

  13. Bile salts of the coelacanth, Latimeria chalumnae.

    PubMed

    Kihira, K; Akashi, Y; Kuroki, S; Yanagisawa, J; Nakayama, F; Hoshita, T

    1984-12-01

    Bile salts of the coelacanth, Latimeria chalumnae, Smith, have been analyzed and shown to have three bile alcohols, latimerol, 5 alpha-cyprinol, and 5 alpha-cholestane-3 beta, 7 alpha,-12 alpha,25,26-pentol, two C24 bile acids, chenodeoxycholic acid and cholic acid, one C26 bile acid, probably 3 beta, 7 alpha, 12 alpha-trihydroxy-27-nor-5 alpha-cholestan-26-oic acid, and two C27 bile acids, 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-cholestan-26-oic acid and 3 beta,7 alpha,12 alpha-trihydroxy-5 alpha-cholestan-26-oic acid as determined by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry.

  14. Chemical composition and biological activity of Nepeta parnassica oils and isolated nepetalactones.

    PubMed

    Gkinis, Giorgos; Tzakou, Olga; Iliopoulou, Dimitra; Roussis, Vassilios

    2003-01-01

    Essential oils of Nepeta parnassica, collected at different developmental stages, were analyzed by means of GC/MS. From the fifty-five identified constituents in samples A and B, representing 94.8% and 98.7% of the oils respectively, 4a(alpha),7alpha,7alpha(beta)-nepetalactone (22.0%), 1,8-cineole (21.1%), alpha-pinene (9.5%) and 4a(alpha),7,beta,7alpha(beta)-nepetalactone (7.9%) were the major components of sample A (vegetative stage), whereas in sample B (flowering stage) the main contributors were 1,8-cineole (34.6%), 4a(alpha),7alpha,7a(alpha)-nepetalactone (17.3%), alpha-pinene (11.4%) and 4a(alpha),7alpha,7alpha(beta)-nepetalactone (8.9%). The oils were tested on human health important insects such as the Pogonomyrmex sp. ants and the Culex pipiens molestus mosquitoes with promising results on insect repellency/toxicity.

  15. Androgens downregulate miR-21 expression in breast cancer cells underlining the protective role of androgen receptor

    PubMed Central

    Donà, Ada; Rizza, Pietro; Aquila, Saveria; Avena, Paola; Lanzino, Marilena; Pellegrino, Michele; Vivacqua, Adele; Tucci, Paola; Morelli, Catia; Andò, Sebastiano; Sisci, Diego

    2016-01-01

    Although the protective role of androgen receptor (AR) in breast cancer (BC) is well established, the mechanisms involved remains largely unexplored. MicroRNAs play fundamental roles in many biological processes, including tumor cell development and metastasis. Herein, we report that androgens reduce BC cells proliferation acting as a negative modulator of the onco-miRNA-21. The synthetic androgen miboleron (Mib) decreases BC cell proliferation induced by miR-21 over-expression and AR knockdown evidenced the requirement of AR in the down-regulation of miR-21 expression. These effects seem to be a general mechanism occurring in BC tissues. Chromatin immune-precipitation (ChIP) analysis disclosed the binding of AR to a specific ARE sequence in miR-21 proximal promoter and recognizes the recruitment of HDAC3 as component for AR-mediated transcriptional repression. Such event is associated to a significantly reduced PolII binding in Mib treated extracts confirming that activated AR is a transcriptional repressor of miR-21 expression, providing further insight into the protective role of androgens in breast cancer cells. Collectively, our data and the widespread AR expression in primary and metastatic breast tumours, suggest a careful examination of the therapeutic potential of androgens also in potentiating the effectiveness of anti-oestrogen adjuvant therapies. PMID:26862856

  16. (Fluorine-18 labeled androgens and progestins: Imaging agents for tumors of the prostate and breast)

    SciTech Connect

    Katzenellenbogen, J.A.

    1990-09-20

    The objective of this project is to develop fluorine-18 labeled steroids which possess high binding affinity and selectivity for androgen and progesterone receptors and can be used as positron-emission tomographic imaging agents for prostate tumors and breast tumors, respectively. These novel diagnostic agents may enable an accurate estimation of tumor dissemination (metastasis of prostate cancer and lymph node involvement of breast cancer) and an in vivo determination of the endocrine responsiveness of these tumors. Thus, they will provide essential information for the selection of alternative therapies (the extent of surgical ablation, radiation and chemotherapy vs hormonal therapy, etc.), thereby improving the management of prostate and breast cancer patients. Specific aims of the program include: synthesize fluorine-substituted progestins from the following high affinity classes: R5020 (promegestone), norgestrel, RU486, and retroprogestins; synthesize fluorine-substituted androgens from the following high affinity classes: mibolerone, R1881 (metribolone) and 2-oxametribolone; evaluate the receptor binding and non-specific binding of these fluorosteroids by in vitro binding assays; develop and optimize fluoride ion substitution reactions suitable for the rapid, efficient, and convenient preparation of these fluorosteroids in high specific activity, F-18 labeled form; and evaluate the target tissue uptake of the F-18 labeled androgens and progestins in experimental animals. We have synthesized several new fluorine-substituted androgens (1--6) over the past year. Their structures and binding affinity for the androgen receptor (RBA) are listed in this paper. 6 refs.

  17. Androgens downregulate miR-21 expression in breast cancer cells underlining the protective role of androgen receptor.

    PubMed

    Casaburi, Ivan; Cesario, Maria Grazia; Donà, Ada; Rizza, Pietro; Aquila, Saveria; Avena, Paola; Lanzino, Marilena; Pellegrino, Michele; Vivacqua, Adele; Tucci, Paola; Morelli, Catia; Andò, Sebastiano; Sisci, Diego

    2016-03-15

    Although the protective role of androgen receptor (AR) in breast cancer (BC) is well established, the mechanisms involved remains largely unexplored. MicroRNAs play fundamental roles in many biological processes, including tumor cell development and metastasis. Herein, we report that androgens reduce BC cells proliferation acting as a negative modulator of the onco-miRNA-21.The synthetic androgen miboleron (Mib) decreases BC cell proliferation induced by miR-21 over-expression and AR knockdown evidenced the requirement of AR in the down-regulation of miR-21 expression. These effects seem to be a general mechanism occurring in BC tissues.Chromatin immune-precipitation (ChIP) analysis disclosed the binding of AR to a specific ARE sequence in miR-21 proximal promoter and recognizes the recruitment of HDAC3 as component for AR-mediated transcriptional repression. Such event is associated to a significantly reduced PolII binding in Mib treated extracts confirming that activated AR is a transcriptional repressor of miR-21 expression, providing further insight into the protective role of androgens in breast cancer cells.Collectively, our data and the widespread AR expression in primary and metastatic breast tumours, suggest a careful examination of the therapeutic potential of androgens also in potentiating the effectiveness of anti-oestrogen adjuvant therapies.

  18. Precise conditional immortalization of mouse cells using tetracycline-regulated SV40 large T-antigen.

    PubMed

    Anastassiadis, Konstantinos; Rostovskaya, Maria; Lubitz, Sandra; Weidlich, Stefanie; Stewart, A Francis

    2010-04-01

    Cellular immortalization provides a way for expansion and subsequent molecular characterization of rare cell types. Ideally, immortalization can be achieved by the reversible expression of immortalizing proteins. Here, we describe the use of conditional immortalization based on a modified tetracycline-regulated system for the expression of SV40 large T-antigen in embryonic stem (ES) cells and mice. The modified system relies on a codon improved reverse tetracycline transactivator (irtTA) fused to the ligand-binding domain (LBD) of the androgen receptor (irtTA-ABD) or of a mutated glucocorticoid receptor (irtTA-GBD*). Induction of T-antigen is conferred only after addition of two ligands, one to activate the LBD (mibolerone for irtTA-ABD or dexamethasone for irtTA-GBD*) and one to activate the tetracycline transactivator (doxycycline). In ES cells, changes in gene expression upon large T induction were limited and reversible upon deinduction. Similarly, expression of T-antigen was very tightly regulated in mice. We have isolated and expanded bone marrow mesenchymal stem cells that could be genetically manipulated and maintained their differentiation properties after several passages of expansion under conditions that induce the expression of large T-antigen. 2010 Wiley-Liss, Inc.

  19. Studies of targeting and intracellular trafficking of an anti-androgen doxorubicin-formaldehyde conjugate in PC-3 prostate cancer cells bearing androgen receptor-GFP chimera.

    PubMed

    Cogan, Peter S; Koch, Tad H

    2004-11-04

    The synthesis of a doxorubicin-formaldehyde conjugate bound to the nonsteroidal anti-androgen cyanonilutamide, via a cleavable tether, and binding of the construct to cell free androgen receptor (AR) as a function of tether design were previously reported. Cyanonilutamide bearing a linear alkyne tether bound to the AR better than other designs. Fluorescence microscopy studies of binding of the lead targeted drug, as well as various tethered cyanonilutamides, to the AR and subsequent trafficking of the resulting AR complex in live PC3 prostate cancer cells transfected with AR-green fluorescent protein (GFP) chimera are now described. Cyanonilutamide and cyanonilutamide bonded to a linear alkyne tether caused translocation of AR-GFP to the nucleus. In general, the ability of tethered cyanonilutamides to cause translocation paralleled their binding affinity for the AR. However, a noncleavable form of the lead cyanonilutamide-doxorubicin-formaldehyde conjugate bound to AR-GFP but the resulting complex did not translocate to the nucleus. Binding was apparent from the drugs inhibition of Mibolerone-induced translocation. Direct observation of anthraquinone fluorescence of targeted drug in PC3 cells showed initial cytosolic localization, independent of AR expression, with predominant nuclear localization after sufficient time for release of drug from the targeting moiety. The results indicate that doxorubicin-formaldehyde conjugate bonded to cyanonilutamide via a cleavable linear tether enters PC3 cells, resides in cytosol, binds to the AR if present, and ultimately releases doxorubicin or a doxorubicin derivative to the nucleus.

  20. Acid phosphatase activity: a marker of androgen action in prostate explant cultures.

    PubMed

    Shao, T C; Kong, A Y; Cunningham, G R

    1987-01-01

    Acid phosphatase activity in rat ventral prostate explants has been assayed to determine if this parameter could serve as a specific and quantitative marker of androgen action in this in vitro model. Dihydrotestosterone (10 nM) caused an absolute increase in both total (42.5 +/- 2.9 vs control 27.1 +/- 4.0 nmoles p-nitrophenol generated in 30 min/micrograms DNA, P less than .01) and tartrate-resistant acid phosphatase activity (34.1 +/- 1.5 vs control 17.2 +/- 2.8 U/micrograms DNA, P less than .05), and this effect was maximal on the 4th day of culture. This was the time when explant weight and DNA content tended to fall or only to be maintained by androgen. Similar changes were observed with the potent synthetic androgen, mibolerone. The addition of either the antiandrogen cyproterone acetate or flutamide in a 100-fold excess to that of androgen caused significant inhibition in acid phosphatase activity. No significant change was observed at low concentrations of estradiol or progesterone, and only minimal and inconsistent increases in activity were noted at high concentrations. No increase was noted when cortisol, cyproterone acetate, or flutamide was added to the media. We conclude that measurement of acid phosphatase activity in cultured explants of rat ventral prostate provides a biochemical marker of androgenicity that is more specific than measurement of [3H]-thymidine incorporation.

  1. The microbiological transformation of two 15beta-hydroxy-ent-kaurene diterpenes by Gibberella fujikuroi.

    PubMed

    Fraga, Braulio M; Guillermo, Ricardo; Hernández, Melchor G

    2004-01-01

    The incubation of 15beta-hydroxy-3-oxo-ent-kaur-16-ene (1) with the fungus Gibberella fujikuroi afforded 11beta-hydroxy-3,15-dioxo-ent-kaurane (6), 11beta,15beta-dihydroxy-3-oxo-ent-kaur-16-ene (8), 7beta,11beta,15beta-trihydroxy-3-oxo-ent-kaur-16-ene (9), 7alpha,11beta-dihydroxy-3,15-dioxo-ent-kaurane (7), and 7alpha,11beta,15beta-trihydroxy-3-oxo-ent-kaur-16-ene (10). The incubation of 15beta-hydroxy-ent-kaur-2,16-diene (3) with the same fungus yielded 7alpha,11beta-dihydroxy-15-oxo-ent-kaur-2-ene (12), 7alpha,11beta,15beta-trihydroxy-ent-kaur-2,16-diene (13), 7beta,15beta-dihydroxy-ent-kaur-2,16-dien-19,6-olide (14), 1beta,7beta,15beta-trihydroxy-ent-kaur-2,16-dien-19-oic acid (15), 7alpha,11beta,16alpha-trihydroxy-15-oxo-ent-kaur-2-ene (17), and 7alpha,15beta,17-trihydroxy-11beta,16beta-epoxy-ent-kaur-2-ene (19). These results indicated that a 3-oxo group in ent-kaur-16-ene derivatives inhibits the oxidation at C-19, typical of the biosynthetic pathway of gibberellins and kaurenolides, while a 2,3-double bond or a 15beta-OH does not. In both substrates a 15beta-alcohol directs hydroxylations at C-11(beta) and C-7(alpha), while in those with a 2,3-double bond the functionalization of C-1(beta) is favored.

  2. Steroidal inhibitors as chemical probes of the active site of aromatase.

    PubMed

    Brueggemeir, R W; Moh, P P; Ebrahimian, S; Darby, M V

    1993-03-01

    Androstenedione analogs containing 7 alpha-substituents have proven to be potent inhibitors of aromatase in human placental microsomes, in MCF-7 mammary cell cultures, and in JAr choriocarcinoma cells. Recent investigations have focused on the use of mechanism-based inhibitors, such as 7 alpha-substituted 1,4-androstadienediones, to biochemically probe the active site of aromatase. Inhibition kinetics were determined under initial velocity conditions using purified human placental cytochrome P450arom protein in a reconstituted system. Derivatives of 1,4-androstadiene-3,17-dione and 1,4,6-androstatriene-3,17-dione exhibited high affinity in the purified enzyme system. 7 alpha-(4'-Amino)phenylthio-1,4-androstadiene-3,17-dione, abbreviated 7 alpha-APTADD, demonstrated rapid time-dependent, first-order inactivation of reconstituted aromatase activity only in the presence of NADPH. The apparent Kinact for 7 alpha-APTADD is 11.8 nM, the first-order rate of inactivation is 2.72 x 10(-3) sec-1, and the half-time of inactivation at infinite inhibitor concentration is 4.25 min. The values for the rate constant and half-time of inactivation are similar to those observed in the placental microsomal assay system. Further studies were performed with radioiodinated 7 alpha-(4'-iodo)phenylthio-1,4-androstadienedione, 7 alpha-IPTADD, and the reconstituted aromatase system. Incubations with [125I] 7 alpha-IPTADD were followed by protein precipitation, solvent extraction, and column chromatography. Analysis of the isolated cytochrome P450arom by gel electrophoresis and autoradiography demonstrated the presence of only one radioactive band, which corresponded to the protein staining band for cytochrome P450arom. HPLC radiochromatographic analysis of the isolated cytochrome P450aroM confirmed the presence of only one radioactive peak coeluting with the u.v. peak for cytochrome P450arom. Peptide mapping analysis by reverse-phase HPLC of digested inhibitor-cytochrome P450arom complex

  3. Steroids from the leaves of Chinese Melia azedarach and their cytotoxic effects on human cancer cell lines.

    PubMed

    Wu, Shi-Biao; Ji, Yan-Ping; Zhu, Jing-Jing; Zhao, Yun; Xia, Gang; Hu, Ying-He; Hu, Jin-Feng

    2009-09-01

    Three new (1-3) and several known (4-6) steroids were isolated from the leaves of Chinese Melia azedarach. The structures of the new compounds were elucidated by means of spectroscopic methods including 2D NMR techniques and mass spectrometry to be (20S)-5,24(28)-ergostadiene-3beta,7alpha,16beta,20-tetrol (1), (20S)-5-ergostene-3beta,7alpha,16beta,20-tetrol (2), and 2alpha,3beta-dihydro-5-pregnen-16-one (3). The cytotoxicities of the isolated compounds against three human cancer cell lines (A549, H460, U251) were evaluated; only compounds 1, 2, and (20S)-5-stigmastene-3beta,7alpha,20-triol (4) were found to show significant cyctotoxic effects with IC(50)s from 12.0 to 30.1 microg/mL.

  4. In vivo and vitro studies on formation of bile acids in patients with Zellweger syndrome. Evidence that peroxisomes are of importance in the normal biosynthesis of both cholic and chenodeoxycholic acid.

    PubMed Central

    Kase, B F; Pedersen, J I; Strandvik, B; Björkhem, I

    1985-01-01

    The last step in bile acid formation involves conversion of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into cholic acid and 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) into chenodeoxycholic acid. The peroxisomal fraction of rat and human liver has the highest capacity to catalyze these reactions. Infants with Zellweger syndrome lack liver peroxisomes, and accumulate 5 beta-cholestanoic acids in bile and serum. We recently showed that such an infant had reduced capacity to convert a cholic acid precursor, 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol into cholic acid. 7 alpha-Hydroxy-4-cholesten-3-one is a common precursor for both cholic acid and chenodeoxycholic acid. Intravenous administration of [3H]7 alpha-hydroxy-4-cholesten-3-one to an infant with Zellweger syndrome led to a rapid incorporation of 3H into biliary THCA but only 10% of 3H was incorporated into cholic acid after 48 h. The incorporation of 3H into DHCA was only 25% of that into THCA and the incorporation into chenodeoxycholic acid approximately 50% of that in cholic acid. The conversion of intravenously administered [3H]THCA into cholic acid in another infant with Zellweger syndrome was only 7%. There was a slow conversion of THCA into 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-C29-dicarboxylic acid. The pool size of both cholic- and chenodeoxycholic acid was markedly reduced. Preparations of liver from two patients with Zellweger syndrome had no capacity to catalyze conversion of THCA into cholic acid. There was, however, a small conversion of DHCA into chenodeoxycholic acid and into THCA. It is concluded that liver peroxisomes are important both for the conversion of THCA into cholic acid and DHCA into chenodeoxycholic acid. PMID:4077985

  5. High abundance androgen receptor in goldfish brain: characteristics and seasonal changes

    SciTech Connect

    Pasmanik, M.; Callard, G.V.

    1988-08-01

    Testosterone (T) exerts its actions in brain directly via androgen receptors or, after aromatization to estradiol, via estrogen receptors. Brain aromatase activity in teleost fish is 100-1000 times greater than in mammals and would be expected to significantly reduce the quantity of androgen available for receptor binding. Experiments were carried out on the goldfish Carassius auratus to determine if androgen receptors are present in teleost brain and whether their physicochemical properties reflect elevated aromatase. Cytosolic and nuclear extracts were assayed with the use of (/sup 3/H)T and charcoal, Sephadex LH-20, or DNA-cellulose chromatography to separate bound and free steroids. Binding activity was saturable and had an equally high affinity for T and 5 alpha-dihydrotestosterone. Although mibolerone was a relatively weak competitor, the putative teleost androgen 11-ketotestosterone, methyltrienolone (R1881), estradiol, progesterone, and cortisol were poor ligands. Characteristics that distinguish this receptor from a steroid-binding protein in goldfish serum are the presence of binding activity in both nuclear and cytosolic extracts, a low rate of ligand-receptor dissociation, electrophoretic mobility, sedimentation properties in low vs. high salt, and tissue distribution. DNA cellulose-adhering and nonadhering forms were detected, but these did not differ in other variables measured. Although goldfish androgen receptors resembled those of mammals in all important physicochemical characteristics, they were unusually abundant compared to levels in rat brain, but comparable to levels in prostate and other male sex hormone target organs. Moreover, there were seasonal variations in total receptors, with a peak at spawning (April) 4- to 5-fold higher than values in reproductively inactive fish.

  6. Steroid-mediated inhibition of cAMP induced de novo synthesis of cytochrome P-450/sub 17 / in Leydig cell cultures

    SciTech Connect

    Hales, D.B.; Sha, L.; Payne, A.H.

    1987-05-01

    The present study was designed to investigate the mechanism by which testosterone (T), produced during cAMP induction of P-450/sub 17 /, modulates the rate of its de novo synthesis. Purified Leydig cells (LC) were maintained in culture for 7 days prior to the initiation of treatment. De novo synthesis was determined by TVS-methionine incorporation, immunoprecipitation with specific antibody, separation by SDS-gel electrophoresis and quantitation by laser densitometry. Treatment of LC with 0.05 mM 8-Br-cAMP (cA) results in a time-dependent increase in the rate of de novo synthesis of P-450/sub 17 / which is increased 2 fold when T production is inhibited by aminoglutethimide (AG). The addition of increasing concentrations of the androgen receptor antagonist, hydroxyflutamide (1-10 M), to cA treated LC enhances the rate of synthesis similar to that seen in cA-treated LC in which T production was inhibited by AG. The addition of increasing concentrations of T (0.05-5 M) or the androgen agonist, mibolerone (1-5 M), to cA + AG treated LC causes a dose-dependent reversal of the AG-enhanced increase in the rate of cA-induced de novo synthesis of P-450/sub 17 /. Addition of estradiol (1 M) or dexamethasone (1 M) was without effect. These data indicate that T produced during cA induction of P-450/sub 17 / negatively regulates the rate of synthesis of this cytochrome P-450 enzyme by an androgen receptor mediated mechanism.

  7. Proteomic-coupled-network analysis of T877A-androgen receptor interactomes can predict clinical prostate cancer outcomes between White (non-Hispanic) and African-American groups.

    PubMed

    Zaman, Naif; Giannopoulos, Paresa N; Chowdhury, Shafinaz; Bonneil, Eric; Thibault, Pierre; Wang, Edwin; Trifiro, Mark; Paliouras, Miltiadis

    2014-01-01

    The androgen receptor (AR) remains an important contributor to the neoplastic evolution of prostate cancer (CaP). CaP progression is linked to several somatic AR mutational changes that endow upon the AR dramatic gain-of-function properties. One of the most common somatic mutations identified is Thr877-to-Ala (T877A), located in the ligand-binding domain, that results in a receptor capable of promiscuous binding and activation by a variety of steroid hormones and ligands including estrogens, progestins, glucocorticoids, and several anti-androgens. In an attempt to further define somatic mutated AR gain-of-function properties, as a consequence of its promiscuous ligand binding, we undertook a proteomic/network analysis approach to characterize the protein interactome of the mutant T877A-AR in LNCaP cells under eight different ligand-specific treatments (dihydrotestosterone, mibolerone, R1881, testosterone, estradiol, progesterone, dexamethasone, and cyproterone acetate). In extending the analysis of our multi-ligand complexes of the mutant T877A-AR we observed significant enrichment of specific complexes between normal and primary prostatic tumors, which were furthermore correlated with known clinical outcomes. Further analysis of certain mutant T877A-AR complexes showed specific population preferences distinguishing primary prostatic disease between white (non-Hispanic) vs. African-American males. Moreover, these cancer-related AR-protein complexes demonstrated predictive survival outcomes specific to CaP, and not for breast, lung, lymphoma or medulloblastoma cancers. Our study, by coupling data generated by our proteomics to network analysis of clinical samples, has helped to define real and novel biological pathways in complicated gain-of-function AR complex systems.

  8. The use of androgen receptor amino/carboxyl-terminal interaction assays to investigate androgen receptor gene mutations in subjects with varying degrees of androgen insensitivity.

    PubMed

    Ghali, Shereen A; Gottlieb, Bruce; Lumbroso, Rose; Beitel, Lenore K; Elhaji, Youssef; Wu, Jian; Pinsky, Leonard; Trifiro, Mark A

    2003-05-01

    Five mutations in the ligand-binding domain (LBD) of the human androgen receptor (hAR) found in patients with varying degrees of androgen insensitivity syndrome (AIS) were investigated for their effects on receptor dynamics. These were Arg(871)Gly (mild), Ser(814)Asn (partial), Glu(772)Ala (partial), Val(866)Met (complete), and Arg(774)Cys (complete). Previous analysis showed that the mutant receptors exhibited near-normal kinetics, except Arg(774)Cys, which had severely reduced androgen binding, and Val(866)Met, which showed increased equilibrium dissociation constant (K(d)) and elevated dissociation rate (k) values. Ser(814)Asn exhibited ligand-selective k values, i.e. increased for dihydrotestosterone and mibolerone, but normal for methyltrenolene. Using mammalian two-hybrid assays, hAR amino/carboxyl (N/C)-terminal interactions of the mutant receptors were analyzed in the presence and absence of the hAR coactivator transcription intermediary factor 2 (TIF2). The mutations conferred decreased hAR N/C-terminal interaction, i.e. mild (approximately 1.5-fold), partial (2-fold), and complete (10-fold), that mirrored the degree of AIS. All mutant LBDs showed a 2- to 3-fold increase in N/C-terminal interactions when TIF2 was cotransfected, although of a magnitude still less than that of wild-type LBD with TIF2. The ligand-selective properties of the Ser(814)Asn mutant were also clearly reflected by the N/C-terminal interactions. Thus, measurement of N/C-terminal interactions may assist in the molecular analysis of mutant hARs associated with AIS.

  9. A new antibacterial sesquiterpene from Premna oligotricha.

    PubMed

    Habtemariam, S; Gray, A I; Waterman, P G

    1993-01-01

    A novel sesquiterpene, 7 alpha-hydroxy-6, 11-cyclofarnes-3(15)-en-2-one [1], has been isolated from the aerial parts of Premna oligotricha (Verbenaceae) using an antimicrobial bioassay-guided isolation procedure. The sesquiterpene was identified on the basis of spectroscopic data and showed weak activity against Gram-positive bacteria Bacillus pumilus, Bacillus subtilis, Staphylococcus aureus, and Streptococcus faecalis.

  10. [Chemical investingation of Chinese mangrove Sonneratia apetala II].

    PubMed

    Ji, Qing-fei; Lin, Wen-han; Li, Jun; Li, Wei; Kazuo, Koike; Tamotsu, Nikaido; Fu, Hong-zheng

    2005-08-01

    Isolation and structural elucidation of the constituents of China mangrove Sonneratia apetala. chromatography methods were used for isolation of compounds, spectroscopic methods were used for structural identifyication. seven known compounds named (+/-) symgaresinol, betulinic acid, lupeol, lupeone, stigmast-5-ene-3beta, 7alpha-diol, beta-alpha myrin hexadecaneate, physcoion were isolated. these known compouns were unreported previously from this plant.

  11. Transport of resistance-inducing sterols in phloem sap of barley.

    PubMed

    Lehrer, A T; Dugassa-Gobena, D; Vidal, S; Seifert, K

    2000-01-01

    After root application of [7alpha-3H]-7beta-hydroxysitosterol and [3alpha,6beta-3H2]-6alpha-hydroxylathosterol these sterols could be detected in the leaves and phloem sap feeding aphids. These results imply that the phloem sap is a sterol transport system in barley plants.

  12. Ozonation of cholesterol in the presence of ethanol: identification of a cytotoxic ethoxyhydroperoxide molecule.

    PubMed

    Tagiri-Endo, Misako; Nakagawa, Kiyotaka; Sugawara, Tatsuya; Ono, Kaori; Miyazawa, Teruo

    2004-03-01

    Cholesterol ozonation was carried out in ethanol-containing aqueous or nonaqueous solvent, and the ozonized products were analyzed by chemiluminescence detection-HPLC with on-line electrospray MS (HPLC-CL-MS) and characterized on the basis of NMR and FABMS. After the ozonolysis of cholesterol in water/ethanol (aqueous system) as well as in chloroform/ethanol (nonaqueous system), a unique ethoxyhydroperoxide molecule (7alpha-ethoxy-3beta-hydroxy-5alpha-B-homo-6-oxacholestane-5-hydroperoxide, termed "7alpha-ethoxy-5-OOH") appeared as main ozonation product. In addition to structural analysis, we confirmed the remarkable cytotoxicity of 7alpha-ethoxy-5-OOH toward human lung adenocarcinoma A549 cells and found that its cytotoxicity is superior to that of the commonly known autoxidized cholesterol (3beta-hydroxycholest-5-ene-7-one). Hence, 7alpha-ethoxy-5-OOH is a toxic molecule of primary importance, arising during cholesterol ozonation in the presence of ethanol.

  13. Neurosteroid hydroxylase CYP7B: vivid reporter activity in dentate gyrus of gene-targeted mice and abolition of a widespread pathway of steroid and oxysterol hydroxylation.

    PubMed

    Rose, K; Allan, A; Gauldie, S; Stapleton, G; Dobbie, L; Dott, K; Martin, C; Wang, L; Hedlund, E; Seckl, J R; Gustafsson, J A; Lathe, R

    2001-06-29

    The major adrenal steroid dehydroepiandrosterone (DHEA) enhances memory and immune function but has no known dedicated receptor; local metabolism may govern its activity. We described a cytochrome P450 expressed in brain and other tissues, CYP7B, that catalyzes the 7alpha-hydroxylation of oxysterols and 3beta-hydroxysteroids including DHEA. We report here that CYP7B mRNA and 7alpha-hydroxylation activity are widespread in rat tissues. However, steroids related to DHEA are reported to be modified at positions other than 7alpha, exemplified by prominent 6alpha-hydroxylation of 5alpha-androstane-3beta,17beta-diol (A/anediol) in some rodent tissues including brain. To determine whether CYP7B is responsible for these and other activities we disrupted the mouse Cyp7b gene by targeted insertion of an IRES-lacZ reporter cassette, placing reporter enzyme activity (beta-galactosidase) under Cyp7b promoter control. In heterozygous mouse brain, chromogenic detection of reporter activity was strikingly restricted to the dentate gyrus. Staining did not exactly reproduce the in situ hybridization expression pattern; post-transcriptional control is inferred. Lower level staining was detected in cerebellum, liver, and kidney, and which largely paralleled mRNA distribution. Liver and kidney expression was sexually dimorphic. Mice homozygous for the insertion are viable and superficially normal, but ex vivo metabolism of DHEA to 7alpha-hydroxy-DHEA was abolished in brain, spleen, thymus, heart, lung, prostate, uterus, and mammary gland; lower abundance metabolites were also eliminated. 7alpha-Hydroxylation of 25-hydroxycholesterol and related substrates was also abolished, as was presumed 6alpha-hydroxylation of A/anediol. These different enzyme activities therefore derive from the Cyp7b gene. CYP7B is thus a major extrahepatic steroid and oxysterol hydroxylase and provides the predominant route for local metabolism of DHEA and related molecules in brain and other tissues.

  14. Human hepatoblastoma cells (HepG2) and rat hepatoma cells are defective in important enzyme activities in the oxidation of the C27 steroid side chain in bile acid formation.

    PubMed

    Farrants, A K; Nilsson, A; Pedersen, J I

    1993-12-01

    We have examined the ability of HepG2 human hepatoblastoma cells and 7800 C1 Morris rat hepatoma cells to convert 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) to cholic acid and chenodeoxycholic acid, respectively. Cell extracts from both these cell lines could neither form cholic acid from THCA nor from the activated form, THCA-CoA. This suggests that both cell lines are defective in two enzyme activities involved in the pathway, the microsomal THCA-CoA ligase and the peroxisomal THCA-CoA oxidase. Furthermore, we show that the subsequent enzymes are active in the conversion to bile acids, because the product of the THCA-CoA oxidase, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-24-enoyl-coenzyme A (delta 24-THCA-CoA) or delta 24-THCA in the presence of THCA-CoA ligase, are converted to cholic acid by both cell lines. HepG2 cells were able to slowly form chenodeoxycholic acid and cholic acid from 5 beta-cholestane-3 alpha, 7 alpha-diol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, respectively, in 24- and 96-h incubations. The rate of cholic acid formation was lower than the rate for chenodeoxycholic acid and there was a clear accumulation of THCA. 7800 C1 Morris cells had no ability to form cholic acid or chenodeoxycholic acid after 96 h incubation. We conclude that these two cell lines have defects in two enzyme activities involved in the peroxisomal oxidation in bile acid formation, the microsomal THCA-CoA ligase and the peroxisomal THCA-CoA oxidase.

  15. The Effect of Finasteride and Dutasteride on the Growth of WPE1-NA22 Prostate Cancer Xenografts in Nude Mice

    PubMed Central

    Opoku-Acheampong, Alexander B.; Nelsen, Michelle K.; Unis, Dave; Lindshield, Brian L.

    2012-01-01

    Background 5α-reductase 1 (5αR1) and 5α-reductase 2 (5αR2) convert testosterone into the more potent androgen dihydrotestosterone. 5αR2 is the main isoenzyme in normal prostate tissue; however, most prostate tumors have increased 5αR1 and decreased 5αR2 expression. Previously, finasteride (5αR2 inhibitor) treatment begun 3 weeks post-tumor implantation had no effect on Dunning R3327-H rat prostate tumor growth. We believe the tumor compensated for finasteride treatment by increasing tumor 5αR1 expression or activity. We hypothesize that finasteride treatment would not significantly alter tumor growth even if begun before tumor implantation, whereas dutasteride (5αR1 and 5αR2 inhibitor) treatment would decrease tumor growth regardless of whether treatment was initiated before or after tumor implantation. Methodology/Principal Findings Sixty 8-week-old male nude mice were randomized to Control, Pre- and Post-Finasteride, and Pre- and Post-Dutasteride (83.3 mg drug/kg diet) diet groups. Pre- and post-groups began their treatment diets 1–2 weeks prior to or 3 weeks after subcutaneous injection of 1×105 WPE1-NA22 human prostate cancer cells, respectively. Tumors were allowed to grow for 22 weeks; tumor areas, body weights, and food intakes were measured weekly. At study's conclusion, prostate and seminal vesicle weights were significantly decreased in all treatment groups versus the control; dutasteride intake significantly decreased seminal vesicle weights compared to finasteride intake. No differences were measured in final tumor areas or tumor weights between groups, likely due to poor tumor growth. In follow-up studies, proliferation of WPE1-NA22 prostate cancer cells and parent line RWPE-1 prostate epithelial cells were unaltered by treatment with testosterone, dihydrotestosterone, or mibolerone, suggesting that these cell lines are not androgen-sensitive. Conclusion The lack of response of WPE1-NA22 prostate cancer cells to androgen treatment may

  16. Discordant measures of androgen-binding kinetics in two mutant androgen receptors causing mild or partial androgen insensitivity, respectively.

    PubMed

    Shkolny, D L; Beitel, L K; Ginsberg, J; Pekeles, G; Arbour, L; Pinsky, L; Trifiro, M A

    1999-02-01

    We have characterized two different mutations of the human androgen receptor (hAR) found in two unrelated subjects with androgen insensitivity syndrome (AIS): in one, the external genitalia were ambiguous (partial, PAIS); in the other, they were male, but small (mild, MAIS). Single base substitutions have been found in both individuals: E772A in the PAIS subject, and R871G in the MAIS patient. In COS-1 cells transfected with the E772A and R871G hARs, the apparent equilibrium dissociation constants (Kd) for mibolerone (MB) and methyltrienolone are normal. Nonetheless, the mutant hAR from the PAIS subject (E772A) has elevated nonequilibrium dissociation rate constants (k(diss)) for both androgens. In contrast, the MAIS subject's hAR (R871G) has k(diss) values that are apparently normal for MB and methyltrienolone; in addition, the R871G hAR's ability to bind MB resists thermal stress better than the hAR from the PAIS subject. The E772A and R871G hARs, therefore, confer the same pattern of discordant androgen-binding parameters in transfected COS-1 cells as observed previously in the subjects' genital skin fibroblasts. This proves their pathogenicity and correlates with the relative severity of the clinical phenotype. In COS-1 cells transfected with an androgen-responsive reporter gene, trans-activation was 50% of normal in cells containing either mutant hAR. However, mutant hAR-MB binding is unstable during prolonged incubation with MB, whereas normal hAR-MB binding increases. Thus, normal equilibrium dissociation constants alone, as determined by Scatchard analysis, may not be indicative of normal hAR function. An increased k(diss) despite a normal Kd for a given androgen suggests that it not only has increased egress from a mutant ligand-binding pocket, but also increased access to it. This hypothesis has certain implications in terms of the three-dimensional model of the ligand-binding domain of the nuclear receptor superfamily.

  17. Androgen responsive adult human prostatic epithelial cell lines immortalized by human papillomavirus 18.

    PubMed

    Bello, D; Webber, M M; Kleinman, H K; Wartinger, D D; Rhim, J S

    1997-06-01

    Prostate cancer and benign tumors of the prostate are the two most common neoplastic diseases in men in the United States, however, research on their causes and treatment has been slow because of the difficulty in obtaining fresh samples of human tissue and a lack of well characterized cell lines which exhibit growth and differentiation characteristics of normal prostatic epithelium. Non-neoplastic adult human prostatic epithelial cells from a white male donor were immortalized with human papillomavirus 18 which resulted in the establishment of the RWPE-1 cell line. Cells from the RWPE-1 cell line were further transformed by v-Ki-ras to establish the RWPE-2 cell line. The objectives of this study were to: (1) establish the prostatic epithelial origin and androgen responsiveness of RWPE-1 and RWPE-2 cell lines; (2) examine their response to growth factors; and (3) establish the malignant characteristics of the RWPE-2 cell line. Immunoperoxidase staining showed that both RWPE-1 and RWPE-2 cells express cytokeratins 8 and 18, which are characteristic of luminal prostatic epithelial cells, but they also coexpress basal cell cytokeratins. These cell lines show growth stimulation and prostate specific antigen (PSA) and androgen receptor (AR) expression in response to the synthetic androgen mibolerone, which establishes their prostatic epithelial origin. Both cell lines also show a dose-dependent growth stimulation by EGF and bFGF and growth inhibition when exposed to TGF-beta, however, the transformed RWPE-2 cells are less responsive. RWPE-1 cells neither grow in agar nor form tumors when injected into nude mice with or without Matrigel. However, RWPE-2 cells form colonies in agar and tumors in nude mice. In the in vitro invasion assay, RWPE-1 cells are not invasive whereas RWPE-2 cells are invasive. Nuclear expression of p53 and Rb proteins was heterogeneous but detectable by immunostaining in both cell lines. The RWPE-1 cells, which show many normal cell

  18. Effect of coprophagy on bile acid metabolism in the rabbit.

    PubMed

    Yahiro, K; Setoguchi, T; Katsuki, T

    1979-12-01

    The effect of coprophagy on the 7 alpha-dehydroxylation of biliary bile acids was studied in the rabbit. Bile acid composition of bile and intestinal contents was analyzed by gas-liquid chromatography and thin layer chromatography. Biliary bile acid composition of normal rabbits (n = 5) was: deoxycholic acid, 95.3 +/- 1.0SE % and cholic acid, 2.3 +/- 1.1SE %. When coprophagy was prevented, significant alterations were observed in biliary bile acid composition, including a considerable decrease in deoxycholic acid (82.5 +/- 2.8SE %, p less than 0.01) and a marked increase in cholic acid (15.2 +/- 3.0SE %, p less than 0.002). These results indicate that coprophagy is a factor causing an increase of the 7 alpha-dehydroxylated bile acid, deoxycholic acid (and lithocholic acid when the animals were fed chenodeoxycholic acid) in rabbit bile.

  19. Bile salts of the green turtle Chelonia mydas (L.)

    PubMed Central

    Haslewood, G A; Ikawa, S; Tökés, L; Wong, D

    1978-01-01

    1. Bile salts of the green turtle Chelonia mydas (L.) were analysed as completely as possible. 2. They consist of taurine conjugates of 3 alpha, 7 alpha, 12 alpha, 22 xi-tetrahydroxy-5 beta-cholestan-26-oic acid (tetrahydroxysterocholanic acid) and 3 alpha 12 alpha, 22 xi-trihydroxy-5 beta-cholestan-26-oic acid, with minor amounts of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5beta-cholan-24-oic acid (cholic acid), 3alpha, 12 alpha-dihydroxy-5beta-cholan-24-oic acid (deoxycholic acid) and possibly other bile acids. 3. Cholic acid and deoxycholic acid represent the first known examples of bile acids common to chelonians and other animal forms: they may indicate independent evolution in chelonians to C24 bile acids. 4. The discovery of a 7-deoxy C27 bile acid is the first evidence that C27 bile acids or their conjugates have an enterohepatic circulation. PMID:656052

  20. Ergosteroids: induction of thermogenic enzymes in liver of rats treated with steroids derived from dehydroepiandrosterone.

    PubMed

    Lardy, H; Partridge, B; Kneer, N; Wei, Y

    1995-07-03

    Dehydroepiandrosterone (DHEA), an intermediate in the biosynthesis of testosterone and estrogens, exerts several physiological effects not involving the sex hormones. When fed to rats it induces the thermogenic enzymes mitochondrial sn-glycerol-3-phosphate dehydrogenase and cytosolic malic enzyme in their livers. Animals and humans, and their excised tissues, are known to hydroxylate DHEA at several positions and to interconvert 7 alpha-hydroxy-DHEA, 7 beta-hydroxy-DHEA, 7-oxo-DHEA, and the corresponding derivatives of androst-5-enediol. We report here that these 7-oxygenated derivatives are active inducers of these thermogenic enzymes in rats and that the 7-oxo derivatives are more active than the parent steroids. We postulate that the 7 alpha-hydroxy and 7-oxo derivatives are on a metabolic pathway from DHEA to more active steroid hormones. These 7-oxo steroids have potential as therapeutic agents because of their increased activity and because they are not convertible to either testosterone or estrogens.

  1. Lipoxygenase inhibitory constituents of the fruits of noni (Morinda citrifolia) collected in Tahiti.

    PubMed

    Deng, Shixin; Palu, 'Afa K; West, Brett J; Su, Chen X; Zhou, Bing-Nan; Jensen, Jarakae C

    2007-05-01

    A phytochemical study of the fruits of noni (Morinda citrifolia) collected in Tahiti led to the isolation of two new lignans, (+)-3,4,3',4'-tetrahydroxy-9,7'alpha-epoxylignano-7 alpha,9'-lactone (1) and (+)-3,3'-bisdemethyltanegool (2), as well as seven known compounds, (-)-pinoresinol (3), (-)-3,3'-bisdemethylpinoresinol (4), quercetin (5), kaempferol (6), scopoletin (7), isoscopoletin (8), and vanillin. The structures of 1 and 2 were determined by spectroscopic techniques. Compounds 3, 6, and 8 were isolated for the first time from noni fruit. Compounds 1-8 were shown to inhibit 5- and/or 15-lipoxygenase, with IC50 values ranging from 0.43 to 16.5 microM. Compound 5 exhibited weak inhibitory activity toward cyclooxygenase-2.

  2. Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway.

    PubMed

    Lehmann, J M; Kliewer, S A; Moore, L B; Smith-Oliver, T A; Oliver, B B; Su, J L; Sundseth, S S; Winegar, D A; Blanchard, D E; Spencer, T A; Willson, T M

    1997-02-07

    Accumulation of cholesterol causes both repression of genes controlling cholesterol biosynthesis and cellular uptake and induction of cholesterol 7alpha-hydroxylase, which leads to the removal of cholesterol by increased metabolism to bile acids. Here, we report that LXRalpha and LXRbeta, two orphan members of the nuclear receptor superfamily, are activated by 24(S), 25-epoxycholesterol and 24(S)-hydroxycholesterol at physiologic concentrations. In addition, we have identified an LXR response element in the promoter region of the rat cholesterol 7alpha-hydroxylase gene. Our data provide evidence for a new hormonal signaling pathway that activates transcription in response to oxysterols and suggest that LXRs play a critical role in the regulation of cholesterol homeostasis.

  3. Novel cytotoxic oxygenated C29 sterols from the Colombian marine sponge Polymastia tenax.

    PubMed

    Santafé, Gilmar; Paz, Vanessa; Rodríguez, Jaime; Jiménez, Carlos

    2002-08-01

    Three new sterols, 5alpha,6alpha-epoxy-24R-ethylcholest-8(14)-en-3beta,7alpha-diol (1), 5alpha,6alpha-epoxy-24R-ethylcholest-8-en-3beta,7alpha-diol (2), and 3beta-hydroxy-24R-ethylcholesta-5,8-dien-7-one (3), have been isolated from the marine sponge Polymastia tenax, collected in the Colombian Caribbean, and their structures established on the basis of extensive NMR and MS studies. Compounds 1 and 2 showed antiproliferative activity toward A-549, HT-29, H-116, MS-1, and PC-3 tumor cells in the range 0.5-10 microg/mL.

  4. Quantitative analysis of geraniol, nerol, linalool, and alpha-terpineol in wine.

    PubMed

    Pedersen, D Sejer; Capone, Dimitra L; Skouroumounis, George K; Pollnitz, Alan P; Sefton, Mark A

    2003-02-01

    A mixture of [(2)H(7)]-geraniol, [(2)H(7)]-nerol, [(2)H(7)]-linalool and [(2)H(7)]-alpha-terpineol was prepared for use as internal standards in a rapid and accurate analytical method, employing gas chromatography-mass spectrometry (GC/MS), to determine the concentration of geraniol, nerol, linalool and alpha-terpineol in wine. The method avoids the possible formation, degradation and interconversion of these compounds during their analysis.

  5. alpha-globin gene deletion and point mutation analysis among in Iranian patients with microcytic hypochromic anemia.

    PubMed

    Garshasbi, Masoud; Oberkanins, Christian; Law, Hai Yang; Neishabury, Maryam; Kariminejad, Roxana; Najmabadi, Hossein

    2003-10-01

    We tested 67 Iranian individuals, presenting with low mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) levels, normal hemoglobin electrophoresis and iron status, for the presence of twelve common alpha-thalassemia gene deletions and point mutations. Five different mutations (-alpha(3.7), -alpha(4.2), --MED, -(alpha)20.5, Hb Constant Spring) were identified in a total of 43 cases

  6. Synthesis of (17 alpha,20E/Z)iodovinyl testosterone and 19-nortestosterone derivatives as potential radioligands for androgen and progesterone receptors.

    PubMed

    Ali, H; Rousseau, A J; van Lier, J E

    1994-05-01

    To develop androgen and progesterone receptor-based radioligands for SPECT imaging we synthesized several radioiodinated 17 alpha-iodovinyl testosterone and 19-nortestosterone analogs and evaluated their biological properties. The synthesis of these compounds proceeds via the (17 alpha,20E/Z)stannyl intermediates and involves addition of tri-n-butyltin hydride to the 17 alpha-ethynyl group of the steroid using either azobisiso butyronitrile or triethylborane as a catalyst. The stannyl derivatives are stereospecifically converted to the corresponding (17 alpha,20E/Z)iodovinyl derivatives using molecular iodine, or to the [125I]iodovinyl analogs using [125I]NaI and H2O2. Androgen and progesterone receptor (AR and PgR) binding affinities were measured via a competitive in vitro binding assay. In general 19-nortestosterone derivatives showed higher receptor affinities as compared to the testosterone derivatives. In the latter series the highest PgR binding affinities were observed with the (17 alpha,20Z)iodovinyl-19-nortestosterone (IVNT) (92 vs 100 for R5020) followed by the 7 alpha-methyl analog, whereas the highest AR binding affinity was observed with the 7 alpha-Me-(17 alpha,20Z)IVNT (54 vs 100 for 5 alpha-dihydrotestosterone). These derivatives were also labeled with 125I and evaluated for their in vivo target organ uptake (prostate and estrogen-primed uterus). The highest PgR-mediated target tissue uptake was observed with the (17 alpha,20Z)-[125I]IVNT and its 7 alpha-methyl derivatives whereas only one derivative, the 7 alpha-Me-(17 alpha,20Z)-[125I]IVNT, showed AR-mediated dorsal prostate retention. Although some of the IVNT derivatives have interesting binding properties, the lack of in vivo selectivity does suggest that the 123I-labeled analogs are unlikely to be suitable for imaging of AR and PgR-rich tissues.

  7. Hydroperoxysterols from the tunicate Eudistoma sp.

    PubMed

    Sung, Ping-Jyun; Lin, Mei-Ru; Chen, Jih-Jung; Lin, Shen-Fan; Wu, Yang-Chang; Hwang, Tsong-Long; Fang, Lee-Shing

    2007-04-01

    Two 27C hydroperoxysterols, 7beta-hydroperoxycholesterol (1) and its stereoisomer 7alpha-hydroperoxycholesterol (2), were isolated from the lipophilic extracts of a Formosan tunicate belonging to the genus Eudistoma. The structures of sterols 1 and 2 were elucidated on the basis of extensive spectral data analyses. Cytotoxicity of sterols 1 and 2 against a limited panel of cancer cell lines is also described. Sterol 1 show weak inhibitory effects on human neutrophil elastase release.

  8. Comparative phytochemical investigation of Salvia miltiorrhiza and Salvia triloba.

    PubMed

    Liu, J; Zapp, J; Becker, H

    1995-10-01

    From the aerial parts of Chinese Salvia miltiorrhiza Bunge a novel sesquiterpene (1), which possesses a rare substitution pattern, together with seven known abietane type diterpenes has been isolated. Structure 1 was elucidated on the basis of spectral analysis, including 2D-NMR spectroscopy, especially long range 1H-13C correlation (COLOC). At the same time royleanone, 7alpha-acetoxyroyleanone, and sugiol were identified for the first time from the roots of Salvia triloba L. f.

  9. Treatment of refractory melasma with the MedLite C6 Q-switched Nd:YAG laser and alpha arbutin: a prospective study.

    PubMed

    Polnikorn, Niwat

    2010-06-01

    To evaluate the effectiveness of a Q-switched Nd:YAG laser (MedLite C6; HOYA ConBio, Fremont, CA, USA) and 7% alpha arbutin solution (Skin Advance Laboratory, Japan) in the treatment of melasma. This was a prospective study of 35 refractory melasma cases treated with 10 weekly laser sessions, two monthly follow-up treatments and topical 7% alpha arbutin solution. Clinical photographs and severity grading on a 5-point scale were carried out by an independent observer at each visit. At 6 months, 30% of study subjects received results in the excellent clearance category (> 81% reduction of melasma) and 36.7% received good (51-80% reduction) clearance. Mild and transient side effects included discomfort during treatment, erythema, whitening of fine hair and urticaria. Three cases of mottling hypo-pigmentation (8.57%) and two cases of recurrence of melasma (5.71%) were recorded. Combination therapy with the MedLite C6 and 7% alpha arbutin solution is an effective and well-tolerated treatment for refractory melasma.

  10. Electrospray ionization mass spectrometry and ion mobility analysis of the 20S proteasome complex.

    PubMed

    Loo, Joseph A; Berhane, Beniam; Kaddis, Catherine S; Wooding, Kerry M; Xie, Yongming; Kaufman, Stanley L; Chernushevich, Igor V

    2005-07-01

    Mass spectrometry and gas phase ion mobility [gas phase electrophoretic macromolecule analyzer (GEMMA)] with electrospray ionization were used to characterize the structure of the noncovalent 28-subunit 20S proteasome from Methanosarcina thermophila and rabbit. ESI-MS measurements with a quadrupole time-of-flight analyzer of the 192 kDa alpha7-ring and the intact 690 kDa alpha7beta7beta7alpha7 are consistent with their expected stoichiometries. Collisionally activated dissociation of the 20S gas phase complex yields loss of individual alpha-subunits only, and it is generally consistent with the known alpha7beta7beta7alpha7 architecture. The analysis of the binding of a reversible inhibitor to the 20S proteasome shows the expected stoichiometry of one inhibitor for each beta-subunit. Ion mobility measurements of the alpha7-ring and the alpha7beta7beta7alpha7 complex yield electrophoretic diameters of 10.9 and 15.1 nm, respectively; these dimensions are similar to those measured by crystallographic methods. Sequestration of multiple apo-myoglobin substrates by a lactacystin-inhibited 20S proteasome is demonstrated by GEMMA experiments. This study suggests that many elements of the gas phase structure of large protein complexes are preserved upon desolvation, and that methods such as mass spectrometry and ion mobility analysis can reveal structural details of the solution protein complex.

  11. Stimulation of follicular melanogenesis in the mouse by topical and injected melanotropins.

    PubMed

    Levine, N; Lemus-Wilson, A; Wood, S H; Abdel Malek, Z A; Al-Obeidi, F; Hruby, V J; Hadley, M E

    1987-09-01

    The effects of melanocyte-stimulating hormone (alpha-MSH) and related analogs on follicular melanogenesis in the mouse (C57BL/6JA gamma) were studied. [Nle4, D-Phe7]-alpha-MSH and the related fragment analogues Ac-[Nle4, D-Phe7]-alpha-MSH4-11-NH2 and Ac-[Nle4, D-Phe7]-alpha-MSH4-10-NH2, stimulated the conversion of pheomelanogenesis to eumelanogenesis when subcutaneously injected at concentrations 100-fold lower than the native hormone, alpha-MSH. In addition, the melanotropin analogs stimulated follicular eumelanogenesis when applied topically to the skin of mice. The melanotropins were transdermally delivered to the systemic circulation as evidenced by the fact that eumelanogenesis was stimulated in hair follicles in areas distant from the site of topical application. These results demonstrate that peptide hormone analogs can be transported across the skin. The unique actions of the melanotropin analogs may relate to the fact that these peptides are nonbiodegradable and thus exert prolonged actions on melanocytes. These compounds may prove important for studies on normal integumental melanogenesis and for the treatment of hypopigmentary disorders in humans.

  12. New allo-cedrane type sesquiterpene hemiketals and further sesquiterpene lactones from fruits of Illicium floridanum.

    PubMed

    Schmidt, T J; Müller, E; Fronczek, F R

    2001-04-01

    Three new tetracyclic sesquiterpene hemiketals possessing the very rare allo-cedrane carbon skeleton, debenzoyl-7-deoxo-1 alpha,7 alpha-dihydroxytashironin, debenzoyl-7-deoxo-7 alpha-hydroxytashironin, and debenzoyl-7-deoxo-7 alpha-hydroxy-3-oxotashironin (1--3), were isolated from the fruits of Illicium floridanum ELLIS (American Star Anise, Star bush). Their structures were elucidated by mass and NMR spectroscopic analyses. The molecular structure of 1, including absolute stereochemistry, was determined by single-crystal X-ray diffraction of its monohydrate. Only one compound of this type, tashironin, has previously been isolated from Illicium tashiroi. Furthermore, a new sesquiterpene lactone of the seco-prezizaane type, 3,4-anhydro-13,14-dihydroxyfloridanolide (4), and the known anislactone B (5) were isolated. The occurrence of further allo-cedrane sesquiterpenes in another Illicium species confirms the hypothesis of previous authors that this type of compound is a biogenetic precursor of the typical seco-prezizaane sesquiterpene lactones found in this genus. Moreover, regarding their co-occurrence with anislactone B as reported here, they may also be considered precursors for the anislactone skeletal type.

  13. Expression of mouse Fbxw7 isoforms is regulated in a cell cycle- or p53-dependent manner

    SciTech Connect

    Matsumoto, Akinobu; Onoyama, Ichiro; Nakayama, Keiichi I. . E-mail: nakayak1@bioreg.kyushu-u.ac.jp

    2006-11-10

    Fbxw7 is the F-box protein component of an SCF-type ubiquitin ligase that contributes to the ubiquitin-dependent degradation of cell cycle activators and oncoproteins. Three isoforms ({alpha}, {beta}, and {gamma}) of Fbxw7 are produced from mRNAs with distinct 5' exons. We have now investigated regulation of Fbxw7 expression in mouse tissues. Fbxw7{alpha} mRNA was present in all tissues examined, whereas Fbxw7{beta} mRNA was detected only in brain and testis, and Fbxw7{gamma} mRNA in heart and skeletal muscle. The amount of Fbxw7{alpha} mRNA was high during quiescence (G phase) in mouse embryonic fibroblasts (MEFs) and T cells, but it decreased markedly as these cells entered the cell cycle. The abundance of Fbxw7{alpha} mRNA was unaffected by cell irradiation or p53 status. In contrast, X-irradiation increased the amount of Fbxw7{beta} mRNA in wild-type MEFs but not in those from p53-deficient mice, suggesting that radiation-induced up-regulation of p53 leads to production of Fbxw7{beta} mRNA. Our results thus indicate that expression of Fbxw7 isoforms is differentially regulated in a cell cycle- or p53-dependent manner.

  14. Antimicrobial and antioxidant activity of the essential oil and methanol extract of Nepeta cataria.

    PubMed

    Adiguzel, Ahmet; Ozer, Hakan; Sokmen, Munevver; Gulluce, Medine; Sokmen, Atalay; Kilic, Hamdullah; Sahin, Fikrettin; Baris, Ozlem

    2009-01-01

    Catnip (Nepeta cataria) is an important medicinal herb belonging to the mint family, Lamiaceae. In this study, the in vitro antimicrobial and antioxidant activities of the essential oil and methanol extract from Nepeta cataria, and its essential oil composition were investigated. The essential oil, which has 4aalpha,7alpha,7abeta-nepetalactone (70.4%), 4aalpha,7alpha,7abeta-nepetalactone (6.0%), thymol (2.3%), and 4aalpha,7alpha, 7abeta3-nepetalactone (2.5%), as main components, exhibited activity against eleven bacteria, and twelve fungi and a yeast, C. albicans; with Minimum Inhibitory Concentrations (MIC) values ranging from 12.50 to 250 microl/ml; the methanol extract showed weaker activity. The samples were also subjected to a screening for their possible antioxidant activities by using 2.2-diphenyl-1-picrylhydrazyl (DPPH) and beta-carotene/linoleic acid assays. In DPPH assay, the extract showed slight antioxidant activity whereas the essential oil remained inactive. In the latter case, both the extract and the essential oil exerted weak activity having inhibiton ratios of linoleic acid oxidation at 16.4% and 27.0%, respectively. The weak antioxidative nature of the extract could be attributed to the low phenolic content, estimated as gallic acid equivalent at 22.6 +/- 2.07 microg/ml or 2.26%. In both systems, antioxidant capacity of BHT was determined in parallel experiments.

  15. ATP-dependent transport of bile acid intermediates across rat liver peroxisomal membranes.

    PubMed

    Une, Mizuho; Iguchi, Yusuke; Sakamoto, Tomoko; Tomita, Takashi; Suzuki, Yasuyuki; Morita, Masashi; Imanaka, Tsuneo

    2003-08-01

    The bile acid intermediate 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid (THCA) is converted to cholic acid exclusively in peroxisomes by the oxidative cleavage of the side chain. To investigate the mechanism by which the biosynthetic intermediates of bile acids are transported into peroxisomes, we incubated THCA or its CoA ester (THC-CoA) with isolated intact rat liver peroxisomes and analyzed their oxidation products, cholic acid and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoic acid. The oxidation of both THCA and THC-CoA was dependent on incubation time and peroxisomal proteins, and was stimulated by ATP. THC-CoA was efficiently oxidized to cholic acid and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoic acid as compared with THCA, suggesting that THC-CoA is the preferred substrate for transport into peroxisomes. The oxidation of THC-CoA was significantly inhibited by sodium azide, verapamile, and N-ethylmaleimide. Furthermore, the stimulatory effect of ATP on the oxidation was not replaced by GTP or AMP. In addition, the ATP-dependent oxidation of THC-CoA was markedly inhibited by pretreatment of peroxisomes with proteinase K when peroxisomal matrix proteins were not degraded. These results suggest that an ATP-dependent transport system for THC-CoA exists on peroxisomal membranes.

  16. Combination of chromatographic and spectroscopic methods for the isolation and characterization of polar guaianolides from Achillea asiatica.

    PubMed

    Glasl, S; Gunbilig, D; Narantuya, S; Werner, I; Jurenitsch, J

    2001-11-30

    Four polar guaianolides, 8alpha-angeloxy-2alpha,4alpha, 10beta-trihydroxy-6betaH,7alphaH, 11betaH-1(5)-guaien- 12,6alpha-olide; 8alpha-angeloxy-1beta,2beta:4beta,5beta-diepoxy- 10beta-hydroxy-6betaH,7alphaH,11betaH-12,6alpha-guaianolide; 8alpha-angeloxy-4alpha, 10beta-dihydroxy-2-oxo-6betaH, 7alphaH, 11betaH- 1(5)-guaien- 12,6alpha-olide and 8-desacetyl-matricarin, were isolated from Achillea asiatica and characterized by TLC, MS, IR, HPLC and diode array detection. Purified extracts were separated by means of flash chromatography. HPLC separations were achieved using different methanol-water gradients as mobile phase and LiChrospher 100-RP8 5 microm or Zorbax SB-C8 3.5 microm as stationary phases. The chromatographical data are compared to those of the proazulene 8alpha-tigloxy-artabsin which shows antiinflammatory effects. By means of these characteristics the identification of the guaianolides with potential antiphlogistic properties is also possible from other sources.

  17. Bile acids. 38. Conversion of 5 -cholestane-3 ,7 -diol to allo bile acids by the rat.

    PubMed

    Noll, B W; Doisy, E A; Elliott, W H

    1973-07-01

    5alpha-[4-(14)C, 3alpha-(3)H]Cholestane-3beta,7alpha-diol was prepared from individual samples of 5alpha-[3alpha-(3)H]cholestane-3beta,7alpha-diol and 5alpha-[4-(14)C]cholestane-3beta,7alpha-diol, each derived from 3beta-acetoxycholest-5-en-7-one. Bile was collected for 11 days from adult male rats, with cannulated bile ducts, that had received intraperitoneally 0.90-0.92 mg of the doubly labeled diol. Bile from the first 10 hr, containing 63% of the administered (14)C and 6% of the (3)H, was hydrolyzed, and the bile acids were separated by acetic acid partition chromatography. Allochenodeoxycholic and allocholic acids contained at least 20.6% and 48.6%, respectively, of the (14)C retained in the biliary acids. Small amounts of (14)C (2.5% and 1.9%, respectively) were present in the 3beta isomers of these acids, but the tritium content totaled more than half of that found in the bile acid fraction. No evidence was obtained for presence of the extensive quantities of the allomuricholates.

  18. Inhibitory effect of an oxygenated cholesterol on the induction and progression of DMBA-induced mammary carcinomas in the rat.

    PubMed

    Iversen, O H; Kolberg, A; Smith-Kielland, I; Stabursvik, A

    1986-01-01

    In vivo studies on the effect of two stereoisomeric 7,22-dihydroxycholesterols on tumor development were conducted in the Charles Huggins animal cancer model (DMBA-induced mammary cancer in the Sprague-Dawley female rat). Three groups of DMBA-treated animals were fed a 9:1 mixture of (22R)-cholest-5-ene-3 beta,7 beta,22-triol and (22R)-cholest-5-ene-3 beta,7 alpha,22-triol in the drinking water in a calculated dose of 250 micrograms per animal per day. One group (A) received the sterols throughout the experimental period of 35 weeks, another group (B) during the first 12 weeks only, and a third group (C) only during weeks 13 through 35 after DMBA injection. Tumor rates and tumor yields were calculated, and statistical assessment by accepted methods demonstrated a very significant inhibitory effect on tumor development in Groups A and B, as compared with Group C. The results indicate a growth-inhibitory effect during the induction period of carcinoma development. The influence on neoplastic growth of (22R)-cholest-5-ene-3 beta,7 alpha,22-triol, (22R)-cholest-5-ene-3 beta,7 beta,22-triol, and (22R)-cholest-5-ene-3 beta,22-diol-7-one was examined in suspension cultures of Ehrlich ascites tumor cells. The 7 alpha-hydroxy compound proved to be ineffective, whereas the latter two substances displayed a strong cytotoxic effect.

  19. The enzymic and chemical synthesis of ursodeoxycholic and chenodeoxycholic acid from cholic acid.

    PubMed

    Sutherland, J D; Macdonald, I A; Forrest, T P

    1982-01-01

    Three approaches to the synthesis of ursodeoxycholic acid (UDC) from cholic acid have been investigated: (i) oxidation of cholic acid to 3 alpha, 7 alpha-dihydroxy-12 keto-5 beta-cholanoic acid (12K-CDC) with Clostridium group P 12 alpha-hydroxysteroid dehydrogenase (HSDH), isomerization of 12K-CDC to 3 alpha, 7 beta-dihydroxy-12 keto-5 beta-cholanoic acid (12K-UDC) with Clostridium absonum 7 alpha- and 7 beta-HSDH and reduction of 12K-UDC by Wolff-Kishner to UDC; (ii) isomerization of cholic acid to ursocholic acid (UC) by C. absonum 7 alpha- and 7 beta-HSDH, oxidation of UC to 12K-UDC with Clostridium group P 12 alpha-HSDH and Wolff-Kishner reduction of 12K-UDC to UDC; (iii) oxidation of cholic acid to 12K-CDC by Clostridium group P 12 alpha-HSDH, Wolff-Kishner reduction of 12K-CDC to chenodeoxycholic acid (CDC) and isomerization of CDC to UDC using whole cell cultures of C. absonum. In the first two approaches (using cell free systems) the yields of desired product were relatively low primarily due to the formation of various side products. The third method proved the most successful giving an overall yield of 37% (UDC) whose structure was verified by mass spectroscopy of the methyl ester.

  20. Origin of oxysterols in hepatic bile of patients with biliary infection.

    PubMed

    Yoshida, Tadashi; Matsuzaki, Yasushi; Haigh, W Geoffrey; Fukushima, Sugano; Ikezawa, Kazuto; Tanaka, Naomi; Lee, Sum P

    2003-10-01

    Oxysterols are ubiquitous in the body and are potential cytotoxic agents in addition to being metabolic regulators. Although bile contains high concentrations of cholesterol, oxysterol concentrations in bile and the effect of infection on oxysterol levels have not been measured, nor has their origin been studied. The purpose of this study was to determine if infection of the biliary tract was associated with increased concentrations of oxysterols in the bile and, if so, which oxysterols showed a significant change. Hepatic bile was obtained from eight patients with biliary tract disease by means of a naso-biliary catheter. Oxysterols were extracted and purified by solid-phase extraction, derivatized and measured by gas chromatography-mass spectrometry. The following were quantified in hepatic bile: 7-alpha-hydroxycholesterol, 7-beta-hydroxycholesterol, cholestan-3-beta,5-alpha,6-beta-triol, 25-hydroxycholesterol, 26-hydroxycholesterol, 7-ketocholesterol, and 7-alpha-hydroxy-4-cholesten-3-one. Total oxysterols in hepatic bile ranged from 0.133 mumol/L to 7.748 mumol/L (1.47 +/- 2.55 mumol/L). Levels of 7-alpha-hydroxycholesterol and 7-beta-hydroxycholesterol were increased in infected bile (14.2 +/- 15.1 x 10(-3)% of cholesterol vs 1.9 +/- 0.5 x 10(-3)% of cholesterol, p < 0.05, and 22.0 +/- 25.0 x 10(-3)% of cholesterol vs 1.6 +/- 1.2 x 10(-3)% of cholesterol, p < 0.05, respectively). Serum C-reactive protein levels correlated positively with biliary levels of 7-alpha-hydroxycholesterol (R = 0.948), 7-beta-hydroxycholesterol (R = 0.976), cholestan-3-beta,5-alpha,6-beta-triol (R = 0.823), 7-alpha-hydroxy-4-cholesten-3-one (R = 0.846,) and 7-ketocholesterol (R = 0.973). Different oxysterols were found in gallstones, chiefly 3-keto-cholest-4-ene (624 +/- 316 parts per million [ppm] of dry weight), 3-keto-cholesta-4,6-diene (240 +/- 329 ppm) and 7-keto-cholesterol (77 +/- 81 ppm). Incubation of human leukocytes with model bile in the presence of bacterial

  1. Defective peroxisomal cleavage of the C27-steroid side chain in the cerebro-hepato-renal syndrome of Zellweger.

    PubMed Central

    Kase, B F; Björkhem, I; Hågå, P; Pedersen, J I

    1985-01-01

    Based on in vitro work with rat liver, we recently suggested that the peroxisomal fraction is most important for the oxidation of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into cholic acid. The cerebro-hepato-renal syndrome of Zellweger is a fatal recessive autosomal disorder, the most characteristic histological feature of which is a virtual absence of peroxisomes in liver and kidneys. This disease offers a unique opportunity to evaluate the relative importance of peroxisomes in bile acid biosynthesis. A child with Zellweger syndrome was studied in the present work. In accordance with previous work, there was a considerable accumulation of THCA, 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid (24-OH-THCA), 3 alpha, 7 alpha, 12 alpha-trihydroxy-27-carboxymethyl-5 beta-cholestan-26-oic acid (C29-dicarboxylic acid), and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid in serum. In addition, a tetrahydroxylated 5 beta-cholestanoic acid with all the hydroxyl groups in the steroid nucleus was found. 3H-Labeled 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol was administered intravenously together with 14C-labeled cholic acid. There was a rapid incorporation of 3H in THCA and a slow incorporation into cholic acid. The specific radioactivity of 3H in THCA was about one magnitude higher than that in cholic acid. The conversion was evaluated by following the increasing ratio between 3H and 14C in biliary cholic acid. The rate of incorporation of 3H in cholic acid was considerably less than previously reported in experiments with healthy subjects, and the maximal conversion of the triol into cholic acid was only 15-20%. About the same rate of conversion was found after oral administration of 3H-THCA. Both in the experiment with 3H-5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and with 3H-THCA, there was an efficient incorporation of 3H in the above unidentified tetrahydroxylated 5 beta-cholestanoic acid. There was

  2. Microbial metabolism of steviol and steviol-16alpha,17-epoxide.

    PubMed

    Yang, Li-Ming; Hsu, Feng-Lin; Chang, Shwu-Fen; Cheng, Juei-Tang; Hsu, Ju-Yin; Hsu, Chung-Yi; Liu, Pan-Chun; Lin, Shwu-Jiuan

    2007-02-01

    Steviol (2) possesses a blood glucose-lowering property. In order to produce potentially more- or less-active, toxic, or inactive metabolites compared to steviol (2), its microbial metabolism was investigated. Incubation of 2 with the microorganisms Bacillus megaterium ATCC 14581, Mucor recurvatus MR 36, and Aspergillus niger BCRC 32720 yielded one new metabolite, ent-7alpha,11beta,13-trihydroxykaur-16-en-19-oic acid (7), together with four known related biotransformation products, ent-7alpha,13-dihydroxykaur-16-en-19-oic acid (3), ent-13-hydroxykaur-16-en-19-alpha-d-glucopyranosyl ester (4), ent-13,16beta,17-trihydroxykauran-19-oic acid (5), and ent-13-hydroxy-7-ketokaur-16-en-19-oic acid (6). The preliminary testing of antihyperglycemic effects showed that 5 was more potent than the parent compound (2). Thus, the microbial metabolism of steviol-16alpha,17-epoxide (8) with M. recurvatus MR 36 was continued to produce higher amounts of 5 for future study of its action mechanism. Preparative-scale fermentation of 8 yielded 5, ent-11alpha,13,16alpha,17-tetrahydroxykauran-19-oic acid (10), ent-1beta,17-dihydroxy-16-ketobeyeran-19-oic acid (11), and ent-7alpha,17-dihydroxy-16-ketobeyeran-19-oic acid (13), together with three new metabolites: ent-13,16beta-dihydroxykauran-17-acetoxy-19-oic acid (9), ent-11beta,13-dihydroxy-16beta,17-epoxykauran-19-oic acid (12), and ent-11beta,13,16beta,17-tetrahydroxykauran-19-oic acid (14). The structures of the compounds were fully elucidated using 1D and 2D NMR spectroscopic techniques, as well as HRFABMS. In addition, a GRE (glucocorticoid responsive element)-mediated luciferase reporter assay was used to initially screen the compounds 3-5, and 7 as glucocorticoid agonists. Compounds 4, 5 and 7 showed significant effects.

  3. Bile acid synthesis is increased in Chilean Hispanics with gallstones and in gallstone high-risk Mapuche Indians.

    PubMed

    Gälman, Cecilia; Miquel, Juan Francisco; Pérez, Rosa Maria; Einarsson, Curt; Ståhle, Lars; Marshall, Guillermo; Nervi, Flavio; Rudling, Mats

    2004-03-01

    Gallstone disease is an important, costly health-care problem in Western societies. It is still unclear whether hepatic lipid regulatory enzymes play primary or secondary roles in gallstone formation. In this study, the aim was to investigate whether the synthesis of bile acids and cholesterol is increased in gallstone disease and to test whether such a metabolic change, if present, might occur before gallstone formation. A total of 125 Chilean Hispanic women (80 without gallstones and 45 with gallstones) matched for age and body mass index were investigated, along with 40 Chilean Mapuche Indian women (20 without gallstones and 20 with gallstones), a population group in which the prevalence for gallstone disease is very high. Fasting blood plasma samples were assayed for 7 alpha-hydroxy-4-cholesten-3-one and lathosterol, 2 strong indicators for hepatic bile acid and body cholesterol synthesis, respectively. Plasma 7 alpha-hydroxy-4-cholesten-3-one levels, corrected for plasma cholesterol, were significantly increased by 50% in Hispanic women with gallstones as compared with gallstone-free Hispanics (P < 0.006). As compared with Hispanic women without gallstones, plasma 7 alpha-hydroxy-4-cholesten-3-one levels were increased by > or =100% (P < 0.002) in Mapuche Indian women, independently of whether gallstones were present. Plasma lathosterol, corrected for plasma cholesterol, was significantly increased by 22% in Hispanic women with gallstones and in Mapuche Indian women compared with Hispanic women. The results indicate that the synthesis of bile acids and cholesterol is induced in gallstone disease and precedes gallstone development. These inductions presumably occur as a response to an increased intestinal loss of bile acids.

  4. Different patterns of metabolism determine the relative anabolic activity of 19-norandrogens.

    PubMed

    Sundaram, K; Kumar, N; Monder, C; Bardin, C W

    1995-06-01

    Testosterone, the principal androgen secreted by Leydig cells, exerts a wide range of actions including growth of the male reproductive tract (androgenic effects) and growth of non-reproductive tissues such as muscle, kidney, liver, and salivary gland (anabolic effects). As androgenic steroids were discovered some were found to have relatively more anabolic than androgenic activity. The results reviewed in this report suggest that these differences result, in part, from the differential metabolism of the steroids in individual tissues and the varied activities of the individual metabolites. In the accessory sex organs (e.g. the prostate) testosterone is 5 alpha-reduced to dihydrotestosterone (DHT) which, due to its higher affinity for androgen receptors (AR), amplifies the action of testosterone. In contrast, when 19-nortestosterone (NT) is 5 alpha-reduced, its affinity for AR decreases, resulting in a decrease in its androgenic potency. However, their anabolic potency remains unchanged since significant 5 alpha-reduction of the steroids does not occur in the muscle. 7 alpha-methyl-19-nortestosterone (MENT) does not get 5 alpha-reduced due to steric hindrance from the 7 alpha-methyl group. Therefore, the androgenic potency of MENT is not amplified as happens with testosterone. These metabolic differences are responsible for the increased anabolic activity of NT and MENT compared to testosterone. Part of the biological effects of testosterone are mediated by its aromatization to estrogens. The fact that MENT is also aromatized to 7 alpha-methyl estradiol, a potent estrogen, in vitro by human placental and rat ovarian aromatase suggests that some of the anabolic actions of MENT may be mediated by this estrogen.

  5. Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana

    SciTech Connect

    Noma, Y.; Kihira, K.; Kuramoto, T.; Hoshita, T.

    1988-03-01

    Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana, was studied. (24-14C)-24-Dehydro-26-deoxy-5 beta-ranol (3 alpha,7 alpha,12 alpha-trihydroxy-27-nor-5 beta-cholestan-24-one) was chemically synthesized from (24-14C)cholic acid and incubated with bullfrog liver homogenate fortified with NADPH. 24-Dehydro-26-deoxy-5 beta-ranol was shown to be converted into both 26-deoxy-5 beta-ranol and 24-epi-26-deoxy-5 beta-ranol ((24S)- and (24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrols) in addition to 5 beta-ranol ((24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol), which is the major bile alcohol of the bullfrog. (24-3H)-26-Deoxy-5 beta-ranol and (24-3H)-24-epi-26-deoxy-5 beta-ranol were prepared from 24-dehydro-26-deoxy-5 beta-ranol by reduction with sodium (3H) borohydride and administered respectively to two each of four bullfrogs by intraperitoneal injection. After 24 h, labeled 5 beta-ranol was isolated from the bile of the bullfrogs that received (24-3H)-26-deoxy-5 beta-ranol. In contrast little if any radioactivity could be detected in 5 beta-ranol or its 24-epimer after administration of (24-3H)-24-epi-26-deoxy-5 beta-ranol.

  6. Effects of simulated nuclear fuel particles on the histopathology and CYP enzymes in the rat lung and liver

    SciTech Connect

    Pasanen, M.; Lang, S.; Kojo, A.; Kosma, V.M.

    1995-08-01

    We studied both short-term (3 and 30 days) and long-term (3-24 months) effects of simulated nuclear fuel particles (neutron-activated UO{sub 2}) on the rat lung and liver histopathology and cytochrome P450 (CYP) activities. In the short-term study, after a single intratracheal instillation with neutron-activated particles (administered activity 36 kBq), the lung histology revealed inflammation and a decrease in several lung testosterone hydroxylation levels. Liver exhibited normal histology but hepatic testosterone 7{alpha}-hydroxylase 9T7{alpha}OH was decreased by 30% at 3 days treatment with neutron-activated particles (9.3 kBq). At 30 days after treatment, hepatic T7{alpha}OH and testosterone 15{alpha}-hydroxylase activities were enhanced by 70 and 40%, respectively. At the long-term follow-up, benign and malignant lung tumors were observed but in the livers only slightly increased inflammation was found. At the 1.5-year follow-up (cumulated lung dose 0.4-0.66 Gy, 131 and 182 kBq), decreases in lung testosterone 6{beta}-hydorxylase (60%) and testosterone 6{alpha}-hydroxylase (30%) activities were found. In contrast to lungs, hepatic testosterone 16{alpha}-hydroxylase activity decreased by 60-75% with both nonactivated UO{sub 2} or {beta}-emitting UO{sub 2} particles they have differential effects on CYP enzymes in both the primary target organ (lung) and secondary tissue (liver). 44 refs., 2 figs., 3 tabs.

  7. Effects of CYP7A1 overexpression on cholesterol and bile acid homeostasis.

    PubMed

    Pandak, W M; Schwarz, C; Hylemon, P B; Mallonee, D; Valerie, K; Heuman, D M; Fisher, R A; Redford, K; Vlahcevic, Z R

    2001-10-01

    The initial and rate-limiting step in the classic pathway of bile acid biosynthesis is 7alpha-hydroxylation of cholesterol, a reaction catalyzed by cholesterol 7alpha-hydroxylase (CYP7A1). The effect of CYP7A1 overexpression on cholesterol homeostasis in human liver cells has not been examined. The specific aim of this study was to determine the effects of overexpression of CYP7A1 on key regulatory steps involved in hepatocellular cholesterol homeostasis, using primary human hepatocytes (PHH) and HepG2 cells. Overexpression of CYP7A1 in HepG2 cells and PHH was accomplished by using a recombinant adenovirus encoding a CYP7A1 cDNA (AdCMV-CYP7A1). CYP7A1 overexpression resulted in a marked activation of the classic pathway of bile acid biosynthesis in both PHH and HepG2 cells. In response, there was decreased HMG-CoA-reductase (HMGR) activity, decreased acyl CoA:cholesterol acyltransferase (ACAT) activity, increased cholesteryl ester hydrolase (CEH) activity, and increased low-density lipoprotein receptor (LDLR) mRNA expression. Changes observed in HMGR, ACAT, and CEH mRNA levels paralleled changes in enzyme specific activities. More specifically, LDLR expression, ACAT activity, and CEH activity appeared responsive to an increase in cholesterol degradation after increased CYP7A1 expression. Conversely, accumulation of the oxysterol 7alpha-hydroxycholesterol in the microsomes after CYP7A1 overexpression was correlated with a decrease in HMGR activity.

  8. Molecular characterization of sickle cell anemia in the Northern Brazilian state of Pará.

    PubMed

    De Lemos Cardoso, Greice; Guerreiro, João Farias

    2010-01-01

    To assess alpha+-thalassemia deletion alleles, beta-thalassemia mutations and haplotypes linked to the HBB*S cluster in a sample of 130 unrelated sickle cell anemia (SCA) patients (55% female) from Belém, Pará State, for their possible effects on the patients' survival. -alpha(3.7), -alpha(42), -alpha(20.5), and -(MED) alpha+-thalassemia deletion alleles were investigated using multiplex gap-PCR method. Characterization of beta-thalassemia mutations was made by direct genomic sequencing of the beta-globin gene amplified through polymerase chain reaction (PCR). Haplotypes were determined by analysis of six polymorphic restriction sites [(1) XmnI-5'gammaG, (2) HindIII-gammaG, (3) HindIII-gammaA, (4) HincII-psibeta, (5) HincII-3'psibeta, and (6) HinfI-5'beta] followed by restriction digestion and agarose gel electrophoresis. Twenty-one patients (16%) presented -alpha3.7 thalassemia. Sixteen of those (76%) were heterozygous (-alpha3.7/alphaalpha) and 5 (24%) were homozygous (-alpha3.7/-alpha3.7). -Alpha(4.2), -alpha(20.5) and -(MED) deletions were not found. Nine cases of sickle cell-beta thalassemia were found and four different beta-thal mutations were identified: beta(+) -88 (C>T), 3.8%; beta(+) codon 24 (T > A), 1.5%; beta(+) IVSI-110 (G > A), 0.7% and beta (IVSI-1 (G > A), 0.7%. No differences according to age were observed in -alpha(3.7) deletion, beta-thalassemia and HHB*S haplotypes distribution. Our results suggest that although alpha- and beta-thalassemia and betaS haplotypes may have modulating effect on clinical expression and hematological parameters of SCA, these genetic variables probably have little influence on the subjects' survival.

  9. The neuro-steroid, 3beta androstene 17alpha diol exhibits potent cytotoxic effects on human malignant glioma and lymphoma cells through different programmed cell death pathways.

    PubMed

    Graf, M R; Jia, W; Loria, R M

    2007-09-03

    The neuro-steroids 3beta-androstene-17alpha-diol (17alpha-AED), 3beta-androstene-17beta-diol (17beta-AED), 3beta-androstene-7alpha,-17beta-triol (7alpha-AET) and 3beta-androstene-7beta,-17beta-triol (7beta-AET) are metabolites of dehydroepiandrosterone and are produced in neuro-ectodermal tissue. Both epimers of androstenediols (17alpha-AED and 17beta-AED) and androstenetriols (7alpha-AET and 7beta-AET) have markedly different biological functions of their chemical analogue. We investigated the cytotoxic activity of these neuro-steroids on human T98G and U251MG glioblastoma and U937 lymphoma cells. Proliferation studies showed that 17alpha-AED is the most potent inhibitor, with an IC(50) approximately 15 microM. For T98G glioma, 90% inhibition was achieved with 25 muM of 17alpha-AED. Other neuro-steroids tested only marginally suppressed cell proliferation. Reduced cell adherence and viability could be detected after 18 h of 17alpha-AED exposure. Treatment with 17alpha-AED induced a significant level of apoptosis in U937 lymphoma cells, but not in the glioma cells. Cytopathology of 17alpha-AED-treated T98G cells revealed the presence of multiple cytoplasmic vacuoles. Acridine orange staining demonstrated the formation of acidic vesicular organelles in 17alpha-AED-treated T98G and U251MG, which was inhibited by bafilomycin A1. These findings indicate that 17alpha-AED bears the most potent cytotoxic activity of the neuro-steroids tested, and the effectiveness may depend on the number of hydroxyls and their position on the androstene molecule. These cytotoxic effects may utilize a non-apoptotic pathway in malignant glioma cells.

  10. The chemical composition of the essential oils of Euphorbia caracasana and E. cotinifolia (Euphorbiaceae) from Venezuela.

    PubMed

    Rojas, Janne; Baldovino, Shirley; Vizcaya, Marietta; Rojas, Luis B; Morales, Antonio

    2009-04-01

    The essential oils from leaves of E. caracasana Boiss collected from Miyoi, Pueblo Llano, Mérida State at 1800 m above sea level and leaves of E. cotinifolia L collected from Manzano Alto-Ejido, Mérida State at 1520 m were analyzed by GC/MS. Beta-Caryophyllene (33.7 %), alpha-humulene (18.8 %) and aromadendrene (8.4 %) were the major constituents of E. caracasana oil, whereas those of E. cotinifolia oil were beta-caryophyllene (39.3 %), germacrene-D (21.5 %) and alpha-copaene (9.3 %).

  11. Bile Acid secreted by male sea lamprey that acts as a sex pheromone.

    PubMed

    Li, Weiming; Scott, Alexander P; Siefkes, Michael J; Yan, Honggao; Liu, Qin; Yun, Sang-Seon; Gage, Douglas A

    2002-04-05

    We show that reproductively mature male sea lampreys release a bile acid that acts as a potent sex pheromone, inducing preference and searching behavior in ovulated female lampreys. The secreted bile acid 7alpha,12alpha,24-trihydroxy-5alpha-cholan-3-one 24-sulfate was released in much higher amounts relative to known vertebrate steroid pheromones and may be secreted through the gills. Hence, the male of this fish species signals both its reproductive status and location to females by secreting a pheromone that can act over long distances.

  12. Diterpenoid acids from Grindelia nana.

    PubMed

    Mahmoud, A A; Ahmed, A A; Tanaka, T; Iinuma, M

    2000-03-01

    Two new norditerpenoid acids of the labdane-type (norgrindelic acids), 4,5-dehydro-6-oxo-18-norgrindelic acid (1) and 4beta-hydroxy-6-oxo-19-norgrindelic acid (2), as well as a new grindelic acid derivative, 18-hydroxy-6-oxogrindelic acid (3), were isolated from the aerial parts of Grindelia nana. In addition, the known compounds, 6-oxogrindelic acid, grindelic acid, methyl grindeloate, 7alpha,8alpha-epoxygrindelic acid, and 4alpha-carboxygrindelic acid were also isolated. The structures of the new compounds were characterized on the basis of spectroscopic analysis.

  13. Rapid detection of deletional alpha-thalassemia by an oligonucleotide microarray.

    PubMed

    Zesong, Li; Ruijun, Guo; Wen, Zhang

    2005-12-01

    A simple, fast, and highly reproducible protocol for detection of the three most common deletional alpha-thalassemias (- -SEA, - alpha3.7, -alpha4.2) using an oligonucleotide microarray was developed. PCR products were directly hybridized to the microarrays with different deletion-specific probes. Genotypes were determined by quantitative analysis of the fluorescent signals detected by fluorescence scanning. Blind assays on 400 samples validated the efficiency and specificity of the protocol. This method may be suitable for routine clinical use and population screening for deletional alpha-thalassemia. (c) 2005 Wiley-Liss, Inc.

  14. A new tetranortriterpenoid from Trichilia havanensis.

    PubMed

    Rodríguez, Benjamín; Caballero, Cristina; Ortego, Félix; Castañera, Pedro

    2003-03-01

    A new triterpenoid, 1beta,2beta;21,23-diepoxy-7alpha-hydroxy-24,25,26,27-tetranor-apotirucalla-14,20,22-trien-3-one (1), has been isolated from an acetone extract of seeds of Trichilia havanensis, along with other known limonoids. The structure of 1 was established by spectroscopic methods, particularly by 1D and 2D NMR studies. Compound 1 was tested as an antifeedant agent against Leptinotarsa decemlineata larvae, showing a significant antifeedant activity at 300 ppm.

  15. Estrogenic potencies of resorcylic acid lactones and 17 beta-estradiol in female rats.

    PubMed

    Everett, D J; Perry, C J; Scott, K A; Martin, B W; Terry, M K

    1987-01-01

    Uterotrophic response in sexually immature female rats has been used to rank the relative estrogenic potencies of six resorcylic acid lactones (RALs) and to compare their activities with that of 17 beta-estradiol. On oral administration, the estrogenic potency relative to 17 beta-estradiol is as follows: 7 alpha-zearalenol, 10 times less; zeranol, 150 times less; taleranol, 350 times less; zearalanone, 400 times less; zearalenone, 650 times less; 7 beta-zearalenol, 3500 times less. On subcutaneous administration, zeranol is 500 times less estrogenic than 17 beta-estradiol.

  16. Skeletal muscle and liver oxysterols during fasting and alcohol exposure.

    PubMed

    Adachi, Junko; Kudo, Risa; Asano, Migiwa; Ueno, Yasuhiro; Hunter, Ross; Rajendram, Rajkumar; Martin, Colin; Preedy, Victor R

    2006-01-01

    Oxysterols are cytotoxic agents that have a range of cellular actions, including impairment of albumin synthesis, cell differentiation, and induction of apoptosis. Their regulations by nutritional factors are poorly described. Our objective was to test the hypothesis that the imposition of food withdrawal and alcohol exposure increases tissue oxysterol concentrations. We measured the concentrations of the oxysterols 7alpha-hydroxycholest-5-en-3beta-ol (7alpha-OH), 7beta-hydroxycholest-5-en-3beta-ol (7beta-OH), and 3beta-hydroxycholest-5-en-7-one (7-keto) in liver and skeletal muscle of fed and fasted (food withdrawal for 1 and 2 days) male Wistar rats. Both oxidative (type I; soleus) and glycolytic (type II; plantaris) muscles were analyzed. We also investigated the effects of a nutritional perturbant induced by a short-term bolus of ethanol (75 mmol/kg weight IP administered 2.5 hours before sacrifice). The results showed that in response to fasting there were significant increases in 7alpha-OH, 7beta-OH, and 7-keto in liver and both type I and II skeletal muscle (P < .001 in all instances). For skeletal muscle, the increases were blunted or ameliorated after 2 days when compared with data from rats starved for 1 day. In contrast, the increases in liver after 1 day's fasting were relatively sustained at 2 days. Short-term ethanol increased 7alpha-OH, 7beta-OH, and 7-keto in type I muscle of fed animals only (P < .001 in all instances) with a significant interaction between fasting and alcohol (P < .001 in all instances). For the first time, we have shown that oxysterols can increase in muscle and liver in response to food withdrawal and in response to an immediately imposed nutritional perturbant (ie, alcohol). Increased oxysterols represent elevated oxidative stress and/or disturbances in their formation or clearance. Because of the reported cytotoxic properties of oxysterols, these data are important in understanding cellular pathology because episodic anorexia

  17. Two new mutations in cis on (G)gamma chain of fetal hemoglobin: Hb F-Madrid [G gamma 50(D1)Ser-->Cys] and [G gamma 75(E19)Ile-->Thr].

    PubMed

    Hojas, R; Villegas, A; Ropero, P; Valverde, F; Nogales, A; González, F A; José, C; Villar, L; García, C

    2003-03-01

    We describe a new structural hemoglobin variant of (G)gamma with two amino acid replacements in cis found in the umbilical cord blood of a neonate in Madrid, Spain. The substitutions were identified on exon 2 of the (G)gamma globin gene, at codon 50 (T CT-->T GT) and at codon 75 (A TA-->A CA). We have named it Hb F-Madrid. The father of the propositus was the carrier of the same (G)gamma chain variant and, moreover, molecular study of alpha genes revealed the loss of an alpha gene (-alpha(3.7)/alpha alpha) both in the propositus and his mother.

  18. Two-Dimensional Dynamics of Metal Nanoparticles on the Surface of Thin Polymer Films Studied with Coherent X Rays

    SciTech Connect

    Streit, S.; Gutt, C.; Sternemann, H.; Tolan, M.; Chamard, V.; Robert, A.; Sprung, M.

    2007-01-26

    X-ray photon-correlation spectroscopy is used to measure the dynamic structure factor f(q,{tau}) of gold particles moving on the surface of thin polymer films. Above the glass transition of the polymer the peculiar form f(q,{tau}){approx}exp[-({gamma}{tau}){sup {alpha}}] is found with 0.7<{alpha}<1.5, depending on sample age and temperature. The relaxation rates {gamma} scale linearly with q, excluding a simple Brownian diffusive motion. This type of behavior, already observed in aging bulk soft matter systems, is explained by a power law distribution of particle velocities due to ballistic motion.

  19. Gradient HPLC separation of dehydroepiandrosterone (DHEA) from its metabolites and biological congeners: role of tetrahydrofuran in the chromatographic mechanism.

    PubMed

    Gergely, András; Horváth, Péter; Szász, György; Veress, Gábor

    2009-08-01

    A three-step gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for the separation of dehydroepiandrosterone (DHEA), its sulfate ester (DHEA-S), its three C7-oxidized metabolites (7alphaOH-DHEA, 7betaOH-DHEA, 7-keto-DHEA), and its biosynthetic congeners (androstenedione, testosterone, estradiol, pregnenolone). This new method allows the quantitative characterization of DHEA metabolism and biosynthetic transformation under given physiological, pathological, or therapeutically influenced circumstances. Tetrahydrofuran probably acts as a proton acceptor coadsorbent, while isopropanol behaves as a proton donor during the separation of testosterone, estradiol, and the stereoisomers of 7-OH-DHEA.

  20. Plant growth regulation activity of steviol and derivatives.

    PubMed

    de Oliveira, Brás Heleno; Stiirmer, Júlio César; de Souza Filho, José D; Ayub, Ricardo Antonio

    2008-05-01

    This work describes the preparation of tetracyclic diterpenoids and determination of their plant growth regulator properties. Stevioside (2) was used as starting material and the derivatives 13-hydroxy-ent-kaur-16-en-19-oic acid (steviol, 3), ent-7alpha,13-dihydroxy-kaur-16-en-19-oic acid (4), 13-hydroxy, ent-kaur-16,17-epoxi-19-oic acid (steviol epoxide, 5), 17-hydroxy-16-ketobayeran-19-oic acid (17-hydroxyisosteviol, 6), 17-hydroxy-16-hydroxyiminobayeran-19-oic acid (7), 16-ketobayeran-19-oic acid (isosteviol, 9), 16,17-dihydroxybeyeran-19-oic acid (8), and 16-hydroxyiminobayeran-19-oic acid (isosteviol oxime, 10) were obtained by simple chemical procedures. Another derivative, ent-7alpha,13-dihydroxycaur-15-en-19-oic acid (4), was obtained by biotransformation of steviol (3) by Penicillium citrinum. In order to determine the plant growth regulator activity the compounds were submitted to the lettuce hypocotyl and barley aleurone bioassays. All compounds showed significant activities in both bioassays. Steviol (3) and isosteviol (9) were also tested in field-grown grapes resulting in an increase in berry weight and size.

  1. Urinary metabolomics in Fxr-null mice reveals activated adaptive metabolic pathways upon bile acid challenge.

    PubMed

    Cho, Joo-Youn; Matsubara, Tsutomu; Kang, Dong Wook; Ahn, Sung-Hoon; Krausz, Kristopher W; Idle, Jeffrey R; Luecke, Hans; Gonzalez, Frank J

    2010-05-01

    Farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in synthesis, metabolism, and transport of bile acids and thus plays a major role in maintaining bile acid homeostasis. In this study, metabolomic responses were investigated in urine of wild-type and Fxr-null mice fed cholic acid, an FXR ligand, using ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS). Multivariate data analysis between wild-type and Fxr-null mice on a cholic acid diet revealed that the most increased ions were metabolites of p-cresol (4-methylphenol), corticosterone, and cholic acid in Fxr-null mice. The structural identities of the above metabolites were confirmed by chemical synthesis and by comparing retention time (RT) and/or tandem mass fragmentation patterns of the urinary metabolites with the authentic standards. Tauro-3alpha,6,7alpha,12alpha-tetrol (3alpha,6,7alpha,12alpha-tetrahydroxy-5beta-cholestan-26-oyltaurine), one of the most increased metabolites in Fxr-null mice on a CA diet, is a marker for efficient hydroxylation of toxic bile acids possibly through induction of Cyp3a11. A cholestatic model induced by lithocholic acid revealed that enhanced expression of Cyp3a11 is the major defense mechanism to detoxify cholestatic bile acids in Fxr-null mice. These results will be useful for identification of biomarkers for cholestasis and for determination of adaptive molecular mechanisms in cholestasis.

  2. Antimicrobial constituents from fruits of Ailanthus altissima SWINGLE.

    PubMed

    Zhao, Chun-Chao; Shao, Jian-Hua; Li, Xian; Xu, Jing; Zhang, Peng

    2005-10-01

    A new naturally occurring sterol, compound 5, and six known stigmasterols were isolated from fruits of Ailanthus altissima Swingle by repeated column chromatography and RP-HPLC. Their structures were identified as, 5alpha-stigmastane-3,6-dione (1), 3beta-hydroxystigmast-5-en-7-one (2), stigmast-5-ene-3beta, 7alpha-diol (3), 6alpha-hydroxystigmast-4-en-3-one (4), 5alpha-stigmastane-3beta, 6beta-diol (5), stigmast-4-ene-3beta, 6alpha-diol (6), stigmast-5-ene-3beta, 7alpha, 20xi-triol (7) by spectral analysis and comparison with the published data. These compounds have not been reported from genus Ailanthus, whereas compound 7 was identified by NMR for the first time. In addition, the 95% ethanol extract and compounds from the fruits of Ailanthus altissima SWINGLE were assayed for in vitro antimicrobial activity. The extract was potent active against the assayed bacteria while compounds 3 and 7 exhibited moderate activity.

  3. Dehydroepiandrosterone (DHEA) metabolism in Saccharomyces cerevisiae expressing mammalian steroid hydroxylase CYP7B: Ayr1p and Fox2p display 17beta-hydroxysteroid dehydrogenase activity.

    PubMed

    Vico, Pedro; Cauet, Gilles; Rose, Ken; Lathe, Richard; Degryse, Eric

    2002-07-01

    We have engineered recombinant yeast to perform stereospecific hydroxylation of dehydroepiandrosterone (DHEA). This mammalian pro-hormone promotes brain and immune function; hydroxylation at the 7alpha position by P450 CYP7B is the major pathway of metabolic activation. We have sought to activate DHEA via yeast expression of rat CYP7B enzyme. Saccharomyces cerevisiae was found to metabolize DHEA by 3beta-acetylation; this was abolished by mutation at atf2. DHEA was also toxic, blocking tryptophan (trp) uptake: prototrophic strains were DHEA-resistant. In TRP(+) atf2 strains DHEA was then converted to androstene-3beta,17beta-diol (A/enediol) by an endogenous 17beta-hydroxysteroid dehydrogenase (17betaHSD). Seven yeast polypeptides similar to human 17betaHSDs were identified: when expressed in yeast, only AYR1 (1-acyl dihydroxyacetone phosphate reductase) increased A/enediol accumulation, while the hydroxyacyl-CoA dehydrogenase Fox2p, highly homologous to human 17betaHSD4, oxidized A/enediol to DHEA. The presence of endogenous yeast enzymes metabolizing steroids may relate to fungal pathogenesis. Disruption of AYR1 eliminated reductive 17betaHSD activity, and expression of CYP7B on the combination background (atf2, ayr1, TRP(+)) permitted efficient (>98%) bioconversion of DHEA to 7alpha-hydroxyDHEA, a product of potential medical utility. Copyright 2002 John Wiley & Sons, Ltd.

  4. Steroid and sterol 7-hydroxylation: ancient pathways.

    PubMed

    Lathe, Richard

    2002-11-01

    B-ring hydroxylation is a major metabolic pathway for cholesterols and some steroids. In liver, 7 alpha-hydroxylation of cholesterols, mediated by CYP7A and CYP39A1, is the rate-limiting step of bile acid synthesis and metabolic elimination. In brain and other tissues, both sterols and some steroids including dehydroepiandrosterone (DHEA) are prominently 7 alpha-hydroxylated by CYP7B. The function of extra-hepatic steroid and sterol 7-hydroxylation is unknown. Nevertheless, 7-oxygenated cholesterols are potent regulators of cell proliferation and apoptosis; 7-oxygenated derivatives of DHEA, pregnenolone, and androstenediol can have major effects in the brain and in the immune system. The receptor targets involved remain obscure. It is argued that B-ring modification predated steroid evolution: non-enzymatic oxidation of membrane sterols primarily results in 7-oxygenation. Such molecules may have provided early growth and stress signals; a relic may be found in hydroxylation at the symmetrical 11-position of glucocorticoids. Early receptor targets probably included intracellular sterol sites, some modern steroids may continue to act at these targets. 7-Hydroxylation of DHEA may reflect conservation of an early signaling pathway.

  5. CYP7A1 promoter polymorphism -203A>C affects bile salt synthesis rate in patients after ileal resection.

    PubMed

    Lenícek, Martin; Komárek, Viktor; Zimolová, Miluse; Kovár, Jan; Jirsa, Milan; Lukás, Milan; Vítek, Libor

    2008-12-01

    Cholesterol 7alpha-hydroxylase (CYP7A1) plays a crucial role in cholesterol metabolism and has been implicated in genetic susceptibility to atherosclerosis. Thus, an understanding of its transcriptional regulation is of considerable importance. We evaluated the effect of a common -203A>C polymorphism in the CYP7A1 promoter region on the activity of CYP7A1, estimated as the ratios of serum 7alpha-hydroxycholest-4-en-3-one (C4) to either total or non-HDL-cholesterol. The study was performed on patients after resection of the distal ileum, leading to upregulation of CYP7A1 activity (n = 65). Healthy volunteers served as the control group (n = 66). Whereas higher CYP7A1 activity was associated with the -203A allele in the patient group (C4/cholesterol ratio, 29.0 vs. 14.8 microg/mmol, P = 0.032; C4/non-HDL-cholesterol ratio, 53.3 vs. 21.3 microg/mmol in -203AA and -203CC, P = 0.017, respectively), no differences were observed in the healthy controls. We conclude that under physiological conditions, the -203A>C polymorphism in the CYP7A1 gene promoter region does not seem to have any clinically relevant effect. However, in patients with severe bile salt malabsorption, this polymorphism markedly affects CYP7A1 activity.

  6. Synthesis of a beta-estradiol-biotin chimera that potently heterodimerizes estrogen receptor and streptavidin proteins in a yeast three-hybrid system.

    PubMed

    Hussey, Stephen L; Muddana, Smita S; Peterson, Blake R

    2003-04-02

    Small molecules that dimerize proteins in living cells provide powerful probes of biological processes and have potential as tools for the identification of protein targets of natural products. We synthesized 7-alpha-substituted derivatives of beta-estradiol tethered to the natural product biotin to regulate heterodimerization of estrogen receptor (ER) and streptavidin (SA) proteins expressed as components of a yeast three-hybrid system. Addition of an estradiol-biotin chimera bearing a 19-atom linker to yeast expressing DNA-bound ER-alpha or ER-beta LexA fusion proteins and wild-type SA protein fused to the B42 activation domain activated reporter gene expression by as much as 450-fold in vivo (10 muM ligand). Comparative analysis of lower affinity Y43A (biotin Kd approximately 100 pM) and W120A (biotin Kd approximately 100 nM) mutants of SA indicated that moderate affinity interactions can be readily detected with this system. Comparison of a 7-alpha-substituted estradiol-biotin chimera with a structurally similar dexamethasone-biotin chimera revealed that yeast expressing ER proteins can detect cognate ligands with up to 5-fold greater potency and 70-fold higher activity than yeast expressing analogous glucocorticoid receptor (GR) proteins. This approach may facilitate the identification of protein targets of biologically active small molecules screened against genetically encoded libraries of proteins expressed in yeast three-hybrid systems.

  7. Induction of steroidal hydroxylase activity by plant defence compounds in the filamentous fungus Cochliobolus lunatus

    PubMed

    Vitas; Smith; Plavec; Kesselmeier; Pajic; Ferlan; Zigon; Kelly; Komel

    1999-02-01

    We investigated the hypothesis that the endogenous role of the commercially important inducible steroid hydroxylase cytochrome P450s of fungi was in defense against plant toxophores/secondary metabolites. Two plant defense compounds, the aglycones tomatidine and solanidine, the steroidal glycoalkaloid alpha-tomatine and the triterpene saponin beta-escin were tested as inducers of 11beta/14alpha-steroid hydroxylase in the filamentous fungus Cochliobolus lunatus. The extracts of saponins from the roots of Primula veris and green oat leaves were also tested as inducers of 11beta/14alpha-hydroxylation activity in progesterone biotransformation with the same fungus. Induction of steroid hydroxylase and inhibition of activity in some cases support our hypothesis that their endogenous function is in biochemical defence against secondary metabolites. 4-Pregnene-3,11,20-trione was added as a substrate for biotransformation with C. lunatus. We isolated from culture broth 14alpha-hydroxy-4-pregnene-3,11,20-trione, and the hitherto unreported compounds, 7alpha,14alpha-dihydroxy-4-pregnene-3,11,20-trione and 7alpha-hydroxy-pregna-4,8(14)-diene-3,11,20-trione.

  8. Nepetalactone content and antibacterial activity of the essential oils from different parts of Nepeta persica.

    PubMed

    Shafaghat, Ali; Oji, Khodamali

    2010-04-01

    The essential oils from the flower, leaf, stem and root of Nepeta persica Boiss., analyzed by GC and GC/MS, were shown to contain 4abeta,7alpha,7abeta-nepetalactone (58.5%, 62.3%, 66.2% and 27.1%, respectively), and 4aalpha,7alpha,7abeta-nepetalactone (33.0%, 28.3%, 24.9% and 7.6%, respectively). The other main component of the flower and stem oils was alpha-pinene (3.6% and 4.4%) and of the leaf oil beta-ocimene (3.6%). In the root oil, other main constituents were alpha-pinene (40.4%), alpha-amorphene (5.3%), gamma-cadinene (2.9%), and cis-calamenene (2.5%). Nepetalactone was the major component of the flower, leaf and stem oils, which are thus important sources of nepetalactone. Antibacterial activities of the flower, leaf, stem and root oils were evaluated using the micro-dilution broth method. Inhibitory effects on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Enterococcus faecalis were recorded. The flower, leaf, stem, and root oils had difference activities against the test microorganisms. The antibacterial property of the essential oils might be ascribed to their high content of nepetalactone isomers.

  9. Liquid chromatography-mass spectrometry utilizing multi-stage fragmentation for the identification of oxysterols.

    PubMed

    Karu, Kersti; Hornshaw, Martin; Woffendin, Gary; Bodin, Karl; Hamberg, Mats; Alvelius, Gunvor; Sjövall, Jan; Turton, John; Wang, Yuqin; Griffiths, William J

    2007-04-01

    In humans, the brain accounts for about 20% of the body's free cholesterol, most of which is synthesized de novo in brain. To maintain cholesterol balance throughout life, cholesterol becomes metabolized to 24S-hydroxycholesterol, principally in neurons. In mouse, rat, and probably human, metabolism to 24S-hydroxycholesterol accounts for about 50% of cholesterol turnover; however, the route by which the remainder is turned over has yet to be elucidated. Here, we describe a novel liquid chromatography (LC) multi-stage fragmentation mass spectrometry (MS(n)) methodology for the identification, with high sensitivity (low pg), of cholesterol metabolites in rat brain. The methodology includes derivatization to enhance ionization, exact mass analysis at high resolution to identify potential metabolites, and LC-MS(n) (n=3) to allow their characterization. 24S-hydroxycholesterol was confirmed as a major oxysterol in rat brain, and other oxysterols identified for the first time in brain included 24,25-, 24,27-, 25,27-, 6,24,- 7alpha,25-, and 7alpha,27-dihydroxycholesterols. In addition, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol, two molecules linked to amyloidogenesis of proteins, were characterized in rat brain.

  10. Ergosteroids: induction of thermogenic enzymes in liver of rats treated with steroids derived from dehydroepiandrosterone.

    PubMed Central

    Lardy, H; Partridge, B; Kneer, N; Wei, Y

    1995-01-01

    Dehydroepiandrosterone (DHEA), an intermediate in the biosynthesis of testosterone and estrogens, exerts several physiological effects not involving the sex hormones. When fed to rats it induces the thermogenic enzymes mitochondrial sn-glycerol-3-phosphate dehydrogenase and cytosolic malic enzyme in their livers. Animals and humans, and their excised tissues, are known to hydroxylate DHEA at several positions and to interconvert 7 alpha-hydroxy-DHEA, 7 beta-hydroxy-DHEA, 7-oxo-DHEA, and the corresponding derivatives of androst-5-enediol. We report here that these 7-oxygenated derivatives are active inducers of these thermogenic enzymes in rats and that the 7-oxo derivatives are more active than the parent steroids. We postulate that the 7 alpha-hydroxy and 7-oxo derivatives are on a metabolic pathway from DHEA to more active steroid hormones. These 7-oxo steroids have potential as therapeutic agents because of their increased activity and because they are not convertible to either testosterone or estrogens. PMID:7604042

  11. Biologically active monoiodinated alpha-MSH derivatives for receptor binding studies using human melanoma cells

    SciTech Connect

    Eberle, A.N.; Verin, V.J.; Solca, F.; Siegrist, W.; Kueenlin, C.B.; Bagutti, C.; Stutz, S.; Girard, J. , University Hospital, Basel )

    1991-01-01

    Three different monoiodinated radioligands of alpha-MSH (alpha-melanocyte-stimulating hormone) were compared in a binding assay with human D10 melanoma cells: (Tyr(125I)2)-alpha-MSH, (Tyr(125I)2,NIe4)-alpha-MSH, and (Tyr(125I)2,NIe4,D-Phe7)-alpha-MSH. They were prepared either by the classical chloramine T method or by the Enzymobead method. A simple and rapid purification scheme was developed consisting of a primary separation on reversed-phase C18 silica cartridges immediately after the iodination, followed by HPLC purification before each binding experiment. Biological testing of the three radioligands showed that they all retained high melanotropic activity in the B16 melanin assay and the Anolis melanophore assay. However, in human D10 melanoma cells, (Tyr(125I)2,NIe4)-alpha-MSH led to a high degree of non-specific binding to the cells which could not be displaced by excess alpha-MSH and only partially by (NIe4)-alpha-MSH. The (Tyr(125I)2,NIe4,D-Phe7)-alpha-MSH tracer gave similar results but with a much lower proportion of non-specific binding. On the other hand, (Tyr(125I)2)-alpha-MSH proved to be an excellent radioligand whose non-specific binding to the D10 cells was not higher than 20% of the total binding.

  12. 4-Norleucine, 7-D-phenylalanine-alpha-melanocyte-stimulating hormone: a highly potent alpha-melanotropin with ultralong biological activity.

    PubMed Central

    Sawyer, T K; Sanfilippo, P J; Hruby, V J; Engel, M H; Heward, C B; Burnett, J B; Hadley, M E

    1980-01-01

    alpha-Melanocyte-stimulating hormone (alpha-MSH) reversibly darkens frog skins by stimulating melanosome movement (dispersion) within melanophores. Heat-alkali treatment of alpha-MSH results in prolonged biological activity of the hormone. Quantitative gas chromatographic analysis of the hydrolyzed heat-alkali-treated peptide revealed partial racemization particularly at the 4(methionine) and 7(phenylalanine) positions. [Nle4]-alpha-MSH, a synthetic analogue of alpha-MSH, reversibly darkens frog skins and also exhibits prolonged activity after heat-alkali treatment. Synthesis of [Nle4, D-Phe7]-alpha-MSH provided an analogue with prolonged biological activity identical to that observed with heat-alkali-treated alpha-MSH or [Nle4]-alpha-MSH. [Nle4, D-Phe7]-alpha-MSH was resistant to enzymatic degradation by serum enzymes. In addition, this peptide exhibited dramatically increased biological activity as determined by frog skin bioassay, activation of mouse melanoma adenylate cyclase, and stimulation of mouse melanoma cell tyrosinase activity. This Nle4, D-Phe7 synthetic analogue of alpha-MSH is a very porent melanotropin, 26 times as potent as alpha-MSH in the adenylate cyclase assay. The resistance of the peptide to enzymatic degradation and its extraordinarily potent and prolonged biological activity should make this analogue of alpha-MSH an important molecular probe for studying the melanotropin receptors of both normal and abnormal (melanoma) melanocytes. PMID:6777774

  13. Transformations of DHEA and its metabolites by rat liver.

    PubMed

    Lardy, Henry; Marwah, Ashok; Marwah, Padma

    2002-12-01

    Because dehydroepiandrosterone (DHEA) has a wide variety of weak beneficial effects in experimental animals and humans, we searched for metabolites of this steroid in the hope of finding more active compounds that might qualify for the title "steroid hormone." Incubation of DHEA with rat liver homogenate fortified with energy-yielding substrates resulted in rapid hydroxylation at the 7alpha-position of the molecule and subsequent conversion to other 7-oxygenated steroids in the sequence DHEA --> 7alpha-hydroxyDHEA --> 7-oxoDHEA --> 7beta-hydroxyDHEA, with branching to diols, triols, and sulfate esters. The ability of these metabolites to induce the formation of liver thermogenic enzyme activity increased from left to right in that sequence. A total of 25 different steroids were characterized, and at least six additional structures that are currently under study were produced from DHEA. 7-OxoDHEA is more effective than DHEA in enhancing memory performance in old mice and in reversing the amnesic effects of scopolamine.

  14. Selective conversion by microglia of dehydroepiandrosterone to 5-androstenediol-A steroid with inherent estrogenic properties.

    PubMed

    Jellinck, Peter H; Kaufmann, Martin; Gottfried-Blackmore, Andres; McEwen, Bruce S; Jones, Glenville; Bulloch, Karen

    2007-01-01

    The well-established neuroprotective effect of dehydroepiandrosterone (DHEA) has been attributed to its metabolism in the brain to provide estrogens known to be neuroprotective and to enhance memory and learning in humans and animals. However, our previous work showed that the conversion of DHEA to 4-androstenedione (AD), the precursor of estrone (E(1)) and estradiol (E(2)), is very low in several different types of neural cells, and that the main product is 7alpha-hydroxy-DHEA (7alpha-OH-DHEA). In this study, we found that microglia are an exception and produce mainly 5-androstene-3beta,17beta-diol (Delta(5)-Adiol), a C(19) steroid with estrogen-like activity from DHEA. Virtually, no other products, including testosterone (T) were detected by TLC or HPLC in incubations of (3)H-labeled DHEA with the BV2 microglial cell line. Microglia are important brain cells that are thought to play a house-keeping role during the steady state, and that are crucial to the brain's immune reaction to injury and the healing process. Our findings suggest that the microglia-produced Delta(5)-Adiol might have a role in modulating estrogen-sensitive neuroplastic events in the brain, in the absence of adequate local synthesis of estrone and estradiol.

  15. Formation of C21 bile acids from plant sterols in the rat

    SciTech Connect

    Boberg, K.M.; Lund, E.; Olund, J.; Bjoerkhem, I. )

    1990-05-15

    Formation of bile acids from sitosterol in bile-fistulated female Wistar rats was studied with use of 4-14C-labeled sitosterol and sitosterol labeled with 3H in specific positions. The major part (about 75%) of the 14C radioactivity recovered as bile acids in bile after intravenous administration of (4-14C)sitosterol was found to be considerably more polar than cholic acid, and only trace amounts of radioactivity had chromatographic properties similar to those of cholic acid and chenodeoxycholic acid. It was shown that polar metabolites were formed by intermediate oxidation of the 3 beta-hydroxyl group (loss of 3H from 3 alpha-3H-labeled sitosterol) and that the most polar fraction did not contain a hydroxyl group at C7 (retention of 3H in 7 alpha,7 beta-3H2-labeled sitosterol). Furthermore, the polar metabolites had lost at least the terminal 6 or 7 carbon atoms of the side chain (loss of 3H from 22,23-3H2- and 24,28-3H2-labeled sitosterol). Experiments with 3H-labeled 7 alpha-hydroxysitosterol and 4-14C-labeled 26-hydroxysitosterol showed that none of these compounds was an efficient precursor to the polar metabolites. By analysis of purified most polar products of (4-14C) sitosterol by radio-gas chromatography and the same products of 7 alpha,7 beta-(2H2)sitosterol by combined gas chromatography-mass spectrometry, two major metabolites could be identified as C21 bile acids. One metabolite had three hydroxyl groups (3 alpha, 15, and unknown), and one had two hydroxyl groups (3 alpha, 15) and one keto group. Considerably less C21 bile acids were formed from (4-14C)sitosterol in male than in female Wistar rats. The C21 bile acids formed in male rats did not contain a 15-hydroxyl group. Conversion of a (4-14C)sitosterol into C21 bile acids did also occur in adrenalectomized and ovariectomized rats, indicating that endocrine tissues are not involved.

  16. Can 19-nortestosterone derivatives be aromatized in the liver of adult humans? Are there clinical implications?

    PubMed

    Kuhl, H; Wiegratz, I

    2007-08-01

    Previous studies in postmenopausal women have demonstrated that, after oral administration of norethisterone, a small proportion of the compound is rapidly converted into ethinylestradiol. The shape of the concentration - time curve suggested that this occurred in the liver. The results were confirmed by in vitro investigations with adult human liver tissue. In 2002, it was shown that, after oral treatment of women with tibolone, aromatization of the compound occurred, resulting in the formation of a potent estrogen, 7 alpha-methyl-ethinylestradiol. The result has been called into question, because the adult human liver does not express cytochrome P450 aromatase, which is encoded by the CYP 19 gene. Moreover, it has been claimed that the serum level of 7 alpha-methyl-ethinylestradiol measured by gas chromatography/mass spectrometry was an artifact. Aromatization of steroids is a complex process of consecutive oxidation reactions which are catalyzed by cytochrome P450 enzymes. The conversion of the natural C19 steroids, testosterone and androstenedione, into estradiol-17beta and estrone is dependent on the oxidative elimination of the angular C19-methyl group. This complex key reaction is catalyzed by the cytochrome P450 aromatase, which is expressed in many tissues of the adult human (e.g. ovary, fat tissue), but not in the liver. However, 19-nortestosterone derivatives are characterized by the lack of the C19-methyl group. Therefore, for the aromatization of these synthetic steroids, the action of the cytochrome P450 aromatase is not necessary and the oxidative introduction of double bonds into the A-ring can be catalyzed by other hepatic cytochrome P450 enzymes. The final key process in the formation of a phenolic A-ring, both in natural androgens and 19-nortestosterone derivatives, is the enolization of a 3-keto group to the C2-C3-enol or the C3-C4-enol moiety, which occurs without the action of enzymes. 19-nortestosterone derivatives (norethisterone

  17. Characterization of alpha-amylase inhibitor from Palo Fierro seeds.

    PubMed

    Guzman-Partida, A M; Jatomea-Fino, O; Robles-Burgueño, M R; Ortega-Nieblas, M; Vazquez-Moreno, L

    2007-09-01

    Alpha amylase inhibitor from Palo Fierro seeds (alphaAI-PF) was purified using affinity chromatography on a fetuin-fractogel column followed by anionic exchange chromatography. AlphaAI-PF has a molecular mass of 77kDa with two subunits (15.8 and 17.4 kDa), it is nonglycosylated and has pI of 4.7. AlphaAI-PF inhibited porcine pancreatic alpha-amylase (PPA) (1,4-alpha-D-glucan glucanohydrolase; EC 3.2.1.1), but was almost devoid of inhibitory activity on alpha-amylase extracts from Zabrotes subfasciatus (ZSA). Analysis of alphaAI-PF peptides showed a high homology to alphaAI-1 from Phaseolus vulgaris that also inhibits PPA.

  18. Signatures of inhomogeneity in the early universe

    NASA Astrophysics Data System (ADS)

    Fowler, William A.; Kawano, L. H.; Malaney, R. A.; Kavanagh, R. W.

    We have made a systematic study of the production of elemental CNO in inhomogeneous nucleosynthesis, investigating a much larger region of parameter space than previously studied. We have determined abundances of CNO elements and ascertained the main channels to their production. We have focused in particular on the role played by the Li-7(n, gamma)Li-8(alpha, n)B-11(n, gamma)B-12(beta(-) nu)C-12 reaction sequence: in models with Omega(b) = 1, we show that this sequence provides the main channel to CNO element production of which there is a significant amount; for lower values of Omega(b), there is competition from Li-7(alpha, gamma)B-11 but here there is a concurrent decline in CNO production. From these determinations, CNO element production emerges as a distinct signature of an Omega(b) = 1 inhomogeneous universe.

  19. A rhodopsin-like protein in Cyanophora paradoxa: gene sequence and protein immunolocalization.

    PubMed

    Frassanito, Anna Maria; Barsanti, Laura; Passarelli, Vincenzo; Evangelista, Valtere; Gualtieri, Paolo

    2010-03-01

    Here, we report the DNA sequence of the rhodopsin gene in the alga Cyanophora paradoxa (Glaucophyta). The primers were designed according to the conserved regions of prokaryotic and eukaryotic rhodopsin-like proteins deposited in the GenBank. The sequence consists of 1,272 bp comprised of 5 introns. The correspondent protein, named Cyanophopsin, showed high identity to rhodopsin-like proteins of Archea, Bacteria, Fungi, and Algae. At the N-terminal, the protein is characterized by a region with no transmembrane alpha-helices (80 aa), followed by a region with 7alpha-helices (219 aa) and a shorter 35-aa C-terminal region. The DNA sequence of the N-terminal region was expressed in E. coli and the recombinant purified peptide was used as antigen in hens to obtain polyclonal antibodies. Indirect immunofluorescence in C. paradoxa cells showed a marked labeling of the muroplast (aka cyanelle) membrane.

  20. Oxygenated diterpenes and other constituents from Moroccan Juniperus phoenicea and Juniperus thurifera var. africana.

    PubMed

    Barrero, Alejandro F; Quilez del Moral, José F; Herrador, M Mar; Akssira, Mohamed; Bennamara, Ahmed; Akkad, Said; Aitigri, Mohamed

    2004-09-01

    Six new diterpenic acids isolated as their methyl ester derivatives, i.e., methyl 12-oxo-8alpha,15-dihydroxyabiet-13-en-19-oate, methyl 12-oxo-8alpha-hydroxyabiet-13-en-19-oate, methyl 15-hydroperoxy-8alpha,12alpha-epidioxiabiet-13-en-19-oate, methyl 15-hydroxy-8alpha,12alpha-epidioxiabiet-13-en-19-oate, methyl 15-hydroperoxy-8alpha,14alpha,12alpha,13alpha-diepoxiabietan-13-en-19-oate, and methyl 7alpha,12beta-dihydroxysandaracopimarate, together with two new isovalerate derivatives of p-methoxycinnamyl alcohol and linalool, were isolated from the leaves of Juniperus thurifera var. africana and Juniperus phoenicea, grown in Morocco. The structures of these compounds were established by using spectroscopic techniques, including 2D NMR spectra. The cytotoxicity of the abietane diterpenoids was tested against five cell lines.

  1. Beta-lactamase inactivation by mechanism-based reagents.

    PubMed

    Fisher, J; Belasco, J G; Charnas, R L; Khosla, S; Knowles, J R

    1980-05-16

    The mechanistic pathway followed by the E. coli RTEM beta-lactamase has been studied with a view to clarifying the mode of action of a number of recently discovered inactivators of the enzyme. There is clear evidence that the beta-lactamase-catalysed hydrolysis of the 7-alpha-methoxycephem, cefoxitin, proceeds via an acyl-enzyme intermediate. An analysis of the inactivation reactions of all the known beta-lactam derivatives that result in irreversible loss of enzyme activity permits the identification of three structural features required for a beta-lactamase inactivator. The application of these principles suggests a new group of mechanism-based inactivators of the enzyme: the sulphones of N-acyl derivatives of 6-beta-aminopenicillanic acid that are themselves poor substrates for the enzyme. These sulphones are powerful inactivators of the beta-lactamase.

  2. Solubilization of kaurenoic acid oxidase from microsomal preparations of Gibberella fujikuroi

    SciTech Connect

    Jennings, J.C.; Coolbaugh, R.C. )

    1990-05-01

    The gibberellin biosynthetic pathway from ent-kaurenoic acid was investigated using cell-free preparations from G. fujikuroi. Fungal cells were disrupted under 20,000 psi and centrifuged at 150,000 {times} g. Enzyme activity was assayed using chemically synthesized {sup 14}C-kaurenoic acid, and reaction products were separated and quantified by reverse phase HPLC. Products were identified as ent-7{alpha}-hydroxykaurenoic acid and GA{sub 14} by co-chromatography with standards and GC/MS. The 150,000 {times} g pellet was found to contain kaurenoic acid oxidase which was subsequently solubilized using cholic acid. Further separation of solubilized proteins was obtained by HPLC and FPLC anion exchange and Sepharose 4B-octylamine chromatography.

  3. Cytotoxic terpene hydroperoxides from the aerial parts of Aster spathulifolius.

    PubMed

    Lee, Sung Ok; Choi, Sang Zin; Choi, Sang Un; Kim, Gun Hee; Kim, Young Choong; Lee, Kang Ro

    2006-10-01

    Three new sesquiterpene hydroperoxides, 1-[3-(2-hydroperoxy-3-methylbut-3-en)-4-hydroxyphenyl]ethanone (2), 7beta-hydroperoxy-eudesma-11-en-4-ol (3), and 7alpha-hydroperoxymanool (4), together with three known compounds, germacrone (1), ent-germacra-4(15),5,10(14)-trien-1alpha-ol (5) and teucdiol A (6) were isolated from the aerial parts of Aster spathulifolius (Compositae). Their structures were characterized using chemical and spectroscopic methods. The isolated compounds were tested for their cytotoxicity against five human tumor cell lines in vitro using a SRB method. The two new compounds, 3 and 4, showed moderate cytotoxicity against human cancer cells with ED50 values ranging from 0.24 to 13.27 microg/mL.

  4. A new sesquiterpene hydroperoxide from the aerial parts of Aster oharai.

    PubMed

    Choi, Sang Zin; Lee, Sung Ok; Choi, Sang Un; Lee, Kang Ro

    2003-07-01

    Phytochemical works on the aerial parts of Aster oharai (Compositae) led to the isolation of a new sesquiterpene hydroperoxide, 7alpha-hydroperoxy-3,11-eudesmadiene (2) and seven known compounds, teucdiol B (1), alpha-spinasterol (3), oleanolic acid (4), alpha-spinasterol 3-O-beta-D-glucopyranoside (5), methyl 3,5-di-O-caffeoyl quinate (6), 3,5-di-O-caffeoylquinic acid (7), 3,4-di-O-caffeoylquinic acid (8). The chemical structures of 1-8 were established by chemical and spectroscopic methods. Compound 2 showed cytotoxicity against cultured human tumor cell lines in vitro, SK-OV-3 (ovarian), SK-MEL-2 (skin melanoma), and HCT15 (colon) with ED50 values ranging from 3.86-17.21 microg/mL.

  5. Halohydrins and polyols derived from antirrhinoside: structural revisions of muralioside and epimuralioside

    PubMed

    Franzyk; Jensen; Thale; Olsen

    1999-02-01

    Treatment of the iridoid glucoside antirrhinoside (1) with pyridinium chloride in dimethylformamide gave rise to the two possible trans-halohydrins, linarioside (4) and isolinarioside (5). Pyridinium bromide gave the two analogous bromohydrins. It is shown that the iridoid glucosides 8-epi-muralioside from Linaria arcusangeli and 7,8-epi-antirrhinoside from Linaria dalmatica are both identical with isolinarioside, and therefore, these names are redundant. The structure of muralioside isolated from Cymbalaria muralis is revised to that of its 8-epimer (8), while the structure of an isomeric, new iridoid glucoside from Paulownia tomentosa has been elucidated to be 7beta-hydroxyharpagide (3), the structure originally assigned to 8. In addition, 7alpha-hydroxyharpagide (2), the known product from the base-catalyzed hydrolytic opening of 1, has been isolated from Antirrhinum majus and thus shown to be a natural product.

  6. Antimicrobial activity and chemical composition of the essential oil of Nepeta crispa Willd. from Iran.

    PubMed

    Sonboli, Ali; Salehi, Peyman; Yousefzadi, Morteza

    2004-01-01

    The composition and antimicrobial activity of the essential oil of Nepeta crispa Willd., an endemic species from Iran, was studied. The oil was obtained from the aerial parts of the plant and analyzed by GC and GC/MS. Twenty-three compounds, accounting for 99.8% of the total oil, were identified. The main constituents were 1,8-cineol (47.9%) and 4aalpha,7alpha,7abetanepetalactone (20.3%). The antimicrobial activity of essential oil of N. crispa was tested against seven gram-negative or gram-positive bacteria and four fungi. The results of the bioassays showed the interesting antimicrobial activity, in which the gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, were the most sensitive to the oil. Also, the oil exhibited a remarkable antifungal activity against all the tested fungi.

  7. Antibacterial activity and composition of the essential oil of Nepeta menthoides from Iran.

    PubMed

    Sonboli, Ali; Gholipour, Abbas; Yousefzadi, Morteza; Mojarrad, Mehran

    2009-02-01

    The antibacterial activity and chemical composition of the essential oil obtained from the aerial flowering parts of Nepeta menthoides were analyzed by GC and GC-MS. Twenty-nine compounds representing 97.6% of the total oil were identified. Oxygenated monoterpenes (71.9%) were the principal fraction of the oil with 1,8-cineole (33.8%) and 4aa-7alpha-7aalpha-nepetalactone (23.2%) as the main constituents. The antibacterial activity of the oil and its two main constituents were tested against seven bacteria. High activity of the oil and its two main constituents was demonstrated against all the tested bacteria with MIC values in the range of 1.8 - 7.2, 0.9 - 7.2 and 1.8 - 15 mg/mL, respectively.

  8. [Cholesterol oxidation products in fresh and frozen shrimps, raw and grilled].

    PubMed

    Echarte, M; Conchillo, A; Ansoren, D; Astiasarán, I

    2005-01-01

    Cholesterol oxidation products (COPs) have been related to different toxic effects, being the atherosclerotic process one of the best known. The presence of cholesterol oxides in freshly and frozenly commercialised shrimps, both raw and grilled, was studied. The determination was made by gas chromatography-mass spectrometry (GCMS). Fresh shrimps showed significant amounts of all analysed COPs, except for 7alpha-hydroxycholesterol, accounting in total for 33.15 microg COPs/g fat. In contrast, in frozen commercialised shrimps only 7-ketocholesterol and 7beta-hydroxycholesterol were detected. These results point out the great effectiveness of the commercialisation of this type of products under freezing, in terms of to the minimisation of the COPs formation. The cooking method (grilling) increased the COPs content in both types of shrimps, reaching 55.43 microg COPs/g fat in fresh shrimps and only 13.06 microg COPs/g fat in frozen ones.

  9. Equine cytochrome P450 aromatase exhibits an estrogen 2-hydroxylase activity in vitro.

    PubMed

    Almadhidi, J; Moslemi, S; Drosdowsky, M A; Séralini, G E

    1996-09-01

    Aromatase (estrogen synthetase) is a steroidogenic enzyme complex which catalyzes the conversion of androgens to estrogens (termed aromatization). This enzyme was purified from adult equine testis to homogeneity by five chromatographic steps. The ability of purified and reconstituted equine aromatase to exhibit an estrogen 2-hydroxylase activity was tested and compared to testosterone aromatization. Enzymatic activities were assessed by tritiated water release from labelled estradiol and testosterone. Kinetic analysis of estradiol 2-hydroxylation showed an apparent K(m) of 23 microM and a V(max) of 18 nmol/min/mg, whereas the values for testosterone aromatization were a K(m) of 15.7 nM and a V(max) of 34.6 pmol/min/mg. A specific antiserum raised against purified testicular equine P450arom and known to inhibit aromatase activity [1] was also found to inhibit the estrogen hydroxylase activity of equine placental microsomes in a dose-dependent manner with an IC50 value of 15 microl serum: 0.5 ml incubate. The estrogen hydroxylase activity was inhibited in a dose-dependent manner by two classes of aromatase inhibitors, i.e. steroidal-- (4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione)--and non-steroidal--(fadrozole and miconazole). The IC50 values were approximately 300 and 890 nM for 4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione, and 92 and 285 nM, for fadrozole and miconazole, respectively. Furthermore, 4-hydroxyandrostenedione caused a time-dependent inactivation of estrogen hydroxylase activity. We conclude that equine aromatase is able to use estradiol as a substrate, and converts it to catechol estradiol in vitro, possibly using the active site of aromatization. This is the first demonstration that equine aromatase functions as an estrogen 2-hydroxylase, in addition to transforming androgens into estrogen.

  10. Dihydromorphine-peptide hybrids with delta receptor agonistic and mu receptor antagonistic actions

    SciTech Connect

    Smith, C.B.; Medzihradsky, F.; Woods, J.H.

    1986-03-05

    The actions of two morphine derivatives with short peptide side chains were evaluated upon the contraction of the isolated mouse vas deferens and upon displacement of /sup 3/H-etorphine from rat brain membranes. NIH-9833 (N-(6,14-endoetheno-7,8-dihydromorphine-7-alpha-carbonyl)-L-phenylalanyl-L-leucine ethyl ester HCl) was a potent agonist upon the vas deferens. Its EC50 for inhibition of the twitch was 1.2 +/- 0.1 nM. Both naltrexone (10/sup -7/ M) a relatively nonselective opioid antagonist, and ICI-174864 (10/sup -/' M) a highly selective delta receptor antagonist, blocked the actions of NIH-9833 which indicates that this drug is a delta receptor agonist. In contrast, NIH-9835 (N-(6,14-endoetheno-7,8-dihydromorphine-7-alpha-carbonyl)-L-glycyl-L-phenylalanyl-L-leucine ethyl ester HCl), which differs from NIH-9835 by the presence of a single amino acid residue, was devoid of opioid agonistic activity but was a potent antagonist of the inhibitory actions on the vas deferens of morphine and sufentanil. NIH-9833 and NIH-9835 were potent displacers of /sup 3/H-etorphine from rat cerebral membranes with EC50's of 0.58 nM and 1.7 nM, respectively. The observation that addition of a single glycyl group changes a dihydromorphine-peptide analog from a potent delta receptor agonist to an equally potent mu receptor antagonist suggests that the two receptor sites might be structurally quite similar.

  11. Comparison between novel steroid-like and conventional nonsteroidal antioestrogens in inhibiting oestradiol- and IGF-I-induced proliferation of human breast cancer-derived cells.

    PubMed Central

    de Cupis, A.; Noonan, D.; Pirani, P.; Ferrera, A.; Clerico, L.; Favoni, R. E.

    1995-01-01

    1. This study has two specific aims: (a) to compare the antioestrogenic activity of two steroidal analogues of 17 beta-oestradiol, the 7 alpha-alkylamide, ICI 164,384 and the 7 alpha-alkylsulphinylamide, ICI 182,780, with that of the triphenylethylene-derived compound 4OH-tamoxifen on a pool of human breast cancer cell lines (HBCCL) with a range of hormonal responsiveness and acquired anti-oestrogen resistance and (b) to investigate the ability of such antioestrogens to modulate the potent breast carcinoma growth-stimulatory activity of the 'IGF-I system'. 2. For the chemosensitivity investigations we used a long-term colorimetric and the short-term thymidine incorporation assay; we analysed IGF-I in conditioned media by a radioimmunoassay, IGF-I mRNA in the cells by RT-PCR and molecular species of IGF-I-binding proteins, secreted in conditioned media, by Western ligand blot. IGF-I receptors were assayed on cell monolayers by binding studies and by Scatchard analysis, we calculated KD, Bmax and sites/cell. 3. Our results indicate that ICI 182,780 and ICI 164,384 are 1.5-5.5 fold more potent than 4OH-tamoxifen in inhibiting the basal proliferation of oestrogen-receptor positive (ER+) breast cancer cell lines. Moreover we demonstrate the capacity of ICI 182,780 and ICI 164,384 to reduce, in a time-dependent fashion, oestrogen- and/or IGF-I-stimulated growth of ER+cell lines, possibly by negatively interfering with an IGF-I-like material secretion and IGF-I-receptor number. 4. Our data provide the first evidence that, on ER+human breast carcinoma cell lines, steroidal antioestrogens inhibit cell growth and modulate the IGF-I mitogenic system. The mechanism of this latter effect has yet to be identified. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8581274

  12. Effects of long-term ingestion of difructose anhydride III (DFA III) on intestinal bacteria and bile acid metabolism in humans.

    PubMed

    Minamida, Kimiko; Asakawa, Chikako; Sujaya, I Nengah; Kaneko, Maki; Abe, Ayumi; Sone, Teruo; Hara, Hiroshi; Asano, Kozo; Tomita, Fusao

    2006-02-01

    Changes in the intestinal microbiota of 10 human subjects with long-term ingestion of 3 g/d difructose anhydride III (DFA III; 4 persons, 2 months; 3 persons, 6 months; and 3 persons, 12 months) were examined by denaturing gradient gel electrophoresis (DGGE). According to the answers to questionnaires, the subjects were divided into two groups (constipated and normal). The DGGE profile was different for every individual and each subject had unique profiles of intestinal microbiota. In the DGGE profiles of constipated subjects, the intensities of bands related to Bacteroides spp. increased. Moreover, the DFA III-assimilating bacteria, Ruminococcus sp. were isolated from subjects who ingested DFA III for 12 months. These strains showed 95% similarity of their 16S rDNA sequences with that of Ruminococcus obeum ATCC 29174(T) (X85101) and produced large amounts of acetic acid. DFA III ingestion for 2 months tended to increase total organic acids in feces, and tended to decrease fecal pH and the secondary bile acid (SBA) ratio in total bile acids. The SBA ratio in total bile acids corresponded to fecal pH. The production of SBA was decreased by low pH in vitro. These results indicated that DFA III ingestion in humans tended to lower intestinal pH, inhibited bile acid 7alpha-dehydroxylation activities and also tended to decrease the SBA ratios in total bile acids. Moreover, as another cause for the decrease in the SBA ratio in total bile acids, it was suggested that the number of bile acid 7alpha-dehydroxylating bacteria were decreased by DFA III ingestion.

  13. Chemical composition and antimicrobial activity of the essential oils from Cleome spinosa.

    PubMed

    McNeil, Megil J; Porter, Roy B R; Williams, Lawrence A D; Rainford, Lois

    2010-08-01

    Five different essential oil extractions of the aerial parts of Cleome spinosa Jacq. were examined. The oils obtained by hydrodistillation of the whole aerial parts, aerial parts without flowers (fruit, leaves and stem), flowers, fruits and leaves have been examined by GC-FID and GC-MS. The chemical profiles of the oils reveal the dominance of oxygenated sesqui- and diterpenes, with the exception of the fruit oil, which contained a high content of fatty acids. The most abundant compounds from the whole aerial parts were (Z)-phytol (31.3%), integerrimine (5.5%) and incensole (4.0%). The major compounds from the aerial portion without flowers were caryophyllene oxide (10.5%), (-)-spathulenol (7.5%) and Z-phytol (6.9%). In the flower oil, the main components were 7-alpha-hydroxy manool (23.8%), incensole (9.2%) and sclareol (8.7%). The chief constituents in the fruit oil were tetradecanoic acid (40.6%), (Z)-phytol (6.58%) and sclareol (4.5%). In the leaf oil, (Z)-phytol (19.5%), 7-alpha-hydroxy manool (6.8%) and caryophyllene oxide (4.36%) were the predominant compounds. Antimicrobial activity of the oil obtained from the whole aerial part was evaluated against nine microbial strains using a filter paper disc-diffusion method. The volatile oil showed moderate action against seven of the eight bacteria strains used, with significant inhibitory activity against Streptococcus pyogenes Group A when compared with the standard antibiotics, ampicillin and gentamicin. The fungus, Candida albicans was less sensitive to the essential oil. The oils showed moderate insecticidal activity against Cylas formicarius elegantalus, but possessed no antioxidant activity as indicated by the DPPH method. This represents the first report on the chemical composition of the essential oils from C. spinosa found in Jamaica and the in vitro antioxidant, insecticidal and antimicrobial potential of the oil from the aerial parts.

  14. Hepatic bile acid metabolism in the neonatal hamster: expansion of the bile acid pool parallels increased Cyp7a1 expression levels.

    PubMed

    Burke, Katie T; Horn, Paul S; Tso, Patrick; Heubi, James E; Woollett, Laura A

    2009-07-01

    Intraluminal concentrations of bile acids are low in newborn infants and increase rapidly after birth, at least partly owing to increased bile acid synthesis rates. The expansion of the bile acid pool is critical since bile acids are required to stimulate bile flow and absorb lipids, a major component of newborn diets. The purpose of the present studies was to determine the mechanism responsible for the increase in bile acid synthesis rates and the subsequent enlargement of bile acid pool sizes (BAPS) during the neonatal period, and how changes in circulating hormone levels might affect BAPS. In the hamster, pool size was low just after birth and increased modestly until 10.5 days postpartum (dpp). BAPS increased more significantly ( approximately 3-fold) between 10.5 and 15.5 dpp. An increase in mRNA and protein levels of cholesterol 7alpha-hydroxylase (Cyp7a1), the rate-limiting step in classical bile acid synthesis, immediately preceded an increase in BAPS. In contrast, levels of oxysterol 7alpha-hydroxylase (Cyp7b1), a key enzyme in bile acid synthesis by the alternative pathway, were relatively elevated by 1.5 dpp. farnesyl X receptor (FXR) and short heterodimeric partner (SHP) mRNA levels remained relatively constant at a time when Cyp7a1 levels increased. Finally, although simultaneous increases in circulating cortisol and Cyp7a1 levels occurred, precocious expression of Cyp7a1 could not be induced in neonatal hamsters with dexamethasone. Thus the significant increase in Cyp7a1 levels in neonatal hamsters is due to mechanisms independent of the FXR and SHP pathway and cortisol.

  15. Bacterial bile metabolising gene abundance in Crohn's, ulcerative colitis and type 2 diabetes metagenomes.

    PubMed

    Labbé, Alain; Ganopolsky, Jorge G; Martoni, Christopher J; Prakash, Satya; Jones, Mitchell L

    2014-01-01

    We performed an analysis to determine the importance of bile acid modification genes in the gut microbiome of inflammatory bowel disease and type 2 diabetic patients. We used publicly available metagenomic datasets from the Human Microbiome Project and the MetaHIT consortium, and determined the abundance of bile salt hydrolase gene (bsh), 7 alpha-dehydroxylase gene (adh) and 7-alpha hydroxysteroid dehydrogenase gene (hsdh) in fecal bacteria in diseased populations of Crohn's disease (CD), Ulcerative Colitis (UC) and Type 2 diabetes mellitus (T2DM). Phylum level abundance analysis showed a significant reduction in Firmicute-derived bsh in UC and T2DM patients but not in CD patients, relative to healthy controls. Reduction of adh and hsdh genes was also seen in UC and T2DM patients, while an increase was observed in the CD population as compared to healthy controls. A further analysis of the bsh genes showed significant differences in the correlations of certain Firmicutes families with disease or healthy populations. From this observation we proceeded to analyse BSH protein sequences and identified BSH proteins clusters representing the most abundant strains in our analysis of Firmicute bsh genes. The abundance of the bsh genes corresponding to one of these protein clusters was significantly reduced in all disease states relative to healthy controls. This cluster includes bsh genes derived from Lachospiraceae, Clostridiaceae, Erysipelotrichaceae and Ruminococcaceae families. This metagenomic analysis provides evidence of the importance of bile acid modifying enzymes in health and disease. It further highlights the importance of identifying gene and protein clusters, as the same gene may be associated with health or disease, depending on the strains expressing the enzyme, and differences in the enzymes themselves.

  16. The hepatic response to FGF19 is impaired in patients with nonalcoholic fatty liver disease and insulin resistance.

    PubMed

    Schreuder, Tim C M A; Marsman, Hendrik A; Lenicek, Martin; van Werven, Jochem R; Nederveen, Aart J; Jansen, Peter L M; Schaap, Frank G

    2010-03-01

    Intestinal FGF19 has emerged as a novel endocrine regulator of hepatic bile salt and lipid metabolism. In patients with nonalcoholic fatty liver disease (NAFLD) hepatic lipid metabolism is deranged. A possible role of FGF19 in NAFLD has not been reported yet. In this study, we assessed intestinal FGF19 production and the hepatic response to FGF19 in NAFLD patients with and without insulin resistance [homeostasis model of assessment (HOMA) score > or =2.5 (n = 12) and HOMA score <2.5 (n = 8), respectively]. To this end, NAFLD patients received a standardized oral fat challenge. Postprandial excursions of triglycerides, bile salts, and FGF19 were monitored, and plasma levels of a marker for bile salt synthesis (7alpha-hydroxy-4-cholesten-3-one) were determined. Fasted FGF19 levels were comparable in a control group of healthy volunteers (n = 15) and in NAFLD patients (0.26 +/- 0.28 vs. 0.18 +/- 0.09 ng/ml, respectively, P = 0.94). Postprandial FGF19 levels in both controls and NAFLD patients peaked between 3-4 h and were three times higher than baseline levels. The areas under the postprandial FGF19 curve were similar in controls and in the HOMA score-based NAFLD subgroups. In NAFLD patients with HOMA score <2.5, the postprandial increase in plasma FGF19 was accompanied by a lowering of plasma levels of 7alpha-hydroxy-4-cholesten-3-one (-30%, P = 0.015). This anticipated decline was not observed in insulin-resistant NAFLD patients (+10%, P = 0.22). In conclusion, patients with NAFLD show an unimpaired intestinal FGF19 production. However, the hepatic response to FGF19 is impaired in NAFLD patients with insulin resistance (HOMA score > or =2.5). This impaired hepatic response to FGF19 may contribute to the dysregulation of lipid homeostasis in NAFLD.

  17. Synergistic interaction in simultaneous exposure to Streptomyces californicus and Stachybotrys chartarum.

    PubMed Central

    Huttunen, Kati; Pelkonen, Jukka; Nielsen, Kristian Fogg; Nuutinen, Ulla; Jussila, Juha; Hirvonen, Maija-Riitta

    2004-01-01

    The microbial exposure associated with health complaints in moldy houses consists of a heterogeneous group of components, including both living and dead bacteria, fungi, and their metabolites and active compounds. However, little is known about the interactions between different microbes and their metabolites, although the cytotoxicity and inflammatory potential of certain individual microbes have been reported. In this study, we investigated the inflammatory responses of mouse RAW264.7 macrophages after exposure to six indoor air microbes (Aspergillus versicolor, Penicillium spinulosum, Stachybotrys chartarum, Bacillus cereus, Mycobacterium terrae, and Pseudomonas fluorescens) alone and together with the actinomycete Streptomyces californicus. The production of nitric oxide, levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6), and cytotoxicity were measured. The coexposure to Sta. chartarum and Str. californicus caused a synergistic increase in the production of IL-6 but not other cytokines. In further experiments, the metabolites from Sta. chartarum or from closely related fungi (atranones B and E, satratoxin G, trichodermin, 7-alpha-hydroxytrichodermol, staplabin, and SMTP-7) and the known fungal toxins sterigmatocystin, citrinin, and ochratoxin A were each tested with Str. californicus. The testing revealed a synergistic response in TNF-alpha and IL-6 production after coexposure to Str. californicus with both trichodermin and 7-alpha-hydroxytrichodermol. Finally, the synergistic inflammatory response caused by Str. californicus and trichodermin together was studied by analyzing for the presence of nuclear factor-kappa-B (NF-kappa-B) in nuclear extracts of the exposed cells. The exposure to Str. californicus induced the binding of NF-kappa-B proteins to the NF-kappa-B consensus sequence as well as to the natural NF-kappa-B site of the IL-6 promoter. Adding trichodermin to the exposure did not increase the DNA

  18. Tartary buckwheat sprout powder lowers plasma cholesterol level in rats.

    PubMed

    Kuwabara, Tomoko; Han, Kyu-Ho; Hashimoto, Naoto; Yamauchi, Hiroaki; Shimada, Ken-Ichiro; Sekikawa, Mitsuo; Fukushima, Michihiro

    2007-12-01

    We examined the effects of different types of buckwheat sprouts on the plasma cholesterol concentration, fecal steroid excretion and hepatic mRNA expression related to cholesterol metabolism in rats. Rats were fed a cholesterol-free diet with 5 g of Kitawasesoba common buckwheat sprout powder (KS)/100 g, 5 g of Hokkai T no. 8 tartary buckwheat sprout powder (HS-8)/100 g or 5 g of Hokkai T no. 9 tartary buckwheat sprout powder (HS-9)/100 g of diet for 4 wk. Control rats were fed a diet with alpha-cornstarch instead of sprout powder for 4 wk. There were no significant differences in food intake, body weight, liver weight or cecal contents among the groups. Plasma total cholesterol concentrations in the HS-8 and HS-9 groups were significantly lower than in the control group, whereas there was no significant difference between the KS and control groups. Fecal bile acid excretion and cecal short-chain fatty acid concentrations in the KS, HS-8 and HS-9 groups were significantly greater than in the control group. Furthermore, fecal matter excretion in the KS, HS-8 and HS-9 groups tended to be increased compared to the control group, with that in the HS-8 group being significantly higher than in the control group. Hepatic cholesterol 7alpha-hydroxylase mRNA expression in the KS, HS-8 and HS-9 groups and hepatic HMG-CoA reductase mRNA expression in the HS-9 group were significantly higher than in the control group. The results suggest that tartary buckwheat sprout powder has a serum cholesterol-lowering function by enhancing fecal bile acid excretion through increased fecal matter excretion or the upregulation of hepatic cholesterol 7alpha-hydroxylase mRNA expression in rats.

  19. Molecular diagnosis of alpha-thalassemia by combining real-time PCR with SYBR Green1 and dissociation curve analysis.

    PubMed

    Liu, Jingzhong; Yan, Mei; Wang, Zhangyong; Wang, Lirong; Zhou, Yan; Xiao, Bai

    2006-07-01

    The aim of the study was to set up an automatic molecular diagnostic method for deletional alpha-thalassemia without gel electrophoresis and TaqMan probe. Four real-time polymerase chain reactions (PCRs) with SYBR Green1 and ABI7000 (SYBR-PCR) followed by dissociation curve (DC) analysis were used to detect the --(SEA), - alpha(3.7), -alpha(4.2), and non-deletion-type alleles (alpha alpha or alpha(T)alpha), respectively. Positive results of the SYBR-PCRs were defined by the special shapes of the dissociation curves and the peak height at specific Tm for each predetermined PCR at a specific Tm for each PCR amplicon > or = cutoff values. Molecular diagnosis of alpha-thalassemia was determined by combining all four SYBR-PCR results. The specific Tms for the SYBR-PCR1-4, which was used to detect the --(SEA), - alpha(3.7), -alpha(4.2), and non-deletion-type alleles were 82.5 +/- 1 degrees Celsius, 82.8 +/- 1 degrees Celsius, 81.5 +/- 1 degrees Celsius, and 83.0 +/- 1 degrees Celsius, respectively. The cutoff values of the specific peaks for the positive amplificons were 40, 20, 10, and 70. The C(T) VS log copies of a recombinant plasmid DNA showed a good linear relationship between 10(5) approximately 10(0). Sensitivity of the SYBR-PCR-based method was at least 16 times higher than the multiplex PCR (mPCR)/gel electrophoresis method. Diagnostic outcomes of the 120 alpha-thalassemia cases by using the SYBR-PCR and DC analysis techniques were shown to be the same as that by using the mPCR/gel electrophoresis methods. The SYBR-PCR combined with the DC analysis technique is an alternative assay for the routine molecular diagnosis of alpha-thalassemia.

  20. An evaluation of the allelopathic potential of selected perennial groundcovers: foliar volatiles of catmint (Nepeta x faassenii) inhibit seedling growth.

    PubMed

    Eom, Seok Hyun; Yang, Hyun Seuk; Weston, Leslie A

    2006-08-01

    Six perennial groundcovers including Alchemilla mollis, Nepeta x faassenii, Phlox subulata, Sedum acre, Solidago cutleri, and Thymus praecox were investigated for the allelopathic potential of their respective foliar tissues via evaluation of volatile constituents produced by foliage. These groundcovers were selected for further laboratory evaluation because of superior performance as weed-suppressive groundcovers in previous field experiments. Foliar volatile components of N. x faassenii exhibited the strongest inhibitory effects on seedling growth of curly cress (Lepidium sativum), but S. cutleri also showed allelopathic potential by reducing shoot growth of curly cress seedlings with extracted volatiles. Although A. mollis and P. subulata exhibited strong weed-suppressive traits in past field experiments, weed suppression is apparently associated with either competition for resources or other allelopathic mechanisms rather than an allelopathic effect caused by volatiles. Volatiles of N. x faassenii were further evaluated with gas chromatography coupled to mass spectrometry (GC-MS). A total of 21 chemical constituents were identified in the volatile cocktail; 17 components were identified from a direct crude leaf sample extraction, including sabinene, beta-pinene, beta-myrcene, 2-(2-ethoxyethoxy)-ethanol, 1,8-cineole, ocimene, neryl Acetate, 4aalpha,7alpha,7aalpha-nepetalactone, alpha-copaene, trans-caryophyllene, alloaromadendrene, 4abeta,7alpha,7abeta-nepetalactone, germacrene D, beta-farnesene, chi-cadinene, germacrene B, and beta-sesquiphellandrene. Five additional constituents were identified in a methanolic extract of dried of N. x faassenii foliage, but not the volatile cocktail collected from N. x faassenii foliage. These included methyl benzoate, 2,4-decadienal, neryl acetate, isodihydronepetalactone, and caryophyllene oxide. Three components, 2-(2-ethoxyethoxy)-ethanol, alloaromadendrene, and chi-cadinene, were not only detected in both the volatile

  1. Detection and characterization of cholesterol-oxidized products using HPLC coupled to dopant assisted atmospheric pressure photoionization tandem mass spectrometry.

    PubMed

    Ronsein, Graziella E; Prado, Fernanda M; Mansano, Fernando V; Oliveira, Mauricio C B; Medeiros, Marisa H G; Miyamoto, Sayuri; Di Mascio, Paolo

    2010-09-01

    Oxidation of cholesterol (Ch) by a variety of reactive oxygen species gives rise mainly to hydroperoxides and aldehydes. Despite the growing interest in Ch-oxidized products, the detection and characterization of these products is still a matter of concern. In this work, the main Ch-oxidized products, namely, 3beta-hydroxycholest-5-ene-7alpha-hydroperoxide (7alpha-OOH), 3beta-5alpha-cholest-6-ene-5-hydroperoxide (5alpha-OOH), 3beta-hydroxycholest-4-ene-6alpha-hydroperoxide (6alpha-OOH), 3beta-hydroxycholest-4-ene-6beta-hydroperoxide (6beta-OOH), and 3beta-hydroxy-5beta-hydroxy-B-norcholestane-6beta-carboxaldehyde (ChAld), were detected in the same analysis using high-performance liquid chromatography (HPLC) coupled to dopant assisted atmospheric pressure photoionization tandem mass spectrometry. The use of selected reaction monitoring mode (SRM) allowed a sensitive detection of each oxidized product, while the enhanced product ion mode (EPI) helped to improve the confidence of the analyses. Isotopic labeling experiments enabled one to elucidate mechanistic features during fragmentation processes. The characteristic fragmentation pattern of Ch-oxidized products is the consecutive loss of H(2)O molecules, yielding cationic fragments at m/z 401, 383, and 365. Homolytic scissions of the peroxide bond are also seen. With (18)O-labeling approach, it was possible to establish a fragmentation order for each isomer. The SRM transitions ratio along with EPI and (18)O-labeled experiments give detailed information about differences for water elimination, allowing a proper discrimination between the isomers. This is of special interest considering the emerging role of Ch-oxidized products in the development of diseases.

  2. Determination of cholesterol oxides in processed food using high-performance liquid chromatography-mass spectrometry with atmospheric pressure chemical ionisation.

    PubMed

    Razzazi-Fazeli, E; Kleineisen, S; Luf, W

    2000-10-27

    The present work describes the development and application of an on-line atmospheric pressure ionisation (APCI) LC-MS interface for the simultaneous determination of seven toxicologically relevant cholesterol oxides (7alpha-hydroxycholesterol, 7beta-hydroxycholesterol, 25-hydroxycholesterol, 7-ketocholesterol, 5,6alpha-, 5,6beta-epoxycholesterol and cholestan-3beta,5alpha,6beta-triol). The HPLC method has been optimised to reach better separation of all tested compounds. The influences of APCI parameters (nebulising temperature, cone voltage, source temperature) on signal intensity and fragmentation pattern were investigated for all tested cholesterol oxides compounds. This is the first report on optimisation and determination of two compounds 7alpha-hydroxycholesterol and 5,6beta-epoxycholesterol in processed food using LC-MS. After extraction with hexane, clean-up was carried out using solid-phase extraction on a silica column. For the chromatographic separation of cholesterol oxides an Aquasil C18 column was used with acetonitrile-methanol (60:40) as mobile phase. For the first time we report the use of such a C18 column with a relatively hydrophilic nature for the separation of cholesterol oxides. APCI-MS detection was then applied in selected ion monitoring and positive ion modes by using the molecular ions and the main fragments. The developed method shows good linearity, high repeatability and good recovery for all tested cholesterol oxides. The method was applied for determination of seven selected cholesterol oxidation products in different foodstuffs such as butter, butteroil, lard and egg powder.

  3. Subcellular localization and properties of mouse adrenal C19-steroid 5beta-reductase.

    PubMed Central

    Collins, W; Cameron, E H

    1975-01-01

    The localization and some characteristics of mouse adrenal C19-steroid 5 beta-reductase were determined by the incubation of subcellular fractions of mouse adrenal tissue with [7 alpha-3H]androst-4-ene-3,17-dione. This enzyme was present only in the soluble fraction and was NADPH-dependent, although a small activity in the presence of NADH was also detected. The soluble fraction also contained 3alpha-, 3beta- and a small amount of 17 beta-hydroxy steroid dehydrogenase. These and other steroid-metabolizing enzymes present in the remaining subcelluar fractions are also described briefly. To measure 5 beta-androstane-3,17-dione production by the mouse adrenal soluble fraction, all 5 beta products first had to be oxidized to 5 beta-androstane-3,17-dione, and the recovery of radio-activity between the substrate androst-4-ene-3,17-dione and product 5 beta-androstane-3,17-dione of 96.1 +/-3.2% validated this technique. C19-steroid 5 beta-reductase has a pH optimum of 6.5 and at low substrate concentrations the Km and Vmax. for 5 beta reduction of [7 alpha-3H]androst-4-ene-ene-3,17-dione was 2.22 times 10(-6) "/- 0.48 times 10(-6) M and 450+/- 53 pmol/min per mg of protein respectively. At high substrate concentration, inhibition of the reaction occurred, which was shown to be due to increasing product concentration. PMID:239699

  4. Hamsters predisposed to sucrose-induced cholesterol gallstones (LPN strain) are more resistant to excess dietary cholesterol than hamsters that are not sensitive to cholelithiasis induction.

    PubMed

    Souidi, M; Combettes-Souverain, M; Milliat, F; Eckhardt, E R; Audas, O; Dubrac, S; Parquet, M; Férézou, J; Lutton, C

    2001-06-01

    We compared the effects of cholesterol feeding in male hamsters from two strains with different propensities to sucrose-induced cholelithiasis; Laboratoire de Physiologie de la Nutrition (LPN) hamsters are predisposed to developing biliary cholesterol gallstones, whereas Janvier (JAN) hamsters are not. When fed a basal control diet, LPN hamsters had a lower cholesterolemia (-21%, P = 0.01) than JAN hamsters, and a higher activity of 3-hydroxy-3-methyl glutaryl coenzyme A reductase in liver (+148%, P = 0.018) and intestine (+281%, P < 0.0001). After feeding the same diet enriched with 0.3% cholesterol for 5 wk, cholesterolemia increased more dramatically in JAN hamsters (+235%, P < 0.001) than in LPN hamsters (+108%, P < 0.001), as did the liver concentration of cholesterol, which reached 152.30 +/- 13.00 and 44.41 +/- 9.06 micromol/g, respectively. Only JAN hamsters displayed hepatomegaly, with an increased cholesterol saturation index of the gallbladder bile (+100%, P < 0.01), due to the cholesterol challenge. In liver, cholesterol feeding reduced cholesterol 7alpha-hydroxylase activity and mRNA level, and stimulated sterol 27-hydroxylase and oxysterol 7alpha-hydroxylase activities. Hepatic levels of LDL receptor decreased by approximately 60% in both strains, whereas HDL receptor scavenger class B type 1 (SR-BI) levels were unaffected by dietary cholesterol. The greater resistance of LPN hamsters to the hypercholesterolemic diet can be explained by a lower capacity to store cholesterol in the liver and greater efficiency in reducing the activity of 3-hydroxy-3-methyl glutaryl coenzyme A reductase in response to cholesterol feeding [from 11263 to 261 pmol/(min x organ) in LPN hamsters and from 4530 to 694 pmol/(min x organ) in JAN hamsters]. These results highlight the usefulness of this two-strain model, which offers some analogy with the inverse association between the predisposition to cholelithiasis and the risk of atherosclerosis in humans.

  5. Steroid hormones related to 11beta-hydroxysteroid dehydrogenase type 1 in treated obesity.

    PubMed

    Máčová, L; Sosvorová, L; Vítků, J; Bičíková, M; Hill, M; Zamrazilová, H; Sedláčková, B; Stárka, L

    2015-01-01

    The local concentration of glucocorticoids is intensively regulated by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD 1). Human 11beta-HSD 1 also reversibly catalyzes the inter-conversion of 7alpha-hydroxy- and 7beta-hydroxy-dehydroepiandrosterone (DHEA) into 7-oxo-DHEA. The cohort of 282 obese adolescents, 154 girls (median age 15.31 years, range 14.17-16.68 years) and 128 boys (median age 14.95 years, range 13.87-16.16 years), BMI (Body Mass Index) >90th percentile was examined. In samples collected before and after one month of reductive diet therapy, circulating levels of steroids were analyzed by liquid chromatography-tandem mass spectrometry and radioimmunoassay methods. The model of the treatment efficacy prediction was calculated. A significant reduction in circulating levels of cortisone, E2 and increased levels of 7beta-hydroxy-DHEA after the reductive treatment was observed. Levels of cortisol, DHEA, DHT sustained without any significant change. The predictive Orthogonal Projections to Latent Structures (OPLS) model explained 20.1 % of variability of BMI, z-score change by the basal levels of 7alpha-hydroxy-DHEA, DHEA, cortisol and E2 as the strongest predictors. Reduced levels of circulating cortisone and reduced ratios of oxygenated/reduced metabolites reflect increased reductase activity of 11beta-HSD 1 with reduced BMI, z-score. We hypothesize whether these changes can be attributed to the altered activity of 11beta-HSD 1 in the liver.

  6. Abietane diterpenes induce cytotoxic effects in human pancreatic cancer cell line MIA PaCa-2 through different modes of action.

    PubMed

    Fronza, Marcio; Lamy, Evelyn; Günther, Stefan; Heinzmann, Berta; Laufer, Stefan; Merfort, Irmgard

    2012-06-01

    Abietane diterpenes, especially those containing quinone moieties, are often reported to have cytotoxic effects on cancer cell lines. They deserve greater attention because several cancer chemotherapeutic agents also possess the quinone structural feature. To date, very little is known about their cytotoxic molecular modes of action. In the present study, five diterpenes, 7 alpha-acetoxyroyleanone, horminone, royleanone, 7-ketoroyleanone and sugiol which have been previously isolated from the medicinal plant Peltodon longipes were shown to possess cytotoxic activity against the human pancreatic cancer cell line MIA PaCa-2. 7 alpha-Acetoxyroyleanone, horminone and royleanone were demonstrated to possess alkylating properties using the nucleophile 4-(4-nitrobenzyl)pyridine. However, no clear correlation between the alkylating properties and cytotoxicity of these diterpenes was observed. Furthermore, the relaxation activity of human DNA topoisomerases I and II was found to be influenced by these compounds, with 7-ketoroyleanone and sugiol being the most active. These two diterpenes preferentially inhibited topoisomerase I and exhibited lower IC(50) values than the classical topoisomerase I inhibitor camptothecin. Molecular docking studies revealed possible interactions of diterpenes with topoisomerase I, indicating that these compounds do not form the drug-enzyme-DNA covalent ternary complex as observed with camptothecin. A binding pocket located at the surface of the DNA-interaction site was proposed. Moreover, the ability of the five diterpenes to generate DNA-strand breaks in single cells was confirmed using the alkaline comet assay. As expected, these diterpenes also influenced cell cycle progression and arrested cells in different phases of the cell cycle, primarily the G1/G0 and S-phases. Interestingly, the diterpenes only exhibited a slight ability to induce apoptotic cell death and failed to generate intracellular reactive oxygen species. These results provide

  7. Metabolism of dehydroepiandrosterone by rat hippocampal cells in culture: possible role of aromatization and 7-hydroxylation in neuroprotection.

    PubMed

    Jellinck, P H; Lee, S J; McEwen, B S

    2001-10-01

    The rate of metabolism of the multifunctional neurosteroid, dehydroepiandrosterone (DHEA), by embryonic rat hippocampal cells maintained in culture was compared to that of 4-androstenedione (AD), the immediate precursor of estrone (E1). The experiments were carried out to assess the relative contribution of DHEA, its 7-hydroxylated metabolites and estrogen on their reported effects on memory and neuroprotection. The 3H-labeled steroids of high specific radioactivity were incubated for 1, 8, 24 and 48 h and the putative metabolites extracted from the culture medium with acetone-ethyl acetate before separation by TLC for radioassay. [3H]DHEA (2.0 ng/5x10(5) cells) yielded primarily the 7alpha- and 7beta-hydroxylated steroids in an almost equal ratio under conditions that resembled those used by others to study the protection of neurons by hippocampal astrocytes against excitatory amino acid-induced toxicity. The rate of conversion of DHEA to AD, and particularly to E1, was much lower. With [3H]AD as substrate, significant aromatization to estrogen occurred only after 24 h when most of [3H]DHEA had already been converted to its 7-hydroxylated products and the hydroxylase and aromatase systems would no longer be competing for the same coenzyme (NADPH). The hippocampal cells were still viable after 48 h of incubation with the steroids and were able to oxidize estradiol (E2) to E1 and reduce E1 to E2 and AD to testosterone (T). It is suggested that 7alpha- and 7beta-OHDHEA, the main metabolites formed in the rat hippocampus, might be responsible for some of the functions previously ascribed to estrogens in the brain and the reasons for this proposal are discussed.

  8. Evaluation of an (111)In-DOTA-rhenium cyclized alpha-MSH analog: a novel cyclic-peptide analog with improved tumor-targeting properties.

    PubMed

    Chen, J; Cheng, Z; Owen, N K; Hoffman, T J; Miao, Y; Jurisson, S S; Quinn, T P

    2001-12-01

    The aim of this study was to examine the effect of rhenium-mediated peptide cyclization on melanoma targeting, biodistribution, and clearance kinetics of the alpha-melanocyte-stimulating hormone (alpha-MSH) analog 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) coupled ReO-cyclized [Cys(3,4,10),D-Phe(7)]alpha-MSH(3-13) (DOTA-ReCCMSH). DOTA-ReCCMSH was compared with its reduced nonmetalated linear homolog, DOTA-CCMSH, and an analog in which rhenium cyclization was replaced by disulfide bond cyclization, DOTA-[Cys(4,10),D-Phe(7)]alpha-MSH(4-13) (CMSH). DOTA was also conjugated to the amino terminus of one of the highest-affinity alpha-MSH receptor-binding peptides, [Nle(4),D-Phe(7)]alpha-MSH (NDP), as a linear peptide standard. The DOTA-conjugated alpha-MSH analogs were radiolabeled with (111)In and examined for their in vitro receptor-binding affinity with B16/F1 murine melanoma cells, and their in vivo biodistribution properties were evaluated and compared in melanoma tumor-bearing C57 mice. The tumor uptake values of (111)In-DOTA-ReCCMSH were significantly higher than those of the other closely related (111)In-DOTA-alpha-MSH conjugates. Even at 24 h after injection, a comparison of the tumor uptake values for (111)In-DOTA-coupled ReCCMSH (4.86 +/- 1.52 percentage injected dose [%ID]/g), CCMSH (1.91 +/- 0.56 %ID/g), CMSH (3.09 +/- 0.32 %ID/g), and NDP (2.47 +/- 0.79 %ID/g) highlighted the high tumor retention property of ReCCMSH. Rhenium-coordinated cyclization resulted in less renal radioactivity accumulation of (111)In-DOTA-ReCCMSH (8.98 +/- 0.82 %ID/g) than of (111)In-DOTA-CCMSH (63.2 +/- 15.6 %ID/g), (111)In-DOTA-CMSH (38.4 +/- 3.6 %ID/g), and (111)In-DOTA-NDP (12.0 +/- 1.96 %ID/g) at 2 h after injection and significantly increased its clearance into the urine (92 %ID at 2 h after injection). A high radioactivity uptake ratio of tumor to normal tissue was obtained for (111)In-DOTA-ReCCMSH (e.g., 489, 159, 100, and 49 for blood, muscle, lung, and

  9. Effects of CYP7B1-mediated catalysis on estrogen receptor activation.

    PubMed

    Pettersson, Hanna; Lundqvist, Johan; Norlin, Maria

    2010-09-01

    Most of the many biological effects of estrogens are mediated via the estrogen receptors ERalpha and beta. The current study examines the role of CYP7B1-mediated catalysis for activation of ER. Several reports suggest that CYP7B1 may be important for hormonal action but previously published studies are contradictory concerning the manner in which CYP7B1 affects ERbeta-mediated response. In the current study, we examined effects of several CYP7B1-related steroids on ER activation, using an estrogen response element (ERE) reporter system. Our studies showed significant stimulation of ER by 5-androstene-3beta,17beta-diol (Aene-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-Adiol). In contrast, the CYP7B1-formed metabolites from these steroids did not activate the receptor, indicating that CYP7B1-mediated metabolism abolishes the ER-stimulating effect of these compounds. The mRNA level of HEM45, a gene known to be stimulated by estrogens, was strongly up-regulated by Aene-diol but not by its CYP7B1-formed metabolite, further supporting this concept. We did not observe stimulation by dehydroepiandrosterone (DHEA) or 7alpha-hydroxy-DHEA, previously suggested to affect ERbeta-mediated response. As part of these studies we examined metabolism of Aene-diol in pig liver which is high in CYP7B1 content. These experiments indicate that CYP7B1-mediated metabolism of Aene-diol is of a similar rate as the metabolism of the well-known CYP7B1 substrates DHEA and 3beta-Adiol. CYP7B1-mediated metabolism of 3beta-Adiol has been proposed to influence ERbeta-mediated growth suppression. Our results indicate that Aene-diol also might be important for ER-related pathways. Our data indicate that low concentrations of Aene-diol can trigger ER-mediated response equally well for both ERalpha and beta and that CYP7B1-mediated conversion of Aene-diol into a 7alpha-hydroxymetabolite will result in loss of action.

  10. Tumorigenicity of the optical enantiomers of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides in newborn mice: exceptional activity of (+)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

    PubMed Central

    Buening, M K; Wislocki, P G; Levin, W; Yagi, H; Thakker, D R; Akagi, H; Koreeda, M; Jerina, D M; Conney, A H

    1978-01-01

    The tumorigenicities of benzo[a]pyrene and each optical enantiomer of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides derived from trans-7,8-dihydroxy-7,8-dihydrobenzol[a]pyrene were tested by sequential intraperitoneal injection of mice with 1,2, and 4 nmol, or with 2, 4, and 8 nmol of each compound on the 1st, 8th, and 15th day of life, respectively. The experiment was terminated when the animals were 34--37 weeks old. (+)-7beta, 8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzol[a]pyrene [(+)-BP-7beta,8alpha-diol-9alpha,10alpha-epoxide 2] had exceptional tumorigenicity, whereas benzo[a]-pyrene and the other three optically pure isomers of the benzo[a]pyrene 7,8-diol,9,10-epoxides had little or no activity. These results demonstrate differences in the carcinogenic activities of optically active isomers of a polycyclic hydrocarbon diol epoxide. Eleven percent of control mice had pulmonary tumors, whereas 71% and 100% of the mice treated with a total dose of 7 or 14 nmol of (+)-BP-7beta,8alpha-diol-9alpha,10alpha-epoxide 2, respectively, had pulmonary tumors. Control mice had an average of 0.12 pulmonary tumors per mouse, whereas mice treated with a total dose of 7 or 14 nmol of (+)-BP-7beta,8alpha-diol-9alpha,10alpha-epoxide 2 had 1.72 and 7.67 pulmonary tumors per mouse, respectively. Mice treated with 14 nmol of (-)-BP-7alpha,8beta-diol-9beta,10beta-epoxide 2, (-)-BP-7beta,8alpha-diol-9beta,10beta-epoxide 1, or (+)-BP-7alpha,8beta-diol-9alpha,10alpha-epoxide 1 had 0.13, 0.25, and 0.34 pulmonary tumors per animal, respectively. PMID:281685

  11. Binding characteristics of aromatase inhibitors and phytoestrogens to human aromatase.

    PubMed

    Chen, S; Kao, Y C; Laughton, C A

    1997-04-01

    We have evaluated the binding characteristics of three steroidal inhibitors [4-hydroxyandrostenedione (4-OHA), 7alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione (7alpha-APTADD), and bridge (2,19-methyleneoxy) androstene-3,17-dione (MDL 101,003)], four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)], and two flavone phytoestrogens (chrysin, and 7,8-dihydroxyflavone) to aromatase through a combination of computer modeling and inhibitory profile studies on the wild-type and six aromatase mutants (I133Y, P308F, D309A, T310S, I395F, and I474Y). We have generated two aromatase models based on the x-ray structures of cytochrome P450-cam and cytochrome P450bm3, respectively. A major difference between the cytochrome P450cam-based and cytochrome P450bm3-based models is in the predicted lengths of helices F and G. In the cytochrome P450cam-based model, helices F and G lie antiparallel and extend across the active-site face of the molecule from one edge to the center, so that the carboxyl-terminal residues of helix F and the N-terminal residues of helix G make a major contribution to the structure of the active site. In the cytochrome P450bm3-based model, both helices are longer and so extend almost all the way across the active-site face of the molecule. Considering the size of the androgen substrate, we evaluated our results mainly based on the cytochrome P450cam model. The mutations involved in this study are thought to be at or near the proposed active site pocket. The inhibitory profile analysis has produced very interesting results and provided a molecular basis as to how seven aromatase inhibitors with different structures bind to the active site of aromatase. Furthermore, the investigation reveals that phytoestrogens bind to the active site of aromatase in a different orientation from that in the estrogen receptor.

  12. [Chemical diversity of the biological active ingredients of salvia officinalis and some closely related species].

    PubMed

    Máthé, Imre; Hohmann, Judit; Janicsák, Gábor; Nagy, Gábor; Dora, Rédei

    2007-01-01

    acids, caffeic acid and now phenylethanolids. Diterpenes i.e. 7-methyl carnosoate, rosmanol 7- methylether, sageon from S. officinalis, 7alpha-acetoyroyleanone, 7alpha-hydroxyroyleanone, royleanone, 6,7-dehydroroyleanone from S. tomentosa and candesalvoquinone, candelabroquinone, 12-O-methylcandesalvone, candesalvone B methyl ester and candelabrone have been isolated from Salvia candelabrum. All of the compounds belong to the abietane type of diterpenoids and have pronounced antioxidant effect.

  13. Helium radiogenic clock for dating of archaeological gold

    NASA Astrophysics Data System (ADS)

    Kossolapov, Alexander J.; Ivanov, Alexander P.; Kuznetsov, Pavel B.

    1998-05-01

    The archaeological gold contains usually 10 - 80 ppb U238 as a trace element. Under radioactive decay U238 produces approximately 3.7 alpha particles in 1 mg of metal containing 1 ppb of U238 per one year. The a-particles (as He4) are accumulated inside the metal as gold appears to be extremely retentive for He. When the metal is heated to its melting point all accumulated helium escapes thus establishing 'zero-time' for the He-radiogenic clock. This way the clock starts at the point when the object is manufactured. The new ultrasensitive He-recycling mass-spectrometer providing the sensitivity approximately 2.5(DOT)104 He-atoms has been developed and manufactured for the museum usage. Taking a gold sample of 5 - 10 mg with typical 10 ppb U content in gold one can expect to find 185,000 - 370,000 He atoms in metal manufactured 1000 years ago. These quantities can be easily detected by the instrument thus providing rather reliable authentication (and in some cases -- absolute dating) tool for detection of fakes.

  14. Disruption of the murine alpha1-antitrypsin/PI2 gene.

    PubMed

    Kushi, Atsuko; Akiyama, Kiyotaka; Noguchi, Masato; Edamura, Koji; Yoshida, Takayuki; Sasai, Hitoshi

    2004-10-01

    Alpha-1-antitrypsin (alpha1-AT) is a member of the serine protease inhibitor family regulating numerous proteolytic processes. The genetic disorder, alpha1-AT deficiency, is well known as a cause of hereditary pulmonary emphysema and liver cirrhosis. To create an animal model of human alpha1-AT deficiency, we disrupted the major murine isoform PI2, which is similar to human alpha1-AT and is one of 7 alpha1-AT isoforms found in the mouse. The ability of the serum to inhibit the activities of human leukocyte elastase (HLE) and human chymotrypsin (CYT) was significantly lower in heterozygous mice (alpha1-AT/PI2 -/+) than wild-type (alpha1-AT/PI2 +/+) mice (73.2% vs. 100% for HLE and 67.8% vs.100% for CYT, respectively; P<0.05). The distribution of genotypes among F(2) progeny was not in accordance with Mendelian distribution (P<0.01), as the percentages of wild-type, heterozygotes and homozygotes were 47.8%, 37.3% and 14.9%, respectively. Thus, it is likely that impairment of the protease inhibitor had a critical effect on fetus development. The alpha1-AT/PI2 deficient mouse will be a useful animal model for elucidating the function of alpha1-AT in fetal development, studying the mechanisms of chronic inflammatory disease and evaluating therapeutic candidates for the treatment of inflammatory disease.

  15. The fruit of Acanthopanax senticosus (Rupr. et Maxim.) Harms improves insulin resistance and hepatic lipid accumulation by modulation of liver adenosine monophosphate-activated protein kinase activity and lipogenic gene expression in high-fat diet-fed obese mice.

    PubMed

    Saito, Tetsuo; Nishida, Miyako; Saito, Masafumi; Tanabe, Akari; Eitsuka, Takahiro; Yuan, Shi-Hua; Ikekawa, Nobuo; Nishida, Hiroshi

    2016-10-01

    Obesity-associated insulin resistance is a major risk factor for most metabolic diseases, including dyslipidemia and type 2 diabetes. Acanthopanax senticosus (Rupr. et Maxim.) Harms (Goka) root has been used in traditional Chinese medicine for treatment of diabetes and other conditions; however, little is known about the effects of Goka fruit (GF). Goka fruit is rich in anthocyanin, which has beneficial effects on obesity and insulin resistance via activation of adenosine monophosphate-activated protein kinase (AMPK). We hypothesized that GF can improve obesity-associated insulin resistance. The aim of the present study was to investigate whether GF improves insulin resistance in high-fat diet (HFD)-induced obese mice. High-fat diet mice treated with GF (500 and 1000 mg/kg) for 12 weeks showed an improved glucose tolerance and insulin sensitivity, as well as reduced plasma insulin and liver lipid accumulation. Moreover, GF administration to HFD mice resulted in down-regulation of fatty acid synthase expression and up-regulation of cholesterol 7-alpha-hydroxylase expression in the liver. Notably, AMPK phosphorylation in the liver increased after GF administration. In summary, GF supplementation improved obesity-associated insulin resistance and hepatic lipid accumulation through modulation of AMPK activity and lipid metabolism-associated gene expression.

  16. Cholesterol and Lipoprotein Dynamics in a Hibernating Mammal

    PubMed Central

    Otis, Jessica P.; Sahoo, Daisy; Drover, Victor A.; Yen, Chi-Liang Eric; Carey, Hannah V.

    2011-01-01

    Hibernating mammals cease feeding during the winter and rely primarily on stored lipids to fuel alternating periods of torpor and arousal. How hibernators manage large fluxes of lipids and sterols over the annual hibernation cycle is poorly understood. The aim of this study was to investigate lipid and cholesterol transport and storage in ground squirrels studied in spring, summer, and several hibernation states. Cholesterol levels in total plasma, HDL and LDL particles were elevated in hibernators compared with spring or summer squirrels. Hibernation increased plasma apolipoprotein A-I expression and HDL particle size. Expression of cholesterol 7 alpha-hydroxylase was 13-fold lower in hibernators than in active season squirrels. Plasma triglycerides were reduced by fasting in spring but not summer squirrels. In hibernators plasma β-hydroxybutyrate was elevated during torpor whereas triglycerides were low relative to normothermic states. We conclude that the switch to a lipid-based metabolism during winter, coupled with reduced capacity to excrete cholesterol creates a closed system in which efficient use of lipoproteins is essential for survival. PMID:22195001

  17. Structural and functional characterization of a novel homodimeric three-finger neurotoxin from the venom of Ophiophagus hannah (king cobra).

    PubMed

    Roy, Amrita; Zhou, Xingding; Chong, Ming Zhi; D'hoedt, Dieter; Foo, Chun Shin; Rajagopalan, Nandhakishore; Nirthanan, Selvanayagam; Bertrand, Daniel; Sivaraman, J; Kini, R Manjunatha

    2010-03-12

    Snake venoms are a mixture of pharmacologically active proteins and polypeptides that have led to the development of molecular probes and therapeutic agents. Here, we describe the structural and functional characterization of a novel neurotoxin, haditoxin, from the venom of Ophiophagus hannah (King cobra). Haditoxin exhibited novel pharmacology with antagonism toward muscle (alphabetagammadelta) and neuronal (alpha(7), alpha(3)beta(2), and alpha(4)beta(2)) nicotinic acetylcholine receptors (nAChRs) with highest affinity for alpha(7)-nAChRs. The high resolution (1.5 A) crystal structure revealed haditoxin to be a homodimer, like kappa-neurotoxins, which target neuronal alpha(3)beta(2)- and alpha(4)beta(2)-nAChRs. Interestingly however, the monomeric subunits of haditoxin were composed of a three-finger protein fold typical of curaremimetic short-chain alpha-neurotoxins. Biochemical studies confirmed that it existed as a non-covalent dimer species in solution. Its structural similarity to short-chain alpha-neurotoxins and kappa-neurotoxins notwithstanding, haditoxin exhibited unique blockade of alpha(7)-nAChRs (IC(50) 180 nm), which is recognized by neither short-chain alpha-neurotoxins nor kappa-neurotoxins. This is the first report of a dimeric short-chain alpha-neurotoxin interacting with neuronal alpha(7)-nAChRs as well as the first homodimeric three-finger toxin to interact with muscle nAChRs.

  18. Concentration of chosen oxycholesterols in plasma of pregnant women with pregnancy-induced hypertension.

    PubMed

    Bodzek, Piotr; Janoszka, Beata; Wielkoszyński, Tomasz; Bodzek, Danuta; Sieroń, Aleksander

    2002-02-01

    Solid-phase extraction (SPE) was applied for isolation of oxycholesterols from plasma lipid extract from pregnant women with hypertension and from a control group. Separation of oxycholesterols fraction was performed in an SD II horizontal chamber (Chromdes, Poland) using silica gel and octadecyl RPC18 silica gel TLC plates (Merck and Machery Nagel). Visualization was carried out under UV light after Liebermann-Burchard reaction specific for cholesterol and its derivatives. The oxycholesterols (5-cholestene-3beta-ol-7-one, sum of 5-cholestene-3beta, 7beta-diol and 5-cholestene-3beta, 7alpha-diol and sum of 5alpha,6alpha-epoxycholestan-3beta-ol and 5beta, 6beta-epoxycholestan-3beta-ol) were quantified by chromatograms scanning in reflectance and fluorescence mode using a CS 9301 densitometer (Shimadzu). The total concentration of the investigated oxycholesterols in the plasma of pregnant women was up to 5000 ng/mL and was statistically significantly higher in women with pregnancy induced hypertension (PIH).

  19. Predicted unfolding order of the 13 alpha-helices in the catalytic domain of glucoamylase from Aspergillus awamori var. X100 by molecular dynamics simulations.

    PubMed

    Liu, Hsuan-Liang; Wang, Wen-Chi

    2003-01-01

    The unfolding mechanism of the 13 alpha-helices in the catalytic domain of Aspergillus awamori var. X100 glucoamylase was investigated by 200 ps molecular dynamics simulations in explicit water with temperature jump technique. Rather than a simultaneous event, the unfolding of these 13 alpha-helices followed a random ordered mechanism as alpha8-->alpha1-->alpha11-->alpha7-->alpha10-->alpha3-->alpha12-->alpha13-->alpha4-->alpha5-->alpha9-->alpha6-->alpha2. No significant relationships were found between the unfolding order and the length and the hydrophobicity of the helix. alpha-Helix 8 located in the inner region of the catalytic domain was predicted to be the first helix to unfold, indicating that the destruction of the secondary structure motif was initiated from the inner region of the catalytic domain. The dynamic behavior of these alpha-helices induced by increased kinetic energy during the unfolding process is considered to be similar to the expansion and compression of a series of springs under the influence of mechanical stress.

  20. Novel Vaccine Candidates against Brucella melitensis Identified through Reverse Vaccinology Approach.

    PubMed

    Vishnu, Udayakumar S; Sankarasubramanian, Jagadesan; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

    2015-11-01

    Global health therapeutics is a rapidly emerging facet of postgenomics medicine. In this connection, Brucella melitensis is an intracellular bacterium that causes the zoonotic infectious disease, brucellosis. Presently, no licensed vaccines are available for human brucellosis. Here, we report the identification of potential vaccine candidates against B. melitensis using a reverse vaccinology approach. Based on a systematic screening of exoproteome and secretome of B. melitensis 16 M, we identified eight proteins as potential vaccine candidates, including LPS-assembly protein LptD, a polysaccharide export protein, a cell surface protein, heme transporter BhuA, flagellin FliC, 7-alpha-hydroxysteroid dehydrogenase, immunoglobulin-binding protein EIBE, and hemagglutinin. Among these, the roles of BhuA and hemagglutinin in the virulence of Brucella are essential to establish infection. Roles of other proteins in the virulence are yet to be studied. Prediction of protein-protein interactions revealed that these proteins can interact with other proteins involved in virulence, secretion system, metabolism, and transport. From these eight potential vaccine candidates, we predicted three surface exposed novel antigenic epitopes that can induce both B-cell and T-cell immune responses. These peptides can be used for the development of either exclusive peptide vaccines or multi-component vaccines against human brucellosis. Reverse vaccinology is an important strategy for discovery of novel global health therapeutics.

  1. Acid-base titration of melanocortin peptides: evidence of Trp rotational conformers interconversion.

    PubMed

    Fernandez, Roberto M; Vieira, Renata F F; Nakaie, Clóvis R; Lamy, M Teresa; Ito, Amando S

    2005-01-01

    Tryptophantime-resolved fluorescence was used to monitor acid-base titration properties of alpha-melanocyte stimulating hormone (alpha-MSH) and the biologically more potent analog [Nle4, D-Phe7]alpha -MSH (NDP-MSH), labeled or not with the paramagnetic amino acid probe 2,2,6,6-tetramthylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac). Global analysis of fluorescence decay profiles measured in the pH range between 2.0 and 11.0 showed that, for each peptide, the data could be well fitted to three lifetimes whose values remained constant. The less populated short lifetime component changed little with pH and was ascribed to Trp g+ chi1 rotamer, in which electron transfer deactivation predominates over fluorescence. The long and intermediate lifetime preexponential factors interconverted along that pH interval and the result was interpreted as due to interconversion between Trp g- and trans chi1 rotamers, driven by conformational changes promoted by modifications in the ionization state of side-chain residues. The differences in the extent of interconversion in alpha-MSH and NDP-MSH are indicative of structural differences between the peptides, while titration curves suggest structural similarities between each peptide and its Toac-labeled species, in aqueous solution. Though less sensitive than fluorescence, the Toac electron spin resonance (ESR) isotropic hyperfine splitting parameter can also monitor the titration of side-chain residues located relatively far from the probe.

  2. CYP7A1 Gene Polymorphism Located in the 5′ Upstream Region Modifies the Risk of Coronary Artery Disease

    PubMed Central

    Iwanicki, Tomasz; Balcerzyk, Anna; Niemiec, Pawel; Nowak, Tomasz; Ochalska-Tyka, Anna; Krauze, Jolanta; Kosiorz-Gorczynska, Sylwia; Grzeszczak, Wladyslaw; Zak, Iwona

    2015-01-01

    Background. 7-Alpha cholesterol hydroxylase (CYP7A1), the first enzyme of classic conversion pathway leading from cholesterol to bile acids synthesis, is encoded by CYP7A1 gene. Its single nucleotide polymorphisms (SNPs) influence serum lipid levels and may be related to impaired lipid profile leading to coronary artery disease (CAD). The aim of the present study was to analyze the possible association between the rs7833904 CYP7A1 polymorphism and premature CAD. Material and Methods. Serum lipid levels and rs7833904 SNP were determined in 419 subjects: 200 patients with premature CAD and 219 age and sex matched controls. Results. The A allele carrier state was associated with CAD (OR = 1.76, 95% CI; 1.14–2.71, P = 0.014). The effect was even stronger in the male subgroups (OR = 2.16, 95% CI; 1.28–3.65, P = 0.003). There was no effect in the females. Risk factors of CAD and clinical phenotype of atherosclerosis were not associated with genotype variants of the rs7833904 SNP. Lipid profiles also did not differ significantly between individual genotypes. Conclusion. The CYP7A1 rs7833904 polymorphism may modify the risk of CAD. This effect is especially strong in male subjects. The studied polymorphism does not significantly influence serum lipid levels, in the present study. PMID:25944972

  3. Transdermal delivery of a melanotropic peptide hormone analogue

    SciTech Connect

    Dawson, B.V.; Hadley, M.E.; Kreutzfeld, K.; Dorr, R.T.; Hruby, V.J.; Al-Obeidi, F.; Don, S.

    1988-01-01

    We previously reported that topical application of (Nl3/sup 4/,D-Phe/sup 7/)alpha-MSH, a superpotent analogue of alpha-melanocyte stimulating hormone, to mice induces a darkening of follicular melanocytes throughout the skin. We now report that the melanotropin analogue can be delivered across mouse but not rat skin in an in vitro model system. Passage of the analogue from the topically applied vehicle (polyethylene glycol) across the skin into a subcutaneous receiving vessel was demonstrated by both bioassay as well as by radioimmunoassay. The bioassay data demonstrate that percutaneous absorption of the melanotropin did not result in loss of biological activity of the peptide. The differential penetration of the peptide across rodent skin reveals that one cannot predict percutaneous absorption of a substance across the stratum corneum from studies on a single species. The present results are the first to demonstrate, by direct quantitative measurements, that a bioactive peptide can be delivered across the vertebrate integument in vitro. These studies point out the potential of a topically applied melanotropin for tanning of the skin and possibly for treatment of certain hypopigmentary disorders.

  4. Isolation and structure determination of one new metabolite isolated from the red fermented rice of Monascus purpureus.

    PubMed

    Cheng, Ming-Jen; Chen, Jih-Jung; Wu, Ming-Der; Yang, Ping-Shin; Yuan, Gwo-Fang

    2010-06-01

    The n-BuOH-soluble portion of the 95% EtOH extract of red fermented rice fermented with the yellow mutant of the fungus Monascus purpureus BCRC 38113 (Monascaceae) led to the isolation of one new pyran-2-one derivative, namely peroxymonascuspyrone (1), along with nine known compounds, monasfluore A (2), monasfluore B (3), 3-epi-betulinic acid (4), 3-epi-betulinic acid acetate (5), alpha-tocospiro A (6), friedelan-3-one (7), alpha-cadinol (8), anticopalol (9), and spathulenol (10). Interestingly, this is the first report of a naturally occurring pyran-2-one skeleton isolated from Monascus sp. Their structures and relative configurations were elucidated by spectroscopic methods, including 1D- and 2D-NMR ((1)H,(1)H-COSY, HMQC, HMBC and NOESY), as well as low- and high-resolution mass spectrometric analyses. In addition, cytotoxicities against MCF-7, NCI-H460 and SF-268 cancer cell lines were measured in vitro; the results revealed that these metabolites have no cytotoxicity against the selected tumour cells.

  5. A single step solid-phase extraction method for complete separation of sterol oxidation products in food lipids.

    PubMed

    Azadmard-Damirchi, Sodeif; Dutta, Paresh C

    2009-01-02

    One of the crucial steps in determination of sterol oxidation products (SOPs) in foods is their enrichment and purifications by various preparative methods for further analysis by GC and GC-MS. Among the preparative methods, SPE of various adsorbents and solvent systems, are being used most widely. At present, no single step SPE method is suitable to completely separate the SOPs. In this study, a SPE (1g silica) method, suitable for both transesterified and cold saponified oil samples, was developed to separate completely SOPs from other lipid components. This method resulted in high recovery from rapeseed oil of added 5beta,6beta-epoxycholestan-3beta-ol (94-96%), cholest-5-en-3beta-ol-7-one(94%), cholestane-3beta,5alpha,6beta-triol (88-91%), cholest-5-en-3beta,7alpha-diol and 5alpha,6alpha-epoxycholestan-3beta-ol (88-90%). The method has a high sample capacity of up to 1g transesterified or cold-saponified oil sample. The method was tested and applied to different vegetable oils and to monitor the effects of refining processes on POPs in hazelnut oil.

  6. Effect of white wheat bread containing sugar beet fiber on serum lipids and hepatic mRNA in rats fed on a cholesterol-free diet.

    PubMed

    Nakamura, Yumi; Kanazawa, Mizuki; Liyanage, Ruvini; Iijima, Setsuko; Han, Kyu-Ho; Shimada, Ken-ichiro; Sekikawa, Mitsuo; Yamauchi, Akihiro; Hashimoto, Naoto; Ohba, Kiyoshi; Fukushima, Michihiro

    2009-06-01

    We examined the effects of white wheat bread powder (BP) and white wheat bread powder containing sugar beet fiber (BBP) on serum cholesterol. The total cholesterol (-11%, -16%), HDL-cholesterol (-12%, -11%), non-HDL-cholesterol (-9%, -18%) and triacylglycerol (-44%, -58%) concentrations in the BP and BBP groups, respectively, were significantly different from those in the control group. The fecal excretion of neutral sterols in the BP and BBP groups and of acidic sterols in the BBP group was significantly higher than that in the control group. The hepatic cholesterol 7alpha-hydroxylase (CYP7A1) mRNA level in the BP and BBP groups was significantly higher than that in the control group. The cecal total short-chain fatty acid concentrations in the BBP group were significantly higher than those in the control group. These results indicate that the observed changes in serum lipid levels in the BP and BBP groups were due to the increased fecal lipid and CYP7A1 mRNA levels.

  7. Rapid affinity-purification and physicochemical characterization of pumpkin (Cucurbita maxima) phloem exudate lectin.

    PubMed

    Narahari, Akkaladevi; Swamy, Musti J

    2010-04-21

    The chito-oligosaccharide-specific lectin from pumpkin (Cucurbita maxima) phloem exudate has been purified to homogeneity by affinity chromatography on chitin. After SDS/PAGE in the presence of 2-mercaptoethanol, the pumpkin phloem lectin yielded a single band corresponding to a molecular mass of 23.7 kDa, whereas ESI-MS (electrospray ionization MS) gave the molecular masses of the subunit as 24645 Da. Analysis of the CD spectrum of the protein indicated that the secondary structure of the lectin consists of 9.7% alpha-helix, 35.8% beta-sheet, 22.5% beta-turn and 32.3% unordered structure. Saccharide binding did not significantly affect the secondary and tertiary structures of the protein. The haemagglutinating activity of pumpkin phloem lectin was mostly unaffected in the temperature range 4-70 degrees C, but a sharp decrease was seen between 75 and 85 degrees C. Differential scanning calorimetric and CD spectroscopic studies suggest that the lectin undergoes a co-operative thermal unfolding process centred at approx. 81.5 degrees C, indicating that it is a relatively stable protein.

  8. Mid Infrared Hydrogen Recombination Line Emission from the Maser Star MWC 349A

    NASA Technical Reports Server (NTRS)

    Smith, Howard A.; Strelnitski, V.; Miles, J. W.; Kelly, D. M.; Lacy, J. H.

    1997-01-01

    We have detected and spectrally resolved the mid-IR hydrogen recombination lines H6(alpha)(12.372 micrometers), H7(alpha)(19.062 micrometers), H7(beta)(l1.309 micrometers) and H8(gamma)(12.385 micrometers) from the star MWC349A. This object has strong hydrogen maser emission (reported in the millimeter and submillimeter hydrogen recombination lines from H36(alpha) to H21(alpha)) and laser emission (reported in the H15(alpha), H12(alpha) and H10(alpha) lines). The lasers/masers are thought to arise predominantly in a Keplerian disk around the star. The mid-IR lines do not show evident signs of lasing, and can be well modeled as arising from the strong stellar wind, with a component arising from a quasi-static atmosphere around the disk, similar to what is hypothesized for the near IR (less than or equal to 4 micrometers) recombination lines. Since populations inversions in the levels producing these mid-IR transitions are expected at densities up to approximately 10(exp 11)/cu cm, these results imply either that the disk does not contain high-density ionized gas over long enough path lengths to produce a gain approximately 1, and/or that any laser emission from such regions is small compared to the spontaneous background emission from the rest of the source as observed with a large beam. The results reinforce the interpretation of the far-IR lines as true lasers.

  9. FGF15/FGFR4 integrates growth factor signaling with hepatic bile acid metabolism and insulin action.

    PubMed

    Shin, Dong-Ju; Osborne, Timothy F

    2009-04-24

    The current studies show FGF15 signaling decreases hepatic forkhead transcription factor 1 (FoxO1) activity through phosphatidylinositol (PI) 3-kinase-dependent phosphorylation. The bile acid receptor FXR (farnesoid X receptor) activates expression of fibroblast growth factor (FGF) 15 in the intestine, which acts through hepatic FGFR4 to suppress cholesterol-7alpha hydroxylase (CYP7A1) and limit bile acid production. Because FoxO1 activity and CYP7A1 gene expression are both increased by fasting, we hypothesized CYP7A1 might be a FoxO1 target gene. Consistent with recently reported results, we show CYP7A1 is a direct target of FoxO1. Additionally, we show that the PI 3-kinase pathway is key for both the induction of CYP7A1 by fasting and the suppression by FGF15. FGFR4 is the major hepatic FGF receptor isoform and is responsible for the hepatic effects of FGF15. We also show that expression of FGFR4 in liver was decreased by fasting, increased by insulin, and reduced by streptozotocin-induced diabetes, implicating FGFR4 as a primary target of insulin regulation. Because insulin and FGF both target the PI 3-kinase pathway, these observations suggest FoxO1 is a key node in the convergence of FGF and insulin signaling pathways and functions as a key integrator for the regulation of glucose and bile acid metabolism.

  10. Radioimmunoassay of inhibin based on synthetic human inhibin alpha-chain peptide

    SciTech Connect

    Sinosich, M.J.; Sieg, S.; Zakher, A.; Ling, N.; Saunders, D.M.; Rosenwaks, Z.; Hodgen, G.D. )

    1991-01-01

    Polyclonal rabbit antisera were produced against cyclic human inhibin ((Cys6, Tyr7) alpha-(6-30)NH2) peptide, covalently conjugated to bovine serum albumin. The tyrosine residue introduced at position 7 facilitated the oxidative incorporation of radiolabel ({sup 125}I) to yield a tracer with specific activity of 73.9 Ci/g. These reagents were used to develop a homologous equilibrium radioimmunoassay for human inhibin, with polyethylene glycol, 200 g/L, serving as the separation phase. At a detection limit of 2 micrograms/L (n = 7), immunoactive inhibin was detectable in human pre-ovulatory follicular fluid (128 micrograms/L), seminal plasma (2374 micrograms/L), amniotic fluid (66 micrograms/L), and placental extract (347 micrograms/L). We also demonstrated inhibin immunoreactivity in biological fluids from other mammalian species: macaque, chimpanzee, porcine, and bovine, but not rodent (guinea pig). Although the antisera were raised against a nonbioactive inhibin peptide, immunoglobulins fractionated on Protein A-Sepharose neutralized the bioactivity of human ovarian inhibin. Further characterization of inhibin immuno- and bioactivity was undertaken with immobilized heparin, divalent metal cations, and dye ligands. Only heparin-Sepharose distinguished between immuno- and bioactive inhibin.

  11. Fluorescence study of conformational properties of melanotropins labeled with aminobenzoic acid.

    PubMed Central

    Ito, A S; Souza, E S; dos Reis Barbosa, S; Nakaie, C R

    2001-01-01

    The native hormone alpha-melanocyte-stimulating hormone (alpha-MSH) and its more potent analog [Nle(4),D-Phe(7)]alpha-MSH (NDP-alpha MSH), labeled at the amino terminal with the fluorescent aminobenzoic acid (Abz) isomers, were examined by fluorescence methods. We observed energy transfer between the tryptophan(9) residue acting as donor and Abz as acceptor, the transfer being more pronounced to the ortho-form of the acceptor. Within the hypothesis that different peptide conformations coexist in equilibrium during the fluorescence decay, we supposed that the intensity decay was modulated by an acceptor-donor distance distribution function f(r). From the time-resolved fluorescence experimental data, we recovered the distance distribution between Abz and Trp(9), using the CONTIN program, within the framework of the Förster resonance energy transfer model. The methodology proved to be useful to provide quantitative information about conformational dynamics of melanotropins and its dependency on the solvent. In aqueous medium, alpha-MSH has a broad Abz-Trp(9) distance distribution, reflecting the structural flexibility of the peptide. Three different distance populations could be identified in the labeled analog NDP-alpha MSH in water, indicating distinct conformational states for the synthetic peptide, compared with the native hormone. Measurements in trifluoroethanol resulted in the recovery of two Abz-Trp(9) distance populations, both for the native and the analog hormones, reflecting the decrease, induced by the solvent, of the conformational states available to the peptides. PMID:11463659

  12. Oral administration of the Aureobasidium pullulans-derived β-glucan effectively prevents the development of high fat diet-induced fatty liver in mice

    PubMed Central

    Aoki, Shiho; Iwai, Atsushi; Kawata, Koji; Muramatsu, Daisuke; Uchiyama, Hirofumi; Okabe, Mitsuyasu; Ikesue, Masahiro; Maeda, Naoyoshi; Uede, Toshimitsu

    2015-01-01

    Aureobasidium pullulans-derived β-glucan (AP-PG) consisting of a β-(1,3)-linked glucose main chain and β-(1,6)-linked glucose branches is taken as a supplement to improve health. This study demonstrates that oral administration of AP-PG is effective to prevent the development of high-fat diet (HFD)-induced fatty liver in mice. Here, C57BL/6N mice were fed with a normal diet or HFD, and AP-PG diluted in drinking water was administered orally. After 16 weeks, the serological analysis showed that HFD-induced high blood cholesterol and triglyceride levels were reduced by the oral administration of AP-PG. Further, HFD induced-fatty liver was significantly reduced by the oral administration of AP-PG. The triglyceride accumulation in the liver was also significantly reduced in mice administered AP-PG. Liver injury as indicated by an increase in serum alanine aminotransferase (ALT) in the HFD-fed mice was significantly reduced in the mice administered AP-PG orally, and the gene expression of cholesterol 7 alpha-hydroxylase (CYP7A1) which is known to be involved in cholesterol degradation in the liver was significantly increased in the AP-PG administered mice. These results suggest the possibility that the oral administration of AP-PG is effective to prevent the development of non-alcoholic fatty liver disease (NAFLD). PMID:26179949

  13. Ageing Fxr deficient mice develop increased energy expenditure, improved glucose control and liver damage resembling NASH.

    PubMed

    Bjursell, Mikael; Wedin, Marianne; Admyre, Therése; Hermansson, Majlis; Böttcher, Gerhard; Göransson, Melker; Lindén, Daniel; Bamberg, Krister; Oscarsson, Jan; Bohlooly-Y, Mohammad

    2013-01-01

    Nuclear receptor subfamily 1, group H, member 4 (Nr1h4, FXR) is a bile acid activated nuclear receptor mainly expressed in the liver, intestine, kidney and adrenal glands. Upon activation, the primary function is to suppress cholesterol 7 alpha-hydroxylase (Cyp7a1), the rate-limiting enzyme in the classic or neutral bile acid synthesis pathway. In the present study, a novel Fxr deficient mouse line was created and studied with respect to metabolism and liver function in ageing mice fed chow diet. The Fxr deficient mice were similar to wild type mice in terms of body weight, body composition, energy intake and expenditure as well as behaviours at a young age. However, from 15 weeks of age and onwards, the Fxr deficient mice had almost no body weight increase up to 39 weeks of age mainly because of lower body fat mass. The lower body weight gain was associated with increased energy expenditure that was not compensated by increased food intake. Fasting levels of glucose and insulin were lower and glucose tolerance was improved in old and lean Fxr deficient mice. However, the Fxr deficient mice displayed significantly increased liver weight, steatosis, hepatocyte ballooning degeneration and lobular inflammation together with elevated plasma levels of ALT, bilirubin and bile acids, findings compatible with non-alcoholic steatohepatitis (NASH) and cholestasis. In conclusion, ageing Fxr deficient mice display late onset leanness associated with elevated energy expenditure and improved glucose control but develop severe NASH-like liver pathology.

  14. Effects of different rearing and feeding systems on lipid oxidation and antioxidant capacity of freeze-dried egg yolks.

    PubMed

    Pignoli, Giovanni; Rodriguez-Estrada, Maria Teresa; Mandrioli, Mara; Barbanti, Lorenzo; Rizzi, Laura; Lercker, Giovanni

    2009-12-23

    Lipid oxidation and antioxidant capacity of freeze-dried egg yolks produced with two rearing systems (battery cages and free-range) and two types of feedings (conventional and organic) were studied. Nine fresh egg yolks of each crossed treatment were pooled, frozen for a month, freeze-dried, vacuum-packed, and kept at -18 degrees C until analysis. No significant differences were observed in the lipid (58.0-62.1%) and total sterol contents (33.0-35.5 g/kg of lipids) of the freeze-dried egg yolks. Free rearing and conventional feeding systems resulted in significantly higher total tocopherol, alpha-tocopherol, and lutein contents, as compared to the battery cage and the organic feed, respectively. However, no significant differences were found in lipid oxidation (peroxide value = 0.7-0.9 mequiv of O(2)/kg of fat; thiobarbituric reactive substances = 1.0-1.3 mg of malonylaldehyde/kg of sample) and cholesterol oxidation (28.8-43.5 mg of cholesterol oxidation products/kg of lipids; 0.08-0.12% oxidized cholesterol) of freeze-dried egg yolks except for 7alpha-hydroxycholesterol, which was significantly lower in samples obtained with organic feed.

  15. Male sea lampreys, Petromyzon marinus L., excrete a sex pheromone from gill epithelia.

    PubMed

    Siefkes, Michael J; Scott, Alexander P; Zielinski, Barbara; Yun, Sang-Seon; Li, Weiming

    2003-07-01

    During the period when they are producing sperm, male sea lampreys (Petromyzon marinus L.) release a sex pheromone 7alpha, 12alpha, 24-trihydroxy-5alpha-cholan-3-one-24-sulfate (3 keto-petromyzonol sulfate, 3ketoPZS) that induces search and preference behaviors in ovulating females. In this study, we conducted a series of experiments to demonstrate that release of this pheromone into water takes place exclusively through the gills. In a behavioral maze, water conditioned with the anterior region of spermiating males induced an increase of search and preference behaviors in ovulating females. Similar behavior was not elicited by water conditioned by the posterior region. The anterior region washings and whole-body washings from spermiating males also elicited large and virtually identical electro-olfactogram responses from female sea lampreys, while the posterior washings produced negligible responses. Further, mass spectrometry and immunoassay confirmed that virtually all the 3ketoPZS released into water was through the gills. Immunocytochemistry revealed some gill epithelial cells and hepatocytes from spermiating males contained dense immunoreactive 3ketoPZS, but not those from prespermiating males. These results demonstrate that 3ketoPZS is released through the gill epithelia and suggest that this pheromone or its precursor may be produced in the liver.

  16. Novel P2 promoter-derived HNF4{alpha} isoforms with different N-terminus generated by alternate exon insertion

    SciTech Connect

    Huang, Jianmin; Levitsky, Lynne L.; Rhoads, David B.

    2009-04-15

    Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) is a critical transcription factor for pancreas and liver development and functions in islet {beta} cells to maintain glucose homeostasis. Mutations in the human HNF4A gene lead to maturity onset diabetes of the young (MODY1) and polymorphisms are associated with increased risk for type 2 diabetes mellitus (T2DM). Expression of six HNF4{alpha} variants, three each from two developmentally regulated promoters, has been firmly established. We have now detected a new set of HNF4{alpha} variants designated HNF4{alpha}10-12 expressed from distal promoter P2. These variants, generated by inclusion of previously undetected exon 1E (human = 222 nt, rodent = 136 nt) following exon 1D have an altered N-terminus but identical remaining reading frame. HNF4{alpha}10-{alpha}12 are expressed in pancreatic islets (and liver) and exhibit transactivation potentials similar to the corresponding {alpha}7-{alpha}9 isoforms. DNA-binding analyses implied much higher protein levels of HNF4{alpha}10-{alpha}12 in liver than expected from the RT-PCR data. Our results provide evidence for a more complex expression pattern of HNF4{alpha} than previously appreciated. We recommend inclusion of exon 1E and nearby DNA sequences in screening for HNF4{alpha} mutations and polymorphisms in genetic analyses of MODY1 and T2DM.

  17. Effects of dietary alpha- and gamma-linolenic acid on lipid metabolism in young and adult rats.

    PubMed

    Choi, Y S; Sugano, M

    1988-01-01

    The effect of age on lipid metabolism was studied in rats fed diets containing safflower oil (SFO, 78% linoleic acid), evening primrose oil (EPO, 9.4% gamma-linolenic acid and 70% linoleic acid) or the mixture of safflower and linseed oil (SLO, 10.2% alpha-linolenic acid and 68% linoleic acid). The activity of hepatic HMG-CoA reductase declined with age in all groups. In adult rats, the reductase activity was high in the EPO group and low in the SLO group. The activity of hepatic cholesterol 7 alpha-hydroxylase was independent of the diet or age. Hepatic delta 6-desaturase activity was low in adult rats fed EPO. In liver microsomal phospholipids, the percentage of 22:5 n-6 decreased while that of 22:6 n-3 increased with age. The ratio of linoleate metabolites to linoleate was high in the EPO group and low in the SLO group. Liver and serum cholesterol increased with age only in rats fed the SLO diet. Thus, the results indicated an enhanced susceptibility to dietary fats with age.

  18. Efficacy of various natural and synthetic androgens to induce ductal branching morphogenesis in the developing anterior rat prostate.

    PubMed

    Foster, B A; Cunha, G R

    1999-01-01

    The studies presented herein quantitated ductal branching morphogenesis in the anterior prostate (AP) of the newborn rat. Four parameters were measured: epithelial area, epithelial perimeter, node number, and form factor. Nine natural and synthetic androgens were tested for their effectiveness in inducing postnatal prostatic development using 808 newborn rat APs in 68 dose-response experiments. Based on these studies it was shown that testosterone (T) was slightly more effective than dihydrotestosterone (DHT) in supporting ductal branching morphogenesis in the developing rat AP. Furthermore, the activity of T could not be accounted for simply by conversion of T to DHT. Synthetic androgens, 7alpha-methyl-19-nortestosterone and methyltrienolone (R1881), which cannot be 5alpha-reduced to DHT, also induced extensive ductal branching and elicited responses less than those to T and not statistically different from those to DHT. This suggests that although DHT is sufficient for prostatic development, it is not necessary for postnatal ductal branching morphogenesis and growth of the prostate. 5Alpha-androstan-3alpha,17beta-diol was particularly potent in inducing ductal branching, eliciting a response greater than or comparable to those of T and DHT. Androsterone, androstanedione, 5alpha-androstan-3beta,17beta-diol and 5beta-androstan-3alpha,17beta-diol induced ductal branching, but to a lesser extent than either T or DHT. These studies challenge the assumption that DHT is essential for prostatic development, specifically during ductal branching morphogenesis of the neonatal rat prostate.

  19. Emerging drugs for hypogonadism.

    PubMed

    Edelstein, Daniel; Dobs, Adrian; Basaria, Shehzad

    2006-11-01

    Male hypogonadism is a common endocrine problem that affects men of all ages. Recently, there has been a surge in testosterone use among middle-aged and older men who in the past may have been considered to have borderline or even normal testosterone levels. This increasing use of testosterone therapy among men has paralleled the increasing improvements in the development of treatments for male hypogonadism that have been made over the past few decades. Current therapies using transdermal formulations and long-acting injectables such as testosterone undecanoate are quickly replacing the old injectable testosterone esters. In recent years, pharmaceutical sales and prescription data have readily shown a shift in the testosterone marketplace towards greater use of slightly more expensive treatments such as transdermal therapies, which are easier to administer and yield more physiological levels of testosterone. On the horizon are several new compounds in development, such as selective androgen receptor modulators (SARMS), 7alpha-methyl-19-nortestosterone, aromatase inhibitors, clomifene, dihydrotestosterone and human chorionic gonadotropin. Compounds such as SARMs are designed to selectively target androgen receptors in specific tissues (such as bone and muscles), in the hope of dispersing some of the side effects experienced on the prostate, which are presently associated with therapy of exogenous testosterone.

  20. Androgen responsiveness of the pituitary gonadotrope cell line LbetaT2.

    PubMed

    Lawson, M A; Li, D; Glidewell-Kenney, C A; López, F J

    2001-09-01

    Androgens have a profound effect on the hypothalamic-pituitary axis by reducing the synthesis and release of the pituitary gonadotropin LH. The effect on LH is partly a consequence of a direct, steroid-dependent action on pituitary function. Although androgen action has been well studied in vivo, in vitro cell models of androgen action on pituitary gonadotropes have been scarce. Recently, an LH-expressing cell line, LbetaT2, was generated by tumorigenesis targeted to the LH-producing cells of the mouse pituitary. The purpose of these studies was to determine the presence of androgen receptor (AR) and establish its function in this cell line. RT-PCR analysis indicated that the LbetaT2 cell line expresses AR mRNA. Transient transfection assays, using the mouse mammary tumor virus (MMTV) promoter, showed that a functional AR is also present. Testosterone (TEST), dihydrotestosterone (DHT), 7alpha-methyl-19-nortestosterone (MENT), and fluoxymesterone (FLUOXY) increased reporter gene activity in the rank order of potencies MENT>DHT> TEST>FLUOXY. Additionally, activation of MMTV promoter activity by DHT in LbetaT2 cells was diminished by the AR antagonists casodex and 2-hydroxy-flutamide, indicating that the effects of DHT are mediated through AR. In summary, these studies showed that the LbetaT2 cell line is a useful model for the evaluation and molecular characterization of androgen action in pituitary gonadotropes.

  1. Reversal of testicular function after prolonged suppression with an LHRH agonist in rhesus monkeys.

    PubMed

    Sundaram, K; Keizer-Zucker, A; Thau, R B; Bardin, C W

    1987-01-01

    Using subcutaneously implanted osmotic pumps, four male rhesus monkeys were continuously infused for 18 months with 100 micrograms/day of [(imBzl)-D-His6-Pro9-NEt]-LHRH (LHRH-A), a potent agonist of LHRH. After an initial increase, serum testosterone levels declined to 10% of pretreatment levels in three monkeys and the response to electroejaculation was lost. There was a decrease in testicular volume. Androgen replacement in the form of subcutaneous SILASTIC implants releasing 7 alpha-methyl-19-nor-testosterone acetate led to a restoration of ejaculatory response and the electroejaculates were devoid of spermatozoa. Under this treatment regimen (100 micrograms LHRH-A + 100 micrograms androgen daily), azoospermia was essentially maintained in the three monkeys for about 8 months. Withdrawal of LHRH-A and androgen treatment led to a complete restoration of testicular function. Serum testosterone returned to control levels and spermatozoa reappeared in the ejaculates with sperm counts reaching the normal range. Testicular volumes showed a gradual increase. These results indicate that continuous administration of an LHRH agonist together with an androgen can induce an extended period of azoospermia in rhesus monkeys. These results also show that after prolonged suppression (more than one year) of testicular function complete recovery occurs after cessation of treatment.

  2. Acute relaxation of mouse duodenum [correction of duodenun] by estrogens. Evidence for an estrogen receptor-independent modulation of muscle excitability.

    PubMed

    Díaz, Mario; Ramírez, Cristina M; Marin, Raquel; Marrero-Alonso, Jorge; Gómez, Tomás; Alonso, Rafael

    2004-10-06

    17-beta-Estradiol, the stereoisomer 17-alpha-estradiol and the synthetic estrogen diethylstilbestrol (DES), all caused a rapid (<3 min) dose-dependent reversible relaxation of mouse duodenal spontaneous activity, reduced basal tone and depressed the responses to CaCl(2) and KCl. The steroidal antiestrogen 7alpha-[9-[(4,4,5,5,5,-pentafluoropenty)sulphinyl]nonyl]-estra-1,3,5(19)-triene-3,17beta-diol (ICI182,780) failed to either mimic or prevent the effect of 17-beta-estradiol. The effect of estrogens was unrelated to activation of nitric oxide (NO), mitogen-activated protein kinase (MAPK), protein kinase A (PKA), protein kinase G (PKG) or protein kinase C (PKC). Estrogen-induced relaxation was partially reversed by 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-pyridine-3-carboxilic acid methyl ester (BAY-K8644), depolarization, or by application of tetraethylammonium or 4-aminopyridine, but not by glibenclamide, apamin, charybdotoxin, paxilline or verruculogen. The effects of BAY-K8644 and K(+) channel blockers were synergistic, and allowed relaxed tissues to recover spontaneous activity and basal tone. We hypothesize that the rapid non-genomic spasmolytic effect of estrogens on mouse duodenal muscle might be triggered by an estrogen-receptor-independent mechanism likely involving activation of tetraethylamonium- and 4-aminopyridine-sensitive K(+) channels and inhibition of L-type Ca2(+) channels on the smooth muscle cells.

  3. Enantioselective kappa opioid binding sites on the macrophage cell line, P388d sub 1

    SciTech Connect

    Carr, D.J.J.; Blalock, J.E. ); DeCosta, B.R.; Jacobson, A.E.; Rice, K.C. )

    1991-01-01

    A kappa opioid binding site has been characterized on the macrophage cell line, P388d{sub 1}, using the kappa selective affinity ligand, ({sup 3H}(1S,2S)-(-)-trans-2-isothiocyanato-N-methyl-N-(2-(1-phrrolidinyl) cyclohexyl) benzeneacetamide ((-)BD166). The kappa site has a relative molecular mass (Mr) of 38,000 under nonreducing conditions and 42,000 under reducing conditions. Moreover, it exhibits enantioselectivity in that 1S,2S-(-)-trans-3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl) benzeneacetamide ((-)-U-50,488) blocks ({sup 3}H)95{alpha},7{alpha},8{beta})-(-)-N-methyl-N-(7-(1- pyrrolidinyl)-1-oxaspiro-(4,5)-dec-8-yl)benzeneacetamide (U-69,593) binding to P388d{sub 1} cells with an IC{sub 50} = 7.0 nM whereas 1R,2R-(+)-trans-3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl) benzeneacetamide ((+)U-50,488) blocks ({sup 3}H)U-69,593 binding to P388d{sub 1} cells with an IC{sub 50} = 700 nM.

  4. Bile acids: regulation of synthesis.

    PubMed

    Chiang, John Y L

    2009-10-01

    Bile acids are physiological detergents that generate bile flow and facilitate intestinal absorption and transport of lipids, nutrients, and vitamins. Bile acids also are signaling molecules and inflammatory agents that rapidly activate nuclear receptors and cell signaling pathways that regulate lipid, glucose, and energy metabolism. The enterohepatic circulation of bile acids exerts important physiological functions not only in feedback inhibition of bile acid synthesis but also in control of whole-body lipid homeostasis. In the liver, bile acids activate a nuclear receptor, farnesoid X receptor (FXR), that induces an atypical nuclear receptor small heterodimer partner, which subsequently inhibits nuclear receptors, liver-related homolog-1, and hepatocyte nuclear factor 4alpha and results in inhibiting transcription of the critical regulatory gene in bile acid synthesis, cholesterol 7alpha-hydroxylase (CYP7A1). In the intestine, FXR induces an intestinal hormone, fibroblast growth factor 15 (FGF15; or FGF19 in human), which activates hepatic FGF receptor 4 (FGFR4) signaling to inhibit bile acid synthesis. However, the mechanism by which FXR/FGF19/FGFR4 signaling inhibits CYP7A1 remains unknown. Bile acids are able to induce FGF19 in human hepatocytes, and the FGF19 autocrine pathway may exist in the human livers. Bile acids and bile acid receptors are therapeutic targets for development of drugs for treatment of cholestatic liver diseases, fatty liver diseases, diabetes, obesity, and metabolic syndrome.

  5. [Comparison of composition of aetherolea and alcohol extraction from ChangBai Mountain purple common perilla].

    PubMed

    Sun, Jian-Zhong; Yu, Han-Song; Wang, Yu-Hua; Hu, Yao-Hui

    2008-11-01

    To compare the chemical constituents of Aetherolea and Alcohol Extraction of purple common perilla of ChangBai Mountain. We used water vapor distillation and alcohol extraction method, then analyzed identification by thin-layer chromatography and GC-MS. The main chemical compositions of aetherolea were caryophyllen, caryophyllene oxid, ylangene, 4-(2-Methylcyclohex-1-enyl)-but-2-enal and 3,7-dimethyl-1,6-Octadien-3-ol and the main chemical compositions of alcohol extraction were 10-hendeca-acetylene-1-alcohol, n-hexsdecanoic acid (E)-7,11-dimethyl-3-methylene- 1,6,10-Dodecatriene and so on. Other specific compositions including tau. -Muurolol,3H-3a,7-Methanoazulene,2 ,4,5,6,7,8-hexahydro-1,4,9,9-Tetram-ethyl-, [3aR-(3a. alpha, 4. beta 7. alpha)], Astaxanthin and Curan-17-oicacid, 19-acetyl-2,16-didehydro-20-hydroxy-, methyl ester, (19S) have special functions of sterilizing, cancer preventing and immunity improving, which are significantly different from the compositions extracted from other species.

  6. Identification of multiple steroid hydroxylases in Daphnia magna and their modulation by xenobiotics

    SciTech Connect

    Baldwin, W.S.; LeBlanc, G.A. . Dept. of Toxicology)

    1994-07-01

    Steroid hydroxylase activities were characterized in Daphnia magna and evaluated for potential use as biomarkers of xenobiotic exposure. Microsomes prepared from Daphnia magna generated as single NADPH-dependent metabolite of [[sup 14]C] testosterone. However, intact daphnids excreted at least 10 polar metabolites of [[sup 14]C] testosterone into the test medium. Six of these metabolites were identified as 2[alpha]-, 16[beta]-, 6[beta]-, 6[alpha]-, 7[alpha]-, and 15[alpha]-[[sup 14]C]hydroxytestosterone. The unidentified metabolites are also presumed to be hydroxylated products of testosterone, based on their relative migrations during TLC. The inefficient metabolism of [[sup 14]C] testosterone during the in vitro microsomal incubations may have been due to the release of P450 inhibitors during microsome preparation. Exposure of daphnids to the P450 modulators phenobarbital, [beta]-naphthoflavone, piperonyl butoxide, and malathion differentially inhibited the steroid hydroxylase activities. Results from this study indicate that Daphnia magna expresses several P450 enzymes and that these enzymes are differentially modulated by xenobiotic exposure. Steroid hydroxylase activities may serve not only as a biomarker of toxicant exposure, but also as a predictor of toxicant effects involving perturbations of steroid hormone homeostasis.

  7. CYP superfamily perturbation by diflubenzuron or acephate in different tissues of CD1 mice.

    PubMed

    Sapone, A; Pozzetti, L; Canistro, D; Broccoli, M; Bronzetti, G; Potenza, G; Affatato, A; Biagi, G L; Cantelli-Forti, G; Paolini, M

    2005-01-01

    This work aimed to investigate whether the insecticide acephate (125 or 250 mg/kg b.w.) or diflubenzuron (752 or 1075 mg/kg b.w.), two of the most widely used pesticides worldwide, impairs CYP-linked murine metabolism in liver, kidney and lung microsomes after repeated (daily, for three consecutive days) i.p. administration. The regio- and stereo-selective hydroxylation of testosterone was used as multibiomarker of different CYP isoforms. Both gender and tissue specific effects were observed. Lung was the most responsive tissue to induction by lower diflubenzuron dose, as exemplified by the marked increase of testosterone 7alpha-hydroxylation (CYP2A) (up to 13-fold) in males. Higher dose produced a generalized inactivation. At the lower dose acephate induced 6beta- (CYP3A1/2, liver) as well as 2beta- (CYP2B1/2, kidney) hydroxylase activities ( approximately 5 and approximately 4-fold increase, respectively) in males. In females, a marked suppression of the various hydroxylations was observed. At 250 mg/kg of acephate, animals did not survive. Induction of the most affected isoforms was sustained by immunoblotting analysis. Corresponding human CYP modulations might disrupt normal physiological functions related to these enzymes. Furthermore, the co-mutagenic and promoting potential of these pesticides, phenomena linked to CYP upregulation (e.g. increased bioactivation of ubiquitous pollutants and generation of oxygen free radicals) are of concern for a more complete definition of their overall toxicological potential.

  8. Phospholipase C-delta1 and oxytocin receptor signalling: evidence of its role as an effector.

    PubMed

    Park, E S; Won, J H; Han, K J; Suh, P G; Ryu, S H; Lee, H S; Yun, H Y; Kwon, N S; Baek, K J

    1998-04-01

    Although the oxytocin receptor modulates intracellular Ca2+ ion levels in myometrium, the identities of signal molecules have not been clearly clarified. Our previous studies on oxytocin receptor signalling demonstrated that 80 kDa Ghalpha is a signal mediator [Baek, Kwon, Lee, Kim, Muralidhar and Im (1996) Biochem. J. 315, 739-744]. To elucidate the effector in the oxytocin receptor signalling pathway, we evaluated the oxytocin-mediated activation of phospholipase C (PLC) by using solubilized membranes from human myometrium and a three-component preparation containing the oxytocin receptor-Ghalpha-PLC-delta1 complex. PLC-delta1 activity in the three-component preparation, as well as PLC activity in solubilized membranes, was increased by oxytocin in the presence of Ca2+ and activated Ghalpha (GTP-bound Ghalpha). Furthermore the stimulated PLC-delta1 activity resulting from activation of Ghalpha via the oxytocin receptor was significantly attenuated by the selective oxytocin antagonist desGly-NH2d(CH2)5[Tyr(Me)2,Thr4]ornithine vasotocin or GDP. Consistent with these observations, co-immunoprecipitation and co-immunoadsorption of PLC-delta1 in the three-component preparation by anti-Gh7alpha antibody resulted in the PLC-delta1 being tightly coupled to activated Ghalpha on stimulation of the oxytocin receptor. These results indicate that PLC-delta1 is the effector for Ghalpha-mediated oxytocin receptor signalling.

  9. Consumption of barley beta-glucan ameliorates fatty liver and insulin resistance in mice fed a high-fat diet.

    PubMed

    Choi, Joo Sun; Kim, Hyunsook; Jung, Myeong Ho; Hong, Shinhee; Song, Jihyun

    2010-07-01

    Consumption of a diet high in barley beta-glucan (BG) has been shown to prevent insulin resistance. To investigate the mechanism for the effects of barley BG, three groups of male 7-wk-old C57BL/6J mice were fed high-fat diets containing 0, 2, or 4% of barley BG for 12 wk. The 2% BG and 4% BG groups had significantly lower body weights compared with the 0% BG group. The 4% BG group demonstrated improved glucose tolerance and lower levels of insulin-resistance index and glucose-dependent insulinotropic polypeptide. Consumption of the BG diet decreased hepatic lipid content. Mice on the BG diet also demonstrated decreased fatty acid synthase and increased cholesterol 7alpha-hydroxylase gene expression levels. The BG diet promoted hepatic insulin signaling by decreasing serine phosphorylation of insulin receptor substrate 1 and activating Akt, and it decreased mRNA levels of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. In summary, consumption of BG reduced weight gain, decreased hepatic lipid accumulation, and improved insulin sensitivity in mice fed a high-fat diet. Insulin signaling enhanced due to the expression changes of glucose and lipid metabolism genes by BG consumption. Consumption of barley BG could be an effective strategy for preventing obesity, insulin resistance, and the metabolic syndrome.

  10. The phenylpropanoids of Aster flaccidus.

    PubMed

    Liu, Zhen-ling; Liu, Ying-qian; Zhao, Lei; Xu, Jing; Tian, Xuan

    2010-03-01

    Aster flaccidus bge has been used as traditional medicine in northwestern China. Two new phenylpropanoids (1-2) including one lignan: (7'R, 8S)-9'-lariciresinol-(alpha-methyl)-butanoate (1), 5,9-dimethoxyl-7-(alpha-methyl)-butanoxyl-phenyl-2E-propenol-(alpha-methyl)-butanoate (2) isolated from the chloroform extract of the root of Aster flaccidus bge were identified by means of extensive spectroscopic studies: 1D and 2D NMR spectra as well as HRMS analysis. They have not obvious anti-HIV-1 therapeutic activity (TI=1.0-1.1) compared with AZT (TI=55,556) as the result of the determination of their in vitro anti-HIV-1 activity while compound 2 displays strong antitumor activity against BEL 7402 (human liver carcinoma) with cisplatin as a positive control and the effect increases with the measuring-time going on (24 h, IC(50): 106.67+/-8.47 microM - 72 h, IC(50): 50.51+/-6.11 microM). 2009 Elsevier B.V. All rights reserved.

  11. IL-1 regulates the Cyp7a1 gene and serum total cholesterol level at steady state in mice.

    PubMed

    Kojima, Misaki; Ashino, Takashi; Yoshida, Takemi; Iwakura, Yoichiro; Sekimoto, Masashi; Degawa, Masakuni

    2009-02-06

    We examined the role of hepatic interleukin (IL)-1alpha/beta in serum total cholesterol homeostasis using male and female IL-1-knockout (KO) mice and wild-type (WT) mice. Serum total cholesterol level was higher in males than in females in WT and KO mice. The difference between sexes was closely correlated with the difference in gene expression level of cholesterol 7alpha-hydroxylase (Cyp7a1), a rate-limiting enzyme for bile acid synthesis. No significant sex difference in gene expression level of 3-hydroxy-3-methylglutaryl-CoA reductase, a rate-limiting enzyme for cholesterol synthesis, was observed in WT mice. Interestingly, the gene expression level of hepatic Cyp7a1 was lower in KO mice than in sex-matched WT mice, while the serum total cholesterol level was the opposite. The present findings demonstrate that IL-1alpha and IL-1beta are positive regulators for the Cyp7a1 gene in steady-state mice and that Cyp7a1 is one of the factors that mediate the difference in serum total cholesterol level between sexes.

  12. Hypercholesterolemia and changes in lipid and bile acid metabolism in male and female cyp7A1-deficient mice.

    PubMed

    Erickson, Sandra K; Lear, Steven R; Deane, Sean; Dubrac, Sandrine; Huling, Sandra L; Nguyen, Lien; Bollineni, Jaya S; Shefer, Sarah; Hyogo, Hideyuki; Cohen, David E; Shneider, Benjamin; Sehayek, Ephraim; Ananthanarayanan, Meena; Balasubramaniyan, Natarajan; Suchy, Fredrick J; Batta, Ashok K; Salen, Gerald

    2003-05-01

    Cholesterol 7alpha-hydroxylase, a rate-limiting enzyme for bile acid synthesis, has been implicated in genetic susceptibility to atherosclerosis. The gene, CYP7A1, encoding a protein with this activity, is expressed normally only in hepatocytes and is highly regulated. Our cyp7A1 gene knockout mouse colony, as young adults on a chow diet, is hypercholesterolemic. These mice were characterized extensively to understand how cyp7A1 affects lipid and bile acid homeostasis in different tissue compartments and whether gender plays a modifying role. Both male and female cyp7A1-deficient mice had decreased hepatic LDL receptors, unchanged hepatic cholesterol synthesis, increased intestinal cholesterol synthesis and bile acid transporters, and decreased fecal bile acids but increased fecal sterols. In females, cyp7A1 deficiency also caused changes in hepatic fatty acid metabolism, decreased hepatic canalicular bile acid transporter, Bsep, and gallbladder bile composition altered to a lithogenic profile. Taken together, the data suggest that cyp7A1 deficiency results in a proatherogenic phenotype in both genders and leads to a prolithogenic phenotype in females.

  13. The stimulatory effect of LXRalpha is blocked by SHP despite the presence of a LXRalpha binding site in the rabbit CYP7A1 promoter.

    PubMed

    Shang, Quan; Pan, Luxing; Saumoy, Monica; Chiang, John Y L; Tint, G Stephen; Salen, Gerald; Xu, Guorong

    2006-05-01

    The transcription of the cholesterol 7alpha-hydroxylase gene (CYP7A1) is greatly decreased in cholesterol-fed rabbits. To determine whether the molecular structure of the promoter is responsible for this downregulation, we cloned the rabbit CYP7A1 promoter, identified the binding sites for alpha-fetoprotein transcription factor (FTF) and liver X receptor (LXRalpha), and studied the effects of FTF, LXRalpha, and SHP on its transcription. Adding LXRalpha/retinoid X receptor together with their ligands (L/R) to the promoter/reporter construct transfected into HepG2 cells greatly increased its activity. FTF did not increase promoter activity, nor did it enhance the stimulatory effect of L/R. Mutating the FTF binding site abolished the promoter baseline activity. Increasing amounts of SHP abolished the effect of L/R, and FTF enhanced the ability of SHP to decrease promoter activity below baseline levels. Thus, downregulation of CYP7A1 in cholesterol-fed rabbits is attributable secondarily to the activation of farnesoid X receptor, which increases SHP expression to override the positive effects of LXRalpha. Although FTF is a competent factor for maintaining baseline activity, it does not further enhance and may suppress CYP7A1 transcription.

  14. Retinoic acid represses CYP7A1 expression in human hepatocytes and HepG2 cells by FXR/RXR-dependent and independent mechanisms.

    PubMed

    Cai, Shi-Ying; He, Hongwei; Nguyen, Trong; Mennone, Albert; Boyer, James L

    2010-08-01

    Cholesterol 7alpha-hydroxylase (CYP7A1) plays a key role in maintaining lipid and bile salt homeostasis as it is the rate-limiting enzyme converting cholesterol to bile acids. Deficiency of CYP7A1 leads to hyperlipidemia in man and mouse. Hyperlipidemia is often seen in patients when treated with high-dose retinoic acid (RA), but the molecular mechanisms remain elusive. Our present study revealed that CYP7A1 mRNA expression is greatly repressed by RA in both human hepatocytes and HepG2 cells where increased fibroblast growth factor 19 (FGF19) and small heterodimer partner (SHP) expressions were also observed, suggesting farnesoid X receptor (FXR) and retinoid X receptor (RXR) were activated. Promoter reporter assays demonstrate that all-trans RA (atRA) specifically activated FXR/RXR. However, detailed molecular analyses indicate that this activation is through RXR, whose ligand is 9-cis RA. Knocking down of FXR or RXRalpha by small interference RNA (siRNA) in human hepatocytes increased CYP7A1 basal expression, but the repressive effect of atRA persisted, suggesting there are also FXR/RXR-independent mechanisms mediating atRA repression of CYP7A1 expression. Chromatin immunoprecipitation (ChIP) assay and cell transfection results indicate that PGC-1alpha plays a role in the FXR/RXR-independent mechanism. Our findings may provide a potential explanation for hyperlipidemic side effects observed in some patients treated with high-dose RA.

  15. Determination of 7α-OH cholesterol by LC-MS/MS: Application in assessing the activity of CYP7A1 in cholestatic minipigs.

    PubMed

    Yun, Changhong; Yin, Taijun; Shatzer, Katherine; Burrin, Douglas G; Cui, Liwei; Tu, Yifan; Hu, Ming

    2016-07-01

    An LC-MS/MS method was developed and validated to determine 7α-OH cholesterol in liver microsome. This method was convenient and fast with high specificity and sensitivity. Briefly, a gradient elution was performed on a Synergi polar-C18 column (50×4.6mm i.d., 3μm). The mobile phase (consisting of 0.1% HCOOH solution and acetonitrile) eluted in gradient at a flow rate of 1ml/min. MS detection was operated on APCI (+) mode; the MRM transitions for 7α-OH cholesterol and D7-cholesterol (I.S.) were 385.1≥159.1 and 376.4≥266.3, respectively. The linear response range of 7α-OH cholesterol was covered from 1.563 to 100.0ng/ml. All of the validation items meet the requirement of FDA guidance for bioanalytical method validation. This method was applied to enzymatic studies for determination of cholesterol 7alpha-hydroxylation activity catalyzed by CYP7A1 in the cholestatic minipigs liver microsomes.

  16. Chitosan oligosaccharides promote reverse cholesterol transport and expression of scavenger receptor BI and CYP7A1 in mice.

    PubMed

    Zong, Chuanlong; Yu, Yang; Song, Guohua; Luo, Tian; Li, Luqin; Wang, Xinnong; Qin, Shucun

    2012-02-01

    Chitosan oligosaccharides (COS) are beneficial in improving plasma lipids and diminishing atherosclerotic risks. In this study, we examined the effects of COS on reverse cholesterol transport (RCT) in C57BL/6 mice. (3)H-cholesterol-laden macrophages were injected intraperitoneally into mice fed with various dosage of COS (250, 500, 1000 mg/kg mouse weight, respectively) or vehicle by gastric gavages. Plasma lipid level was determined and (3)H-cholesterol was traced in plasma, liver, bile and feces. The effects of COS on hepatic cholesterol 7 alpha-hydroxylase (CYP7A1) and scavenger receptor BI (SR-BI) expression were also investigated. COS administration led to a significant decrease in plasma total cholesterol and low-density lipoprotein (LDL) cholesterol and a significant increase in peritoneal macrophage-derived (3)H-cholesterol in liver and bile as well as in feces. Liver protein expressions of CYP7A1, SR-BI and LDL receptor (LDL-R) were improved in a dosage-dependent manner in COS-administered mice. Our findings provide the first in vivo demonstration of a positive role for COS in RCT pathway and hepatic CYP7A1 and SR-BI expression in mice. Additionally, the LDL cholesterol lowering effect might be relative to hepatic LDL-R expression stimulated by COS in mice.

  17. Dietary chitosan enhances hepatic CYP7A1 activity and reduces plasma and liver cholesterol concentrations in diet-induced hypercholesterolemia in rats.

    PubMed

    Moon, Min-Sun; Lee, Mak-Soon; Kim, Chong-Tai; Kim, Yangha

    2007-01-01

    The present study was performed to elucidate the hypocholesterolemic action of chitosan on the diet-induced hypercholesterolemia in rats. Male Sprague-Dawley rats (n=24) were fed with chitosan-free diet (Control), diets containing 2% or 5% chitosan for 4 weeks. Hypercholesterolemia was induced by adding 1% cholesterol and 0.5% cholic acid to all diets. Body weight gain and food intake of rats did not differ among the groups. The chitosan treated groups showed significant improvement in the plasma concentration of total cholesterol and LDL-cholesterol compared to the control group (p<0.05). Also, the chitosan treated groups decreased the liver concentration of total lipid and total cholesterol compared to the control group (p<0.05). The activity of hepatic cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in the conversion of cholesterol to bile acids, was increased by 123% and 165% for the 2% or 5% chitosan diets, respectively. These findings suggest that enhancement of hepatic CYP7A1 activity may be a mechanism, which can partially account for the hypocholesterolemic effect of dietary chitosan in cholesterol metabolism.

  18. Phase Diagram and Decomposition of 1,1-Diamino-2,2-Dinitroethene (FOX-7)

    NASA Astrophysics Data System (ADS)

    Tao, Yuchuan; Dreger, Zbigniew; Gupta, Yogendra

    2015-06-01

    To understand the reactive behavior of 1,1-diamino-2,2-dinitroethene (FOX-7) at the thermo-mechanical conditions relevant to shock-wave initiation, Raman and FTIR measurements were performed at high-pressures (HP) and high-temperatures (HT). Experiments were performed on single crystals of FOX-7 in a diamond anvil cell to 10 GPa and 800 K to provide the phase diagram and to gain insight into the HP decomposition mechanisms. Previous studies have demonstrated that the ambient structure of FOX-7 (alpha) transforms to beta and gamma phases at higher temperatures, and phase I (2 GPa) and II (4.5 GPa) at higher pressures. In this work, we determined the boundaries between these phases and the decomposition/melting curve. In particular, we found that: (i) both beta and gamma phases exist in a limited P-T domain (>386 K and <1 GPa), (ii) the transition between phase-I and phase-II takes place along the isobar, (iii) the decomposition temperature increases significantly with pressure (~ 25 K / GPa), and (iv) pressure inhibits the decomposition. Using FTIR spectroscopy, we observed that CO2 is the first dominating decomposition product, followed by N2O, NO2, HCN, and HNCO. Pressure effects on reaction kinetics will be presented along with the possible mechanisms of decomposition. Work supported by DOE/NNSA and ONR.

  19. Spironolactone pharmacokinetics and actions in the dog: Influence on the potential spironolactone-digoxin interaction

    SciTech Connect

    Beck, B.L.

    1988-01-01

    This study was designed to determine if administration of spironolactone to achieve steady-state concentrations to dogs at steady-state digoxin concentration resulted in an increased serum digoxin concentration in dogs. Serum digoxin concentration and AcS tolerance were measured in dogs at steady-state spironolactone-digoxin concentration to determine if an increase in serum digoxin concentration resulted from spironolactone administration and if this resulted in increased cardiac effects of digoxin. To determine if a spironolactone-digoxin interaction occurred at the canine myocardial digitalis receptor in vitro, the effects of spironolactone and its metabolites on Na{sup +},K{sup +}-ATPase activity and ({sup 3}H)ouabain binding were studied. Spironolactone, canrenone, or K{sup +} canrenoate did not inhibit canine myocardial K{sup +}-dependent MFPase activity or the concentration-response curve for digoxin inhibition of K{sup +}-dependent MFPase. In contrast, 0.1 mM spironolactone or canrenone competitively antagonized ({sup 3}H)ouabain binding to canine myocardium. Neither 0.24 {mu}M spironolactone, 0.59 {mu}M canrenone, or 1.27 {mu}M 7{alpha}-thiomethylspirolactone, concentrations of spironolactone and its metabolites found after a therapeutic dose of spironolactone in man, altered ({sup 3}H)ouabain binding to canine myocardium.

  20. Enhanced tumour specificity of an anti-carcinoembrionic antigen Fab' fragment by poly(ethylene glycol) (PEG) modification.

    PubMed

    Delgado, C; Pedley, R B; Herraez, A; Boden, R; Boden, J A; Keep, P A; Chester, K A; Fisher, D; Begent, R H; Francis, G E

    1996-01-01

    Polyethylene glycol (PEG) modification of a chimeric Fab' fragment (F9) of A5B7 (alpha-CEA), using an improved coupling method, increases its specificity for subcutaneous LS174T tumours. PEGylation increased the area under the concentration-time curve (AUC0-144) in all tissues but there were significant differences (variance ratio test, F = 27.95, P < 0.001) between the proportional increases in AUC0-144, with the tumour showing the greatest increase. The increase in AUCtumour from F9 to PEG-F9 was similar to the reported increase from Fab' to F(ab')2 while the increase in AUCblood by PEGylation of F9 was only 21% of the reported increase from Fab' to whole IgG. A two sample t-test showed no significant differences between maximal tumour/tissue ratios for PEG-F9 and F9 while the tumour/tissue ratios for PEG-F9 remained high over a longer period, with tumour levels at least double those for F9. PEG-F9 emerges as a new generation antibody with potential advantages for both radioimmunotherapy and tumour imaging. Since there was a reduction in antigen binding, optimisation of PEGylation might further improve tumour specificity. The latter resulted from complex effects on both the entry into and exit rates from tumour and normal tissues in a tissue-specific fashion.

  1. Enhanced tumour specificity of an anti-carcinoembrionic antigen Fab' fragment by poly(ethylene glycol) (PEG) modification.

    PubMed Central

    Delgado, C.; Pedley, R. B.; Herraez, A.; Boden, R.; Boden, J. A.; Keep, P. A.; Chester, K. A.; Fisher, D.; Begent, R. H.; Francis, G. E.

    1996-01-01

    Polyethylene glycol (PEG) modification of a chimeric Fab' fragment (F9) of A5B7 (alpha-CEA), using an improved coupling method, increases its specificity for subcutaneous LS174T tumours. PEGylation increased the area under the concentration-time curve (AUC0-144) in all tissues but there were significant differences (variance ratio test, F = 27.95, P < 0.001) between the proportional increases in AUC0-144, with the tumour showing the greatest increase. The increase in AUCtumour from F9 to PEG-F9 was similar to the reported increase from Fab' to F(ab')2 while the increase in AUCblood by PEGylation of F9 was only 21% of the reported increase from Fab' to whole IgG. A two sample t-test showed no significant differences between maximal tumour/tissue ratios for PEG-F9 and F9 while the tumour/tissue ratios for PEG-F9 remained high over a longer period, with tumour levels at least double those for F9. PEG-F9 emerges as a new generation antibody with potential advantages for both radioimmunotherapy and tumour imaging. Since there was a reduction in antigen binding, optimisation of PEGylation might further improve tumour specificity. The latter resulted from complex effects on both the entry into and exit rates from tumour and normal tissues in a tissue-specific fashion. PMID:8546903

  2. Multicentre study of effects of Org OD 14 on endometrium, vaginal cytology and cervical mucus in post-menopausal and oophorectomized women.

    PubMed

    Punnonen, R; Liukko, P; Cortes-Prieto, J; Eydam, F; Milojevic, S; Trévoux, R; Chryssikopoulos, E; Franchi, F; Luisi, M; Kicovic, P M

    1984-04-01

    A multicentre study covering 69 post-menopausal or oophorectomized women was performed to determine whether Org OD 14 [7 alpha, 17 alpha)-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one) administered orally in a daily dose of 2.5 mg for 90 consecutive days induces endometrial proliferation. The treatment with Org OD 14 was continued in combination with 1 mg/day of lynestrenol from day 91 for 10 days to ascertain whether secretory transformation of the endometrium and subsequent withdrawal bleeding would occur. Endometrial biopsies were obtained before treatment and on day 91. The effects of Org OD 14 on vaginal mucosa and cervical mucus were also evaluated. Org OD 14 did not display any effect on the endometrium in 56 of the study subjects (83.5%). Weak stimulation (initial proliferation) was seen in 11 of the subjects (16.4%) and withdrawal bleeding occurred in only 5 of these after cessation of the combined treatment with lynestrenol. However, moderate 'oestrogenic' effects on vaginal mucosa and cervical mucus were induced in all study subjects.

  3. A combination of transcriptomics and metabolomics uncovers enhanced bile acid biosynthesis in HepG2 cells expressing CCAAT/enhancer-binding protein β (C/EBPβ), hepatocyte nuclear factor 4α (HNF4α), and constitutive androstane receptor (CAR).

    PubMed

    Blazquez, Marina; Carretero, Aitor; Ellis, James K; Athersuch, Toby J; Cavill, Rachel; Ebbels, Timothy M D; Keun, Hector C; Castell, José V; Lahoz, Agustín; Bort, Roque

    2013-06-07

    The development of hepatoma-based in vitro models to study hepatocyte physiology is an invaluable tool for both industry and academia. Here, we develop an in vitro model based on the HepG2 cell line that produces chenodeoxycholic acid, the main bile acid in humans, in amounts comparable to human hepatocytes. A combination of adenoviral transfections for CCAAT/enhancer-binding protein β (C/EBPβ), hepatocyte nuclear factor 4α (HNF4α), and constitutive androstane receptor (CAR) decreased intracellular glutamate, succinate, leucine, and valine levels in HepG2 cells, suggestive of a switch to catabolism to increase lipogenic acetyl CoA and increased anaplerosis to replenish the tricarboxylic acid cycle. Transcripts of key genes involved in bile acid synthesis were significantly induced by approximately 160-fold. Consistently, chenodeoxycholic acid production rate was increased by more than 20-fold. Comparison between mRNA and bile acid levels suggest that 12-alpha hydroxylation of 7-alpha-hydroxy-4-cholesten-3-one is the limiting step in cholic acid synthesis in HepG2 cells. These data reveal that introduction of three hepatocyte-related transcription factors enhance anabolic reactions in HepG2 cells and provide a suitable model to study bile acid biosynthesis under pathophysiological conditions.

  4. Transformation of steroids by Bacillus strains isolated from the foregut of water beetles (Coleoptera: Dytiscidae): II. Metabolism of 3 beta-hydroxypregn-5-en-20-one (pregnenolone).

    PubMed

    Schaaf, O; Dettner, K

    2000-12-15

    The in vitro metabolism of pregnenolone by two Bacillus strains (HA-V6-3 and HA-V6-11) isolated from the foregut of the water beetle Agabus affinis (Payk.) was examined in the course of our studies about a possible participation of gut micro-organisms in the biosynthesis of prothoracic defensive steroids of dytiscids. The transformation products were identified by EI GC--MS of culture extracts after derivatization. The dominating reactions were hydroxylations, with 7 alpha-hydroxypregnenolone as the major product. With considerably lower yields, 7 beta- and 15xi-hydroxypregenolone were formed by both strains, while 11, 17 and 16 alpha-hydroxypregnenolone were produced only by HA-V6-3. The occurrence of 7, 11 alpha- and 7 beta, 11 alpha-dihydroxypregnenolone as well as several minor products containing a 17 alpha-OH group proved the capability of HA-V6-11 to hydroxylate pregenenolone at C(11) and C(17) as well. The monohydroxylated 7-OH-pregnenolones were partly oxidized to 7-oxopregnenolone by both strains. In trace amounts, HA-V6-3 performed 3 beta-acetylation of pregnenolone.

  5. Transformation of steroids by Bacillus strains isolated from the foregut of water beetles (Coleoptera:Dytiscidae): I. Metabolism of androst-4-en-3,17-dione (AD).

    PubMed

    Schaaf, O; Dettner, K

    1998-12-01

    Two Bacillus strains were isolated from the foregut of the water beetle Agabus affinis (Payk.) and tested for their steroid transforming ability. After incubation with androst-4-en-3,17-dione (AD), 13 different transformation products were detected. AD was hydroxylated at C6, C7, C11 and C14, resulting in formation of 6beta-, 7alpha-, 11alpha- and 14alpha-hydroxy-AD. One strain also produced small amounts of 6beta,14alpha-dihydroxy-AD. Partly, the 6beta-hydroxy group was further oxidized to the corresponding 6-oxo steroids. In addition, a specific reduction of the delta4-double bond was observed, leading to the formation of 5alpha-androstane derivatives. In minor yields the carbonyl functions at C3 and C17 were reduced leading to the formation of 3zeta-OH or 17beta-OH steroids. EI mass spectra of the trimethylsilyl and O-methyloxime trimethylsilyl ether derivatives of some transformation products are presented for the first time.

  6. Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells.

    PubMed

    Proskocil, Becky J; Sekhon, Harmanjatinder S; Jia, Yibing; Savchenko, Valentina; Blakely, Randy D; Lindstrom, Jon; Spindel, Eliot R

    2004-05-01

    The role of acetylcholine (ACh) as a key neurotransmitter in the central and peripheral nervous system is well established. However, the role of ACh may be broader because ACh may also function as an autocrine or paracrine signaling molecule in a variety of nonneuronal tissues. To begin to establish ACh of nonneuronal origin as a paracrine hormone in lung, we have examined neonatal and adult monkey bronchial epithelium for the components involved in nicotinic cholinergic signaling. Using immunohistochemistry and RT-PCR, we have demonstrated in lung bronchial epithelial cells (BECs) expression of choline acetyltransferase, the vesicular ACh transporter, the choline high-affinity transporter, alpha7, alpha4, and beta2 nicotinic ACh receptor (nAChR) subunits, and the nAChR accessory protein lynx1. Confocal microscopy demonstrates that these factors are expressed in epithelial cells and are clearly distinct from neighboring nerve fibers. Confirmation of RNA identity has been confirmed by partial sequence analysis of PCR products and by cDNA cloning. Primary culture of BECs confirms the synthesis and secretion of ACh and the activity of cholinesterases. Thus, ACh meets all the criteria for an autocrine/paracrine hormone in lung bronchial epithelium. The nonneuronal cholinergic signaling pathway in lung provides a potentially important target for cholinergic drugs. This pathway may also explain some of the effects of nicotine on fetal development and also provides additional mechanisms by which smoking affects lung cancer growth and development.

  7. Increased serum levels of C21 steroids in female patients with multiple sclerosis.

    PubMed

    Kanceva, R; Stárka, L; Kancheva, L; Hill, M; Veliková, M; Havrdová, E

    2015-01-01

    Multiple sclerosis (MS) is one of the most common neurological diseases. This neurodegenerative autoimmune disease manifests as inflammatory and demyelinating impairment of the central nervous system (CNS). Although some studies demonstrated associations between altered steroidogenesis and pathophysiology of MS as well as the importance of steroids in the pathophysiology of MS, the knowledge concerning the steroid metabolome in female patients is limited. Hence, 51 steroids and steroid polar conjugates were measured in the serum of 12 women with MS, untreated with steroids and 6 age-corresponding female controls with the use of gas chromatography - mass spectrometry (GC-MS). The data were processed using age adjusted ANCOVA, receiver operating characteristics (ROC) analysis and orthogonal projections to latent structures (OPLS). Our data show higher levels of circulating C21 steroids including steroid modulators of ionotropic type A gamma-aminobutyric acid (GABA A) receptors and glutamate receptors. Furthermore, the levels of GABAergic androsterone and 5-androsten-3beta,7alpha,17beta-triol were also higher in the female MS patients. In conclusion, the data demonstrate higher levels of circulating C21 steroids and their polar conjugates and some bioactive C19 steroids in women with MS, which may influence neuronal activity and affect the balance between neuroprotection and excitotoxicity.

  8. Mapping and structural dissection of human 20 S proteasome using proteomic approaches.

    PubMed

    Claverol, Stephane; Burlet-Schiltz, Odile; Girbal-Neuhauser, Elisabeth; Gairin, Jean Edouard; Monsarrat, Bernard

    2002-08-01

    The proteasome, a proteolytic complex present in all eukaryotic cells, is part of the ATP-dependent ubiquitin/proteasome pathway. It plays a critical role in the regulation of many physiological processes. The 20 S proteasome, the catalytic core of the 26 S proteasome, is made of four stacked rings of seven subunits each (alpha7beta7beta7alpha7). Here we studied the human 20 S proteasome using proteomics. This led to the establishment of a fine subunit reference map and to the identification of post-translational modifications. We found that the human 20 S proteasome, purified from erythrocytes, exhibited a high degree of structural heterogeneity, characterized by the presence of multiple isoforms for most of the alpha and beta subunits, including the catalytic ones, resulting in a total of at least 32 visible spots after Coomassie Blue staining. The different isoforms of a given subunit displayed shifted pI values, suggesting that they likely resulted from post-translational modifications. We then took advantage of the efficiency of complementary mass spectrometric approaches to investigate further these protein modifications at the structural level. In particular, we focused our efforts on the alpha7 subunit and characterized its N-acetylation and its phosphorylation site localized on Ser(250).

  9. Influence of dietary sugar on cholesterol and bile acid metabolism in the rat: Marked reduction of hepatic Abcg5/8 expression following sucrose ingestion.

    PubMed

    Apro, Johanna; Beckman, Lena; Angelin, Bo; Rudling, Mats

    2015-06-12

    Previous studies have indicated that dietary intake of sugar may lower bile acid production, and may promote cholesterol gallstone formation in humans. We studied the influence of dietary sucrose on cholesterol and bile acid metabolism in the rat. In two different experiments, rats received high-sucrose diets. In the first, 60% of the weight of standard rat chow was replaced with sucrose (high-sucrose diet). In the second, rats received a diet either containing 65% sucrose (controlled high-sucrose diet) or 65% complex carbohydrates, in order to keep other dietary components constant. Bile acid synthesis, evaluated by measurements of the serum marker 7-alpha-hydroxy-4-cholesten-3-one (C4) and of the hepatic mRNA expression of Cyp7a1, was markedly reduced by the high-sucrose diet, but not by the controlled high-sucrose diet. Both diets strongly reduced the hepatic - but not the intestinal - mRNA levels of Abcg5 and Abcg8. The differential patterns of regulation of bile acid synthesis induced by the two sucrose-enriched diets indicate that it is not sugar per se in the high-sucrose diet that reduces bile acid synthesis, but rather the reduced content of fiber or fat. In contrast, the marked reduction of hepatic Abcg5/8 observed is an effect of the high sugar content of the diets. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Modulation of lipid metabolism by deep-sea water in cultured human liver (HepG2) cells.

    PubMed

    He, Shan; Hao, Jiejie; Peng, Weibing; Qiu, Peiju; Li, Chunxia; Guan, Huashi

    2014-04-01

    It has been found that deep-sea water was associated with lower serum lipid in animal model studies. Herein, we investigated whether DSW exerted a hypolipidemic activity and further elucidated how DSW modulated lipid metabolism in HepG2 cells. Preliminary animal studies showed that DSW exhibited potency to decrease serum total cholesterol, triglycerides, and LDL cholesterol, and increase HDL cholesterol, and the hepatic lipid contents were also significantly lower in the DSW group. When DSW was added to HepG2 cells, it decreased the lipid contents of hepatocyte through the activation of AMP-activated protein kinase, thus inhibiting the synthesis of cholesterol and fatty acid. Besides, LDL receptor was upregulated by activation of sterol regulatory element-binding protein-2. In addition, the levels of apolipoprotein AI and cholesterol 7-alpha-hydroxylase were also raised. Our investigation provided mechanisms by which DSW modulated lipid metabolism and indicated that DSW was worthy of further investigation and could be developed as functional drinking water in the prevention and treatment of hypolipidemic and other lifestyle-related diseases.

  11. BU74, a complex oripavine derivative with potent kappa opioid receptor agonism and delayed opioid antagonism.

    PubMed

    Husbands, Stephen M; Neilan, Claire L; Broadbear, Jillian; Grundt, Peter; Breeden, Simon; Aceto, Mario D; Woods, James H; Lewis, John W; Traynor, John R

    2005-02-21

    In the search for opioid agonists with delayed antagonist actions as potential treatments for substance abuse, the bridged morphinan BU74 (17-cyclopropylmethyl-3-hydroxy-[5beta,7beta,3',5']-pyrrolidino-2'[S]-phenyl-7alpha-methyl-6,14-endoetheno morphinan) (3f) was synthesized. In isolated tissue and [35S]GTPgammaS opioid receptor functional assays BU74 was shown to be a potent long-lasting kappa opioid receptor agonist, delta opioid receptor partial agonist and mu opioid receptor antagonist. In antinociceptive tests in the mouse, BU74 showed high efficacy and potent kappa opioid receptor agonism. When its agonist action had waned BU74 became an antagonist of kappa and mu opioid receptor agonists in the tail flick assay and of delta, kappa and mu opioid receptor agonists in the acetic acid writhing assay. The slow onset, long-duration kappa opioid receptor agonist effects of BU74 suggests that it could be a lead compound for the discovery of a treatment for cocaine abuse.

  12. C/EBPβ in bone marrow is essential for diet induced inflammation, cholesterol balance, and atherosclerosis

    PubMed Central

    Rahman, Shaikh M.; Baquero, Karalee C.; Choudhury, Mahua; Janssen, Rachel C.; de la Houssaye, Becky A.; Sun, Ming; Miyazaki-Anzai, Shinobu; Wang, Shu; Moustaid-Moussa, Naima; Miyazaki, Makoto; Friedman, Jacob E.

    2016-01-01

    Background and Objective Atherosclerosis is both a chronic inflammatory disease and a lipid metabolism disorder. C/EBPβ is well documented for its role in the development of hematopoietic cells and integration of lipid metabolism. However, C/EBPβ's role in atherosclerotic progression has not been examined. We assessed the impact of hematopoietic CEBPβ deletion in ApoE−/− mice on hyperlipidemia, inflammatory responses and lesion formation in the aorta. Methods and Results ApoE−/− mice were reconstituted with bone marrow cells derived from either WT or C/EBPβ−/− mice and placed on low fat or high fat/high cholesterol diet for 11 weeks. Hematopoietic C/EBPβ deletion in ApoE−/− mice reduced blood and hepatic lipids and gene expression of hepatic stearoyl CoA desaturase 1 and fatty acid synthase while expression of ATP binding cassette transporter G1, cholesterol 7-alpha-hydroxylase, and liver X receptor alpha genes were significantly increased. ApoE−/− mice reconstituted with C/EBPβ−/− bone marrow cells also significantly reduced blood cytokine levels and reduced lesion area in aortic sinuses compared with ApoE−/− mice reconstituted with WT bone marrow cells. Silencing of C/EBPβ in RAW264.7 macrophage cells prevented oxLDL-mediated foam cell formation and inflammatory cytokine secretion in conditioned medium. Conclusion C/EBPβ in hematopoietic cells is crucial to regulate diet-induced inflammation, hyperlipidemia and atherosclerosis development. PMID:27072340

  13. CYP7A1 gene polymorphism located in the 5' upstream region modifies the risk of coronary artery disease.

    PubMed

    Iwanicki, Tomasz; Balcerzyk, Anna; Niemiec, Pawel; Nowak, Tomasz; Ochalska-Tyka, Anna; Krauze, Jolanta; Kosiorz-Gorczynska, Sylwia; Grzeszczak, Wladyslaw; Zak, Iwona

    2015-01-01

    7-Alpha cholesterol hydroxylase (CYP7A1), the first enzyme of classic conversion pathway leading from cholesterol to bile acids synthesis, is encoded by CYP7A1 gene. Its single nucleotide polymorphisms (SNPs) influence serum lipid levels and may be related to impaired lipid profile leading to coronary artery disease (CAD). The aim of the present study was to analyze the possible association between the rs7833904 CYP7A1 polymorphism and premature CAD. Serum lipid levels and rs7833904 SNP were determined in 419 subjects: 200 patients with premature CAD and 219 age and sex matched controls. The A allele carrier state was associated with CAD (OR = 1.76, 95% CI; 1.14-2.71, P = 0.014). The effect was even stronger in the male subgroups (OR = 2.16, 95% CI; 1.28-3.65, P = 0.003). There was no effect in the females. Risk factors of CAD and clinical phenotype of atherosclerosis were not associated with genotype variants of the rs7833904 SNP. Lipid profiles also did not differ significantly between individual genotypes. The CYP7A1 rs7833904 polymorphism may modify the risk of CAD. This effect is especially strong in male subjects. The studied polymorphism does not significantly influence serum lipid levels, in the present study.

  14. Inhibition of cholesterol biosynthesis by a water-soluble garlic extract in primary cultures of rat hepatocytes.

    PubMed

    Gebhardt, R

    1991-08-01

    Cultured rat hepatocytes continually synthesize cholesterol form radiolabeled acetate during a 24 h incubation period and export it, presumably as VLDL (very low density lipoprotein) to the culture medium. Mevastatin inhibits cholesterol biosynthesis by 90%. Incubation of the cultures with water-soluble extracts of garlic powder (Kwai, Sapec) diminish cholesterol biosynthesis (20-25%) as well as its export into the medium (30-35%). The IC50-value is 90 micrograms/ml. Between about 0.25 and 10 mg/ml the average maximal inhibition amounts to about 23%. Cytotoxicity of the extracts is apparent at concentrations above 125 mg/ml only. Pure alliin alone, or after incubation with alliinase (conversion to allicin) in concentrations corresponding to its content in the extracts does not exert any inhibition. Replacement of 14C-acetate by 14C-mevalonate omits the inhibitory effect. The activity of HMGCoA (hydroxymethylglutaryl-CoA) reductase is significantly reduced by garlic extracts at 50 micrograms/ml. At higher concentrations fatty acid synthetase, cholesterol 7 alpha-hydroxylase and cholesterol acyltransferase are slightly inhibited. Fatty acid synthetase is the only one of these enzymes which is inhibited by alliin at very high concentrations. These results demonstrate that water-soluble garlic extracts diminish hepatic cholesterol biosynthesis, thus contributing to the reduction of blood cholesterol. The main target site seems to be HMGCoA-reductase. The actual active principle(s) is still unknown. Alliin, however, does not seem to be of major significance.

  15. Bile acid-binding activity of young persimmon (Diospyros kaki) fruit and its hypolipidemic effect in mice.

    PubMed

    Matsumoto, Kenji; Yokoyama, Shin-ichiro; Gato, Nobuki

    2010-02-01

    The hypolipidemic effects and bile acid-binding properties of young persimmon (Diospyros kaki) fruit were examined. In an animal experiment, male C57BL/6.Cr mice (n = 5) were fed an AIN-76-modified high fat diet supplemented with 2% or 5% (w/w) dried young persimmon fruit (YP) for 10 weeks. The intake of YP significantly enhanced fecal bile acid excretion and lowered the concentration of hepatic lipids and plasma cholesterol. Analysis of gene expression in liver tissue showed that 2% or 5% YP up-regulated the expression of the sterol regulatory element-binding protein-2 gene. In the 5% group, there were increased expressions of the genes for cholesterol 7alpha-hydroxylase and the low-density lipoprotein receptor. Next, the bile acid-binding ability of YP was analysed in vitro using cholic acid (CA). In 100-2000 microM CA solutions, 1% (w/v) YP adsorbed approximately 60% of CA, while dried mature persimmon fruit adsorbed approximately 20% of CA. The positive control, cholestyramine, adsorbed approximately 80% of CA in the 100-2000 microM CA solutions. A crude tannin extract from YP, which contained 54.7% condensed tannins, adsorbed approximately 78% of CA in the 2000 microM CA solutions. These results suggest that the ability of YP to bind bile acid contributes to its hypolipidemic effect in mice. (c) 2009 John Wiley & Sons, Ltd.

  16. Scropolioside-D2 and harpagoside-B: two new iridoid glycosides from Scrophularia deserti and their antidiabetic and antiinflammatory activity.

    PubMed

    Ahmed, Bahar; Al-Rehaily, Adnan Jathlan; Al-Howiriny, Tawfeq Abdullah; El-Sayed, Khaled Abdelatee; Ahmad, Mohammad Shamim

    2003-04-01

    Five iridoid glycosides, including the two new compounds scropolioside-D(2) (1) and harpagoside-B (2), were isolated from the aerial parts of Scrophularia deserti DEL (Scrophulariaceae). Their structures were elucidated on the basis of spectral data to be 6-O-[2",4"-di-O-acetyl-3"-O-trans-cinnamoyl)-alpha-L-rhamnopyranosyl]-8 alpha-hydroxymethyl-1 alpha,5 beta,6 alpha,7 alpha,9 beta-pentahydro-7(8)-epoxy-2-oxaind-3-ene-1-O-beta-D-glucopyranoside-6'-O-acetate (1) and 5-O-beta-hydroxy-8-O-beta-trans-cinnamoyl-8 alpha-methyl-1,6,7,9-tetrahydro-2-oxaind-3-ene-1-O-beta-D-glucopyranoside (2), respectively. In addition, three more iridoid glycosides, scropolioside-D (3), koelzioside (4), and 8-O-acetyl-harpagide (5), were also isolated and characterized from this source. The biological activity and the structure activity relationship of the compounds were also studied, and scropolioside-D (3) and harpagoside-B (2) were found to possess significant antidiabetic and antiinflammatory activity, respectively.

  17. The effect of fuel type in unsaturated spent fuel tests

    SciTech Connect

    Finn, P.A.; Gong, M.; Bates, J.K.; Emery, J.W.; Hoh, J.C.

    1994-04-01

    Two well-characterized types of spent nuclear fuel (ATM-103 and ATM-106) were tested under simulated unsaturated conditions with simulated groundwater at 90{degree}C. The actinides present in the leachate were measured after periods of approximately 60, 120, and 275 days. The vessels were acid stripped after 120 and 275 days. Both colloidal and soluble actinide species were detected in the leachates which had pHs ranging from 4 to 7. Alpha spectroscopy studies of filtered and unfiltered leachates showed that large amounts of actinides may be bound in colloids. The uranium phases identified in the colloids were schoepite and soddyite. The actinide release behavior of the two fuels appears to be different. The ATM-106 fuel began to release actinides later than the ATM-103 fuel, but after 275 days, it had released more. The amount of americium released from the two fuels was a higher percentage of the maximum amount of americium present than was the percentage of the simultaneous amount of uranium released.

  18. Different catalytic properties and inhibitor responses of the goldfish brain and ovary aromatase isozymes.

    PubMed

    Zhao, J; Mak, P; Tchoudakova, A; Callard, G; Chen, S

    2001-08-01

    The brain and ovarian aromatase isozymes of goldfish (Carassius auratus) are encoded by different CYP19 genes. This study measured aromatase activity in the goldfish brain tissues. For a direct comparison of the properties of the two aromatase isozymes, Chinese hamster ovary cells were stably transfected with brain- and ovary-derived cDNAs (respectively, p450 arom B and -A) and the properties of the expressed isozymes were compared. The kinetic parameters of the two isozymes were determined using androstenedione and testosterone as substrates and compared to those of human aromatase. Inhibition profile analyses on the two isozymes were performed using seven inhibitors [4-hydroxyandrostenedione, 7 alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione, bridge (2,19-methyleneoxy)androstene-3,17-dione, aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole]. Except for AG, the compounds tested were found to be much stronger inhibitors against the ovary enzyme than the brain enzyme. In addition, the ovary isoform was more sensitive to two phytoestrogens, chrysin and 7,8-dihydroxyflavone, than the brain form. These studies reveal that catalytic properties of the goldfish aromatase isoforms are significantly different from those of human aromatase. In addition, differences in the K(i) values of aromatase inhibitors for the two goldfish isoforms suggest structural variance in the active sites of these isozymes.

  19. Polyphenol-Rich Fraction of Ecklonia cava Improves Nonalcoholic Fatty Liver Disease in High Fat Diet-Fed Mice.

    PubMed

    Park, Eun-Young; Choi, Hojung; Yoon, Ji-Young; Lee, In-Young; Seo, Youngwan; Moon, Hong-Seop; Hwang, Jong-Hee; Jun, Hee-Sook

    2015-11-12

    Ecklonia cava (E. cava; CA) is an edible brown alga with beneficial effects in diabetes via regulation of various metabolic processes such as lipogenesis, lipolysis, inflammation, and the antioxidant defense system in liver and adipose tissue. We investigated the effect of the polyphenol-rich fraction of E. cava produced from Gijang (G-CA) on nonalcoholic fatty liver disease (NAFLD) in high-fat diet (HFD)-fed mice. C57BL6 mice were fed a HFD for six weeks and then the HFD group was administered 300 mg/kg of G-CA extracts by oral intubation for 10 weeks. Body weight, fat mass, and serum biochemical parameters were reduced by G-CA extract treatment. MRI/MRS analysis showed that liver fat and liver volume in HFD-induced obese mice were reduced by G-CA extract treatment. Further, we analyzed hepatic gene expression related to inflammation and lipid metabolism. The mRNA expression levels of inflammatory cytokines and hepatic lipogenesis-related genes were decreased in G-CA-treated HFD mice. The mRNA expression levels of cholesterol 7 alpha-hydroxylase 1 (CYP7A1), the key enzyme in bile acid synthesis, were dramatically increased by G-CA treatment in HFD mice. We suggest that G-CA treatment ameliorated hepatic steatosis by inhibiting inflammation and improving lipid metabolism.

  20. In vivo gibberellin biosynthesis in endosperm of Sechium edule Sw. seeds

    SciTech Connect

    Ceccarelli, N.; Lorenzi, R. )

    1990-07-01

    Biosynthesis of gibberellins (GAs) was studied in vivo in endosperms of Sechium edule Sw. Exogenous ent-({sup 14}C)kaurene was metabolized into four major products: GA{sub 12}, GA{sub 4}, GA{sub 7} and 16,17-dihydro-16-hydroxy-GA{sub 15} alcohol glucoside. Other minor metabolites were also observed including ent-kaurenol and ent-kaurenal. Conversion of ent-({sup 14}C)kaurene to ent-karuenol glucoside by endosperm cell-free preparations in the presence of UDPG was observed. However, the finding was not confirmed in in vivo studies and is probably artifactual. Overall evidence coming from the analysis of endogenous GAs and in vitro and in vivo biosynthetic studies are discussed in relation to the possible existence in the Sechium seeds of a different route, along with the known pathway, branching from ent-kaurene or ent-7-{alpha}-hydroxykaurenoic acid and this also leading to biologically active GAs.

  1. Polyphenol-Rich Fraction of Ecklonia cava Improves Nonalcoholic Fatty Liver Disease in High Fat Diet-Fed Mice

    PubMed Central

    Park, Eun-Young; Choi, Hojung; Yoon, Ji-Young; Lee, In-Young; Seo, Youngwan; Moon, Hong-Seop; Hwang, Jong-Hee; Jun, Hee-Sook

    2015-01-01

    Ecklonia cava (E. cava; CA) is an edible brown alga with beneficial effects in diabetes via regulation of various metabolic processes such as lipogenesis, lipolysis, inflammation, and the antioxidant defense system in liver and adipose tissue. We investigated the effect of the polyphenol-rich fraction of E. cava produced from Gijang (G-CA) on nonalcoholic fatty liver disease (NAFLD) in high-fat diet (HFD)-fed mice. C57BL6 mice were fed a HFD for six weeks and then the HFD group was administered 300 mg/kg of G-CA extracts by oral intubation for 10 weeks. Body weight, fat mass, and serum biochemical parameters were reduced by G-CA extract treatment. MRI/MRS analysis showed that liver fat and liver volume in HFD-induced obese mice were reduced by G-CA extract treatment. Further, we analyzed hepatic gene expression related to inflammation and lipid metabolism. The mRNA expression levels of inflammatory cytokines and hepatic lipogenesis-related genes were decreased in G-CA-treated HFD mice. The mRNA expression levels of cholesterol 7 alpha-hydroxylase 1 (CYP7A1), the key enzyme in bile acid synthesis, were dramatically increased by G-CA treatment in HFD mice. We suggest that G-CA treatment ameliorated hepatic steatosis by inhibiting inflammation and improving lipid metabolism. PMID:26569269

  2. Neuroprotection in focal cerebral ischemia owing to delayed treatment with melanocortins.

    PubMed

    Giuliani, Daniela; Ottani, Alessandra; Mioni, Chiara; Bazzani, Carla; Galantucci, Maria; Minutoli, Letteria; Bitto, Alessandra; Zaffe, Davide; Botticelli, Annibale R; Squadrito, Francesco; Guarini, Salvatore

    2007-09-10

    In gerbils subjected to transient global cerebral ischemia, melanocortin peptides produce long-lasting protection with a broad time window, and through the activation of central nervous system melanocortin MC(4) receptors. Here we aimed to investigate whether melanocortins are neuroprotective also in a rat model of focal cerebral ischemia induced by intrastriatal microinjection of endothelin-1. The vasoconstrictor agent endothelin-1 caused a significant impairment in spatial learning and memory, as well as in sensory-motor orientation and limb use, associated with severe striatal morphological damage including intense neuronal death and an almost complete myelin degradation. Treatment of ischemic rats with a nanomolar dose (340 microg/kg/day i.p. for 11 days, beginning 3 h or 9 h after endothelin-1 microinjection) of the melanocortin analog [Nle(4), D-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) significantly reduced striatal damage, and improved subsequent functional recovery, with all scheduled NDP-alpha-MSH treatments. Pharmacological blockade of melanocortin MC(4) receptors prevented the protective effect of NDP-alpha-MSH. Our findings give evidence that melanocortins are neuroprotective, with a broad time window, also in a severe model of focal cerebral ischemia, and suggest that melanocortin MC(4) receptor agonists could produce neuroprotection in different experimental models of ischemic stroke.

  3. Morphometric examination of native lungs in human lung allograft recipients.

    PubMed

    Wiebe, B M; Burton, C M; Milman, N; Iversen, M; Andersen, C B

    2006-11-01

    The aim of the study was to estimate the degree of lung damage in patients with alpha(1)-antitrypsin (alpha1AT) deficiency, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) at the time of lung transplantation. Using unbiased stereological methods, lung-, bronchial- and vessel-volume, capillary length, and alveolar surface area and densities were estimated in recipient lungs from 21 consecutive patients with pre-transplant diagnoses including COPD (n=7), alpha1AT deficiency (n=6) and CF (n=8). Six unused adult donor lungs served as controls. Information relating to patient demography and pre-transplant lung function was obtained by retrospective chart review. Disease groups differed significantly with respect to demographics and pre-transplant lung function. Total lung volume was similar in all groups. Bronchial volume was significantly larger in CF patients compared to the control group (p<0.0001) and to the other two diagnostic groups: alpha1AT deficiency (p=0.0001) and COPD (p<0.0001). Alveolar surface density and capillary length density were significantly lower in patients with alpha1AT deficiency and COPD compared to controls (p<0.0001, respectively) and to patients with CF (p<0.0002, respectively). There were no correlations between clinical lung function and morphometric measurements. We conclude that unbiased microscopic stereological morphometry is an evolving science with the potential to elucidate pulmonary disease pathogenesis.

  4. Structural reorganization of the interleukin-7 signaling complex

    SciTech Connect

    McElroy, Craig A.; Holland, Paul J.; Zhao, Peng; Lim, Jae-Min; Wells, Lance; Eisenstein, Edward; Walsh, Scott T.R.

    2012-06-29

    We report here an unliganded receptor structure in the common gamma-chain ({gamma}{sub c}) family of receptors and cytokines. The crystal structure of the unliganded form of the interleukin-7 alpha receptor (IL-7R{alpha}) extracellular domain (ECD) at 2.15 {angstrom} resolution reveals a homodimer forming an 'X' geometry looking down onto the cell surface with the C termini of the two chains separated by 110 {angstrom} and the dimer interface comprising residues critical for IL-7 binding. Further biophysical studies indicate a weak association of the IL-7R{alpha} ECDs but a stronger association between the {gamma}{sub c}/IL-7R{alpha} ECDs, similar to previous studies of the full-length receptors on CD4{sup +} T cells. Based on these and previous results, we propose a molecular mechanism detailing the progression from the inactive IL-7R{alpha} homodimer and IL-7R{alpha}-{gamma}{sub c} heterodimer to the active IL-7-IL-7R{alpha}-{gamma}{sub c} ternary complex whereby the two receptors undergo at least a 90{sup o} rotation away from the cell surface, moving the C termini of IL-7R{alpha} and {gamma}{sub c} from a distance of 110 {angstrom} to less than 30 {angstrom} at the cell surface. This molecular mechanism can be used to explain recently discovered IL-7- and {gamma}{sub c}-independent gain-of-function mutations in IL-7R{alpha} from B- and T-cell acute lymphoblastic leukemia patients. The mechanism may also be applicable to other {gamma}{sub c} receptors that form inactive homodimers and heterodimers independent of their cytokines.

  5. Advanced Glycation in macrophages induces intracellular accumulation of 7-ketocholesterol and total sterols by decreasing the expression of ABCA-1 and ABCG-1

    PubMed Central

    2011-01-01

    Background Advanced glycation end products (AGE) alter lipid metabolism and reduce the macrophage expression of ABCA-1 and ABCG-1 which impairs the reverse cholesterol transport, a system that drives cholesterol from arterial wall macrophages to the liver, allowing its excretion into the bile and feces. Oxysterols favors lipid homeostasis in macrophages and drive the reverse cholesterol transport, although the accumulation of 7-ketocholesterol, 7alpha- hydroxycholesterol and 7beta- hydroxycholesterol is related to atherogenesis and cell death. We evaluated the effect of glycolaldehyde treatment (GAD; oxoaldehyde that induces a fast formation of intracellular AGE) in macrophages overloaded with oxidized LDL and incubated with HDL alone or HDL plus LXR agonist (T0901317) in: 1) the intracellular content of oxysterols and total sterols and 2) the contents of ABCA-1 and ABCG-1. Methods Total cholesterol and oxysterol subspecies were determined by gas chromatography/mass spectrometry and HDL receptors content by immunoblot. Results In control macrophages (C), incubation with HDL or HDL + T0901317 reduced the intracellular content of total sterols (total cholesterol + oxysterols), cholesterol and 7-ketocholesterol, which was not observed in GAD macrophages. In all experimental conditions no changes were found in the intracellular content of other oxysterol subspecies comparing C and GAD macrophages. GAD macrophages presented a 45% reduction in ABCA-1 protein level as compared to C cells, even after the addition of HDL or HDL + T0901317. The content of ABCG-1 was 36.6% reduced in GAD macrophages in the presence of HDL as compared to C macrophages. Conclusion In macrophages overloaded with oxidized LDL, glycolaldehyde treatment reduces the HDL-mediated cholesterol and 7-ketocholesterol efflux which is ascribed to the reduction in ABCA-1 and ABCG-1 protein level. This may contribute to atherosclerosis in diabetes mellitus. PMID:21957962

  6. Inhibition and inactivation of equine aromatase by steroidal and non-steroidal compounds. A comparison with human aromatase inhibition.

    PubMed

    Moslemi, S; Seralini, G E

    1997-12-01

    In order to approach the detailed structure-function relationships of aromatase, we studied the inhibitory and inactivatory potencies of several steroidal androstenedione analogues (1: 4-hydroxyandrostenedione, 2: 4-acetoxyandrostenedione and 3: 7 alpha-(4'-amino)phenylthio-4-androstene-3, 17-dione) and non-steroidal imidazole derivatives (4: ketoconazole, 5: miconazole and 6: fadrozole) on equine aromatase in placental microsomes, a well established mammalian model. Human placental microsomes and the purified enzyme from equine testis were also used to compare inhibition by 1 and 2. In equine microsomes, all compounds tested exhibited a competitive inhibition, with Ki values of 4.1, 26 and 1.8 nM for 1, 2 and 3, and of 2400, 1.4 and 4 nM for 4, 5, and 6, respectively. The Km for androstenedione, the substrate mainly used in these studies, was 1.8 +/- 0.13 nM. The three non-steroidal derivatives did not inactivate equine aromatase, but 1 and 2 acted as comparable inactivators to a much higher degree than 3. Compound 1 inhibited in a similar manner (89-94%) purified or equine and human microsomal aromatases, whereas 2 inhibited microsomal aromatase more efficiently in the horse than in man (92% and 33% inhibition, respectively). There was only a 40% inhibition with 2 on the purified equine enzyme, which is no more in the natural membrane environment. The comparisons between equine and human microsomal aromatases allow precise functional and structural differences to be observed with these enzymes.

  7. Pericentric characterization of human chromosome 7 in a melanoma cell line

    SciTech Connect

    Fetni, R.; Lemieux, N.; Richer, C.L.

    1994-09-01

    Cytogenetic analyses of an established melanoma cell line show structural abnormalities involving mainly chromosome 7. Molecular cytogenetic examination of the different abnormalities (i(7q), i(7p), t(7;12)) was used to pinpoint the site of the break and to analyse the possible mechanisms by which isochromosomes 7 could be formed. Human chromosome 7 has been shown to contain two distinct alpha satellite arrays: D7Z1 and D7Z2 which are separated by 1Mb. We confirm the order to be short-arm- D7Z2 - D7Z1 - long arm. Both probes were then used to characterize two different types of isochromosomes 7 (one of the long arms and one of the short arms) and a translocation (7;12). Isochromosome 7q showed a single D7Z1 signal and loss of D7Z2. The unique centromeric structure of i(7q), with only the D7Z1 signal, suggests that a breakpoint occurred within D7Z1. Isochromosome 7p showed two distinct D7Z1 and D7Z2 hybridization signals. The distance observed between the two signals suggests that the breakpoint is in the proximal part of the long arms. This chromosome might be considered as a dicentric isochromosome 7p. Translocation (7;12) showed the two arrays of chromosome 7 {alpha} satellite DNA. The three derived chromosomes appeared to result from independent rearrangements. This observation shows that a variety of breaks may occur in the juxtacentromeric region of a given chromosome. It also shows that the functional centromere of chromosome 7 does not need the presence of D7Z2, since only D7Z1 was conserved in all cases, suggesting the importance of this sequence for the centromeric function.

  8. Immunohistochemistry of adhesion molecules, metalloproteinases and NO-synthases in extravillous trophoblast of tubal pregnancy.

    PubMed

    Dubernard, G; Galtier-Fougairolles, M; Cortez, A; Uzan, S; Challier, J C

    2005-12-12

    Trophoblast invasion in uterine pregnancy is fine-tuned for the remodelling of the uterine wall and its vascularization. Tubal pregnancy, which occurs in a limited number of patients, involves a dramatic trophoblast invasion in a context of a poor decidualization. By studying the histology of the extravillous trophoblast (EVC) in the anchoring villi, the Ki67 labelling, the location of several adhesion markers (cytokeratin-7, alpha1, alpha6, alphaV, beta1, beta4 integrin subunits and E-cadherin, V/E-cadherin), metalloproteinases (MMP-2, 9 and11), NOS2 and 3, we aimed to detect the specificity of tubal compared to intrauterine pregnancies. No difference could be observed between meso or anti-salpingial trophoblast proliferation or invasion using Ki67. Cytokeratin-7 allowed detection of spindle-shape EVCs and we identified some decidualized stromal cells. Integrins alpha1, beta1 and alphaV, and V/E-cadherin were expressed mainly in the distal EVC correspondingly to intrauterine pregnancy, with a poor expression of alpha1. Integrins alpha6 and beta4, E-cadherin were detected in the distal EVC in contrast to uterine pregnancy. MMP-2, 9, 11 were also shown in distal EVC. NOS2 and 3 labelled the perivascular EVC and NOS3 the endothelial cells of the tubal vessels. These changed distributions of adhesion molecules and MMP together with that of the basic and inducible NOS expressions could be related to mechanical effects in superficial implantation or to a failure of decidualization in tubal pregnancies.

  9. Molecular factors involved in the hypolipidemic- and insulin-sensitizing effects of a ginger (Zingiber officinale Roscoe) extract in rats fed a high-fat diet.

    PubMed

    de Las Heras, Natalia; Valero-Muñoz, María; Martín-Fernández, Beatriz; Ballesteros, Sandra; López-Farré, Antonio; Ruiz-Roso, Baltasar; Lahera, Vicente

    2017-02-01

    Hypolipidemic and hypoglycemic properties of ginger in animal models have been reported. However, information related to the mechanisms and factors involved in the metabolic effects of ginger at a hepatic level are limited. The aim of the present study was to investigate molecular factors involved in the hypoglycemic and hypolipidemic effects of a hydroethanolic ginger extract (GE) in the liver of rats fed a high-fat diet (HFD). The study was conducted in male Wistar rats divided into the following 3 groups: (i) Rats fed a standard diet (3.5% fat), the control group; (ii) rats fed an HFD (33.5% fat); and (iii) rats fed an HFD treated with GE (250 mg·kg(-1)·day(-1)) for 5 weeks (HFD+GE). Plasma levels of glucose, insulin, lipid profile, leptin, and adiponectin were measured. Liver expression of glycerol phosphate acyltransferase (GPAT), cholesterol 7 alpha-hydroxylase, peroxisome proliferator-activated receptors (PPAR), PPARα and PPARγ, glucose transporter 2 (GLUT-2), liver X receptor, sterol regulatory element-binding protein (SREBP1c), connective tissue growth factor (CTGF), and collagen I was measured. Data were analyzed using a 1-way ANOVA, followed by a Newman-Keuls test if differences were noted. The study showed that GE improved lipid profile and attenuated the increase of plasma levels of glucose, insulin, and leptin in HFD rats. This effect was associated with a higher liver expression of PPARα, PPARγ, and GLUT-2 and an enhancement of plasma adiponectin levels. Furthermore, GE reduced liver expression of GPAT, SREBP1c, CTGF, and collagen I. The results suggest that GE might be considered as an alternative therapeutic strategy in the management of overweight and hepatic and metabolic-related alterations.

  10. Novel alpha1-adrenergic receptor signaling pathways: secreted factors and interactions with the extracellular matrix.

    PubMed

    Shi, Ting; Duan, Zhong-Hui; Papay, Robert; Pluskota, Elzbieta; Gaivin, Robert J; de la Motte, Carol A; Plow, Edward F; Perez, Dianne M

    2006-07-01

    alpha1-Adrenergic receptor (alpha1-ARs) subtypes (alpha1A, alpha1B, and alpha1D) regulate multiple signal pathways, such as phospholipase C, protein kinase C (PKC), and mitogen-activated protein kinases. We employed oligonucleotide microarray technology to explore the effects of both short- (1 h) and long-term (18 h) activation of the alpha1A-AR to enable RNA changes to occur downstream of earlier well characterized signaling pathways, promoting novel couplings. Polymerase chain reaction (PCR) studies confirmed that PKC was a critical regulator of alpha1A-AR-mediated gene expression, and secreted interleukin (IL)-6 also contributed to gene expression alterations. We next focused on two novel signaling pathways that might be mediated through alpha1A-AR stimulation because of the clustering of gene expression changes for cell adhesion/motility (syndecan-4 and tenascin-C) and hyaluronan (HA) signaling. We confirmed that alpha1-ARs induced adhesion in three cell types to vitronectin, an interaction that was also integrin-, FGF7-, and PKC-dependent. alpha1-AR activation also inhibited cell migration, which was integrin- and PKC-independent but still required secretion of FGF7. alpha1-AR activation also increased the expression and deposition of HA, a glycosaminoglycan, which displayed two distinct structures: pericellular coats and long cable structures, as well as increasing expression of the HA receptor, CD44. Long cable structures of HA can bind leukocytes, which this suggests that alpha1-ARs may be involved in proinflammatory responses. Our results indicate alpha1-ARs induce the secretion of factors that interact with the extracellular matrix to regulate cell adhesion, motility and proinflammatory responses through novel signaling pathways.

  11. Dietary garlic and onion reduce the incidence of atherogenic diet-induced cholesterol gallstones in experimental mice.

    PubMed

    Vidyashankar, Satyakumar; Sambaiah, Kari; Srinivasan, Krishnapura

    2009-06-01

    Mice fed with diet containing 0.5 % cholesterol for 10 weeks resulted in cholesterol supersaturation in gallbladder bile which promoted the formation of cholesterol gallstones (CGS). In this study, dietary hypocholesterolaemic spices, garlic and onion (both raw or heat-processed) were examined for their antilithogenic potential by including at 0.6 and 2.0 % level, respectively, along with lithogenic (LG) diet for 10 weeks. Dietary garlic and onion reduced the CGS incidence by 15-39 %, the effect being maximum in the heat-processed onion group. Dietary garlic and onion markedly reduced biliary cholesterol. The cholesterol:phospholipid ratio which was 1.58 in the LG diet group was reduced to 0.73-0.96 in the garlic and onion groups. The biliary cholesterol saturation index was 0.92, 1.25, 1.09 and 0.86, respectively, in the heat-processed onion, raw garlic, heat-processed garlic and raw onion groups, while it was 1.9 in the LG group. The hydrophobicity index of bile was - 0.08, - 0.079, - 0.032 and - 0.073, respectively, in the heat-processed onion, raw garlic, heat-processed garlic and raw onion groups, while it was +0.054 in the LG group. Hepatic hydroxymethyl glutaryl-CoA reductase activity was lowered in the LG diet-fed group, while dietary garlic or onion countered this alteration and also increased the activities of hepatic cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase. Serum and liver cholesterol were decreased by feeding garlic or onion compared to the LG diet. Thus, dietary Allium spices exerted antilithogenic influence by decreasing the cholesterol hyper-secretion into bile and increasing the bile acid output thus decreasing the formation of lithogenic bile in experimental mice.

  12. Structural basis for substrate specificity differences of horse liver alcohol dehydrogenase isozymes.

    PubMed

    Adolph, H W; Zwart, P; Meijers, R; Hubatsch, I; Kiefer, M; Lamzin, V; Cedergren-Zeppezauer, E

    2000-10-24

    A structure determination in combination with a kinetic study of the steroid converting isozyme of horse liver alcohol dehydrogenase, SS-ADH, is presented. Kinetic parameters for the substrates, 5beta-androstane-3beta,17beta-ol, 5beta-androstane-17beta-ol-3-one, ethanol, and various secondary alcohols and the corresponding ketones are compared for the SS- and EE-isozymes which differ by nine amino acid substitutions and one deletion. Differences in substrate specificity and stereoselectivity are explained on the basis of individual kinetic rate constants for the underlying ordered bi-bi mechanism. SS-ADH was crystallized in complex with 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan -24-acid (cholic acid) and NAD(+), but microspectrophotometric analysis of single crystals proved it to be a mixed complex containing 60-70% NAD(+) and 30-40% NADH. The crystals belong to the space group P2(1) with cell dimensions a = 55.0 A, b = 73.2 A, c = 92.5 A, and beta = 102.5 degrees. A 98% complete data set to 1.54-A resolution was collected at 100 K using synchrotron radiation. The structure was solved by the molecular replacement method utilizing EE-ADH as the search model. The major structural difference between the isozymes is a widening of the substrate channel. The largest shifts in C(alpha) carbon positions (about 5 A) are observed in the loop region, in which a deletion of Asp115 is found in the SS isozyme. SS-ADH easily accommodates cholic acid, whereas steroid substrates of similar bulkiness would not fit into the EE-ADH substrate site. In the ternary complex with NAD(+)/NADH, we find that the carboxyl group of cholic acid ligates to the active site zinc ion, which probably contributes to the strong binding in the ternary NAD(+) complex.

  13. Treatment with alpha-melanocyte stimulating hormone preserves calcium regulatory proteins in rat heart allografts.

    PubMed

    Colombo, Gualtiero; Sordi, Andrea; Lonati, Caterina; Carlin, Andrea; Turcatti, Flavia; Leonardi, Patrizia; Gatti, Stefano; Catania, Anna

    2008-08-01

    Prevention of graft dysfunction is a major objective in transplantation medicine. Previous research on experimental heart transplantation indicated that treatment with the immunomodulatory peptide alpha-melanocyte stimulating hormone (alpha-MSH) improves histopathology, prolongs allograft survival, and reduces expression of the main tissue injury mediators. Because calcium-handling is critical in heart graft function, we determined the effects of transplantation injury and influences of alpha-MSH treatment on representative calcium regulatory proteins in rat heart allografts. Hearts from Brown Norway rats were transplanted heterotopically into MHC incompatible Lewis rats. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), protein kinase C epsilon (PKC epsilon), sarcoplasmic/endoplasmic reticulum calcium-ATPase 2 (SERCA2a), arrestin-beta1 (Arrb1), cholinergic receptor M2 (Chrm2), and inositol 1,4,5-triphosphate receptor 1 (InsP(3)R1) were examined in: (1) non-transplanted donor hearts; (2) allografts from saline-treated rats; and (3) allografts from rats treated with the synthetic alpha-MSH analog Nle4-DPhe7-alpha-MSH (NDP-alpha-MSH) (100 microg i.p. every 12h). Transplantation injury was associated with severe reduction in calcium regulatory protein transcription and expression level. NDP-alpha-MSH administration partly reversed inhibition of protein transcription and almost completely prevented protein loss. Finally, because certain effects of cyclic 3'-5'-adenosine monophosphate (cAMP) signaling on calcium handling in cardiac myocytes depend on activation of exchange protein directly activated by cAMP 1 (Epac1), we determined Epac1 mRNA and protein expression in heart allografts. Transplantation injury markedly reduced Epac1. NDP-alpha-MSH treatment significantly preserved both Epac1 protein and mRNA in the allografts. Administration of alpha-MSH or related melanocortins could reduce transplantation-induced dysfunction through protection of heart calcium

  14. In vitro analysis of integrin expression during chondrogenic differentiation of mesenchymal stem cells and chondrocytes upon dedifferentiation in cell culture.

    PubMed

    Goessler, Ulrich Reinhart; Bieback, Karen; Bugert, Peter; Heller, Tobias; Sadick, Haneen; Hörmann, Karl; Riedel, Frank

    2006-02-01

    Tissue engineering represents a promising method for generating chondrogenic grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation of cells seems unavoidable for multiplication. Stem cells, however, displaying unlimited self-renewal and the capacity to differentiate towards chondrocytes, might be usable after further characterization. As the interactions between the extracellular matrix and the cellular compartment can alter the cellular behaviour, we investigated the expression of integrins using microarray analysis during chondrogenic differentiation of human mesenchymal stem cells (MSC) in comparison with de-differentiating human chondrocytes (HC) harvested during septoplasty. During chondrogenic differentiation of MSC, the fibronectin-receptor (Integrin beta1alpha5), fibronectin and the GPIIb/IIIa-receptor were downregulated. The components of the vitronectin-receptor (Integrin alphavbeta3) and CD47 were constantly expressed and ILK was downregulated. Vitronectin and osteopontin were not expressed by the cells. In HC, Integrin beta1alpha5 in conjunction with the ligand fibronectin were upregulated during dedifferentiation, Integrin alphavbeta3 as well as the GBIIb/IIIa-receptor were activated on day 21 but neither vitronectin nor osteopontin were expressed by the cells. The integrins, beta2, beta4, beta6, beta8 and alpha2, alpha4, alpha6, alpha7, alpha11, were not expressed at any time. ILK, CD47, and ICAP were activated with ongoing dedifferentiation. In conclusion, a candidate for signal-transmission is the fibronectin receptor (integrin alpha5beta1) in conjunction with its ligand fibronectin. Other receptors, e.g. for vitronectin and osteopontin (alphavbeta3), or their ligands do not seem to be involved in signal transmission for dedifferentiation. The GPIIb/IIIa-receptor might assist the process of dedifferentiation. Intracellularly, ILK, ICAP1 and CD47 might be involved in the transduction of integrin-dependent signals.

  15. Structural basis of the resistance of an insect carboxypeptidase to plant protease inhibitors.

    PubMed

    Bayés, Alex; Comellas-Bigler, Mireia; Rodríguez de la Vega, Monica; Maskos, Klaus; Bode, Wolfram; Aviles, Francesc X; Jongsma, Maarten A; Beekwilder, Jules; Vendrell, Josep

    2005-11-15

    Corn earworm (Helicoverpa zea), also called tomato fruitworm, is a common pest of many Solanaceous plants. This insect is known to adapt to the ingestion of plant serine protease inhibitors by using digestive proteases that are insensitive to inhibition. We have now identified a B-type carboxypeptidase of H. zea (CPBHz) insensitive to potato carboxypeptidase inhibitor (PCI) in corn earworm. To elucidate the structural features leading to the adaptation of the insect enzyme, the crystal structure of the recombinant CPBHz protein was determined by x-ray diffraction. CPBHz is a member of the A/B subfamily of metallocarboxypeptidases, which displays the characteristic metallocarboxypeptidase alpha/beta-hydrolase fold, and does not differ essentially from the previously described Helicoverpa armigera CPA, which is very sensitive to PCI. The data provide structural insight into several functional properties of CPBHz. The high selectivity shown by CPBHz for C-terminal lysine residues is due to residue changes in the S1' substrate specificity pocket that render it unable to accommodate the side chain of an arginine. The insensitivity of CPBHz to plant inhibitors is explained by the exceptional positioning of two of the main regions that stabilize other carboxypeptidase-PCI complexes, the beta8-alpha9 loop, and alpha7 together with the alpha7-alpha8 loop. The rearrangement of these two regions leads to a displacement of the active-site entrance that impairs the proper interaction with PCI. This report explains a crystal structure of an insect protease and its adaptation to defensive plant protease inhibitors.

  16. Unconditioned behavioral effects of the powerful kappa-opioid hallucinogen salvinorin A in nonhuman primates: fast onset and entry into cerebrospinal fluid.

    PubMed

    Butelman, Eduardo R; Prisinzano, Thomas E; Deng, Haiteng; Rus, Szymon; Kreek, Mary Jeanne

    2009-02-01

    Salvinorin A is the main active component of the widely available hallucinogenic plant, Salvia divinorum. Salvinorin A is a selective high-efficacy kappa-agonist in vitro, with some unique pharmacodynamic properties. Descriptive reports show that salvinorin A-containing products produce robust behavioral effects in humans. However, these effects have not been systematically characterized in human or nonhuman primates to date. Therefore, the present studies focused on the characterization of overt effects of salvinorin A, such as sedation (operationally defined as unresponsiveness to environmental stimuli) and postural relaxation, previously observed with centrally penetrating kappa-agonists in nonhuman primates. Salvinorin A was active in these endpoints (dose range, 0.01-0.1 mg/kg i.v.) in nonhuman primates (n = 3-5), similar to the synthetic kappa-agonist U69,593 [(+)-(5alpha,7alpha,8beta)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]-dec-8-yl]-benzeneacetamide], used for comparison herein. Salvinorin A effects could be prevented by a clinically available opioid antagonist, nalmefene (0.1 mg/kg), at doses known to block kappa-receptor-mediated effects in nonhuman primates. When injected intravenously, salvinorin A (0.032 mg/kg) could enter the central nervous system (as reflected in cisternal cerebrospinal fluid) within 1 min and reach concentrations that are in the reported range of the affinity (K(i)) of this ligand for brain kappa-receptors. Consistent with this finding, specific translationally viable behavioral effects (e.g., facial relaxation and ptosis) could also be detected within 1 to 2 min of injection of salvinorin A. These are the first studies documenting rapid unconditioned effects of salvinorin A in a primate species, consistent with descriptive reports of rapid and robust effects of this powerful hallucinogen in humans.

  17. The Likelihood of Cognitive Enhancement

    PubMed Central

    Lynch, Gary; Palmer, Linda C.; Gall, Christine M.

    2011-01-01

    Whether drugs that enhance cognition in healthy individuals will appear in the near future has become a topic of considerable interest. We address this possibility using a three variable system (psychological effect, neurobiological mechanism, efficiency vs. capabilities) for classifying candidates. Ritalin and modafinil, two currently available compounds, operate on primary psychological states that in turn affect cognitive operations (attention, memory), but there is little evidence that these effects translate into improvements in complex cognitive processing. A second category of potential enhancers includes agents that improve memory encoding, generally without large changes in primary psychological states. Unfortunately, there is little information on how these compounds affect cognitive performance in standard psychological tests. Recent experiments have identified a number of sites at which memory drugs could, in principle, manipulate the cell biological systems underlying the learning-related long-term potentiation (LTP) effect; this may explain the remarkable diversity of memory promoting compounds. Indeed, many of these agents are known to have positive effects on LTP. A possible third category of enhancement drugs directed specifically at integrated cognitive operations is nearly empty. From a neurobiological perspective, two plausible candidate classes have emerged that both target the fast excitatory transmission responsible for communication within cortical networks. One acts on nicotinic receptors (alpha7, alpha4) that regulate release of the neurotransmitter glutamate while the other (‘ampakines’) allosterically modulates the glutamate receptors mediating the post-synaptic response (EPSCs). Brain imaging in primates has shown that ampakines expand cortical networks engaged by a complex task; coupled with behavioral data, these findings provide evidence for the possibility of generating new cognitive capabilities. Finally, we suggest that

  18. Deployment and background characterization of the Sudbury Neutrino Observatory Neutral Current Detectors

    NASA Astrophysics Data System (ADS)

    Stonehill, Laura C.

    The Sudbury Neutrino Observatory (SNO) is a heavy water Cherenkov detector designed to observe 8B neutrinos from the sun via elastic-scattering interactions with electrons and charged-current and neutral-current interactions with deuterium. SNO is currently taking data in its third phase, in which the detector's capabilities have been enhanced by the addition of the Neutral Current Detectors (NCDs), an array of 3He proportional counters that detect neutrons liberated in neutral-current interactions. The NCDs were designed and constructed to minimize radioactive impurities that can produce backgrounds. A variety of innovative materials and methods were used in the construction and deployment of the NCD array in order to meet the stringent purity requirements. It is important to quantify alpha backgrounds in the NCDs and to verify that the impurity levels are within design specifications. Analysis techniques relying on pulse shape characteristics, time coincidences, and energy spectra were used to identify and measure bulk 238U and 232 Th chain decays and surface 210Po decays in the nickel NCD bodies. The measured bulk impurities in the NCD array are equivalent to 5.7+1.0-0.9 picograms 232Th per gram of nickel and 2.8+0.6-0.8 picograms 238U per gram of nickel. In addition to the 43+6-7 alphas per day in the NCD array from these sources, 210Po surface contamination produces 127 +/- 2 alphas per day, and another 81+5-2 alphas per day are produced by activity in the upper portions of the disequilibrium 238U and 232Th chains. The alpha background studies presented here indicate that the alpha rates in the NCD array are in line with expectations, and will not pose a significant problem to the successful extraction of the neutral-current signal from the NCDs using pulse shape analysis techniques.

  19. Comparative study of serum proteomes in Legg-Calve-Perthes disease.

    PubMed

    Liu, Ruiyu; Fan, Lihong; Yin, Longbin; Wang, Kunzheng; Miao, Wusheng; Song, Qichun; Dang, Xiaoqian; Gao, Hang; Bai, Chuanyi

    2015-10-05

    Legg-Calve-Perthes Disease (LCPD) is an idiopathic osteonecrosis of the developing femoral head complicated by pain and disability of the hip joint. To date, the pathological mechanisms of LCPD are not well-known. This study screened the changes in serum protein expression in patients with LCPD. Age- and sex-matched serum samples from 10 control subjects and 10 patients with LCPD were compared using the isobaric tags for relative and absolute quantification (iTRAQ) technique. Gene ontology analyses, KEGG pathway and functional network analyses were performed. Proteins of interest with large differences in expression, S100-A8, alpha-1-acid glycoprotein 1, haptoglobin and apolipoprotein E, were compared by western blotting. The disease/control ratios showed 26 proteins were significantly differentially expressed (all p < 0.05). Including higher abundances of complement factor H (1.44), complement C4-B (1.45), isocitrate dehydrogenase [NAD] subunit alpha (2.7) alpha-1-acid glycoprotein 1 (1.87), heptoglobin (1.53) and Ig lambda-2 chain C regions (1.46), and lower levels of apolipoprotein E (0.50), apolipoprotein F (0.60), apolipoprotein C-III (0.69), S100-A8 (0.73), S100-A9 (0.75) and prothrombin (0.77) in LCPD than in controls. The alpha-1-acid glycoprotein 1 and haptoglobin increases, and apolipoprotein E and S100-A8 decreases were confirmed by western blot. KEGG pathway analysis revealed these proteins were related to the complement and coagulation cascades, Staphylococcus aureus infection, PPAR signaling, fat digestion and absorption, and vitamin digestion and absorption. Functional network analysis suggested that the proteins were involved in lipid regulation. The complement and coagulation cascades, and abnormal lipid metabolism may be involved in the pathogenesis of LCPD.

  20. A synthesized pheromone induces upstream movement in female sea lamprey and summons them into traps.

    PubMed

    Johnson, Nicholas S; Yun, Sang-Seon; Thompson, Henry T; Brant, Cory O; Li, Weiming

    2009-01-27

    Female insect pheromone blends induce robust tracking responses in males and direct them into traps. In vertebrates, pheromones that induce strong and precise tracking responses in natural habitats have rarely been described. Here, we show in the sea lamprey (Petromyzon marinus), a vertebrate invader of the Laurential Great Lakes, that a synthesized component of the male mating pheromone, 7alpha, 12alpha, 24-trihydroxy-5alpha-cholan-3-one 24-sulfate (3kPZS), when released into a stream to reach concentrations of 10(-14), 10(-13), 10(-12), 10(-11), or 10(-10) M, triggers robust upstream movement in ovulated females drawing approximately 50% into baited traps. Experiments conducted in diverse stream segments demonstrate the level of behavioral response was not affected by habitat conditions and is effective over hundreds of meters. 3kPZS is equally effective at luring ovulated females as the whole pheromone blend released by males between 10(-14) and 10(-11) M. 3kPZS diverts ovulated females away from and disrupts orientation to male washings when applied at concentrations higher than washings. Indeed, a single pheromone compound is able to redirect female sea lampreys away from a natural pheromone source and lure them into traps, which should be more effective than targeting males when applied in population control. Our findings may spur the discovery of other potent and environmentally benign agents to combat biological invasion, a process accelerated by globalization, exacerbated by climate change, and costing the global economy US$ 1.4 trillion of damage annually.

  1. Development and application of an ELISA for a sex pheromone released by the male sea lamprey (Petromyzon marinus L.).

    PubMed

    Yun, Sang-Seon; Scott, Alexander P; Siefkes, Michael J; Li, Weiming

    2002-12-01

    An enzyme-linked immunosorbent assay (ELISA) has been developed for a conjugated bile acid, 7alpha,12alpha,24-trihydroxy-5alpha-cholan-3-one 24-sulfate (commonly referred to as 3-keto petromyzonol sulfate [3kPZS]), a pheromone released by reproductively mature male sea lampreys to attract sexually mature females. A polyclonal antiserum against the pheromone was raised by injecting 3-keto petromyzonol 24-hemisuccinate (3kPZ-HS) conjugated to bovine serum albumin into rabbits. The enzyme label was prepared by conjugating 3kPZ-HS to acetylcholinesterase. The standard curve had a working range of 20 pg-10 ng/well. Intra- and inter-assay variations were less than 5 and 12%, respectively. The antiserum had 100% cross-reaction with 3-keto petromyzonol and 3-keto allocholic acid but less than 0.2% cross-reaction with petromyzonol, allocholic acid, cholic acid, and taurolithocholic acid sulfate. The assay was applied to water which had been conditioned for 4h by either larvae, parasitic juveniles, ovulating females, pre-spermiating males, or spermiating males. Immunoactive material (average 200 ng/ml, which is equivalent to 500 microg animal/h) was only found in water from the reproductively mature males and diluted parallel with the standard curve. Assay of water samples collected from male lampreys in bisected aquaria also established that 99.6% of the immunoactive material emanated from the front end of the fish. This assay has applications in both physiological and ecological aspects of sea lamprey reproduction.

  2. Differential roles for disulfide bonds in the structural integrity and biological activity of kappa-Bungarotoxin, a neuronal nicotinic acetylcholine receptor antagonist.

    PubMed

    Grant, G A; Luetje, C W; Summers, R; Xu, X L

    1998-09-01

    kappa-Bungarotoxin, a kappa-neurotoxin derived from the venom of the banded Krait, Bungarus multicinctus, is a homodimeric protein composed of subunits of 66 amino acid residues containing five disulfide bonds. kappa-Bungarotoxin is a potent, selective, and slowly reversible antagonist of alpha3 beta2 neuronal nicotinic acetylcholine receptors. kappa-Bungarotoxin is structurally related to the alpha-neurotoxins, such as alpha-bungarotoxin derived from the same snake, which are monomeric in solution and which effectively antagonize muscle type receptors (alpha1 beta1 gamma delta) and the homopentameric neuronal type receptors (alpha7, alpha8, and alpha9). Like the kappa-neurotoxins, the long alpha-neurotoxins contain the same five conserved disulfide bonds, while the short alpha-neurotoxins only contain four of the five. Systematic removal of single disulfide bonds in kappa-bungarotoxin by site-specific mutagenesis reveals a differential role for each of the disulfide bonds. Removal of either of the two disulfides connecting elements of the carboxy terminal loop of this toxin (Cys 46-Cys 58 and Cys 59-Cys 64) interferes with the ability of the toxin to fold. In contrast, removal of each of the other three disulfides does not interfere with the general folding of the toxin and yields molecules with biological activity. In fact, when either C3-C21 or C14-C42 are removed individually, no loss in biological activity is seen. However, removing both produces a polypeptide chain which fails to fold properly. Removal of the C27-C31 disulfide only reduces the activity of the toxin 46.6-fold. This disulfide may play a role in specific interaction of the toxin with specific neuronal receptors.

  3. Evaluation of MENT on primary cell cultures from benign prostatic hyperplasia and prostate carcinoma.

    PubMed

    Mendoza, Patricia; Sánchez, Catherine; Contreras, Héctor R; Vergara, Jorge; Acevedo, Cristian; Cabezas, Juan; Huidobro, Christian; Noé, Gabriela; Castellón, Enrique A

    2009-12-01

    7-alpha-Methyl-19-Nortestosterone (MENT) is a synthetic androgen more potent than testosterone (T) and cannot be reduced at 5-alpha position. No important effects of MENT on prostate growth have been reported. However, little is known about the effect of MENT on benign prostatic hyperplasia (BPH) or prostate carcinoma (CaP). We evaluate the effect of MENT, T and dihydrotestosterone (DHT) on secretion, proliferation and gene expression of primary cell cultures from human BPH and CaP. Moreover, the effect of these androgens was examined in the presence of finasteride to determine the influence of the 5-alpha reductase (5-AR) activity on the androgenic potency. BPH and CaP primary cultures were treated with 0, 1, 10 and 100 nM of T, MENT or DHT during 24 and 48 h. Prostate-specific antigen (PSA) was measured by micro particles immunoassay and proliferation rate by spectrophotometric assay (MTT) and by the immunochemical detection of the proliferation marker Ki-67. Gene expression of FGF8b (androgen sensitive gene) was evaluated by semi-quantitative RT-PCR. Results showed that MENT treatments increased PSA secretion and proliferation rate with a potency ranged between T and DHT. Similar effects of MENT were observed in both BPH and CaP cultures. The studies with finasteride showed that in BPH and CaP cells, the conversion of T into DHT significantly contributes to its effect on the proliferation and PSA secretion, and corroborated the resistance of MENT to the 5-AR. The effect of MENT on the gene expression of FGF8b in CaP cells was similar to T and lower than DHT. It is concluded that MENT increases proliferative and secretory activities and gene expression on pathological prostate cells although in less extent than the active metabolite DHT. Furthermore, the fall of endogenous concentration of T during MENT treatment anticipates that this androgen will be of low impact for the prostate.

  4. Inhibition of recovery of spermatogenesis in irradiated rats by different androgens.

    PubMed

    Shetty, Gunapala; Wilson, Gene; Hardy, Matthew P; Niu, Enmei; Huhtaniemi, Ilpo; Meistrich, Marvin L

    2002-09-01

    We previously showed that exogenous testosterone (T) inhibited GnRH-antagonist-stimulated spermatogenic recovery in irradiated rats through an androgen-receptor-mediated action. In the present study, we tested whether the inhibition is attributable to T, a specific androgenic metabolite of T, or a general property of androgens in this system. In addition, we also tested whether estradiol-17beta (E2), a metabolite of T, is similarly inhibitory. Rats irradiated with 5 Gy were treated with a GnRH antagonist during wk 3-7. Neither irradiation nor GnRH-antagonist treatment produced biologically significant changes in the relative intratesticular levels of several androgenic metabolites. Next, groups of rats, irradiated and treated with GnRH antagonist as above, were given various doses of one of the following androgens: T, 5alpha-dihydrotestosterone, 7alpha-methyl-19-nortestosterone, methyltrienolone, or E2. The percentage of tubules showing differentiation (tubule differentiation index) was increased to 68% by the GnRH antagonist, from a value of 0.1% in irradiated-only rats at 13 wk after irradiation. All of the added androgens inhibited spermatogenic recovery, lowering the tubule differentiation index to between 0.4-36%, but no inhibition was observed with the addition of E2. Of all the androgen treatments tested, T (given as daily injections of T propionate) minimally inhibited spermatogenic recovery while maintaining androgen-responsive tissue weights, and might be most useful in clinical studies. Hormonal measurements in androgen-treated rats were most consistent with the androgen inhibition of spermatogenic recovery in irradiated rats being a combined result of a direct inhibitory effect of all androgens on the testis and an indirect effect through the pituitary by raising levels of FSH, which seems to add to the inhibition of spermatogenic recovery.

  5. Pharmacology of the ACAT inhibitor avasimibe (CI-1011).

    PubMed

    Llaverías, Gemma; Laguna, Juan C; Alegret, Marta

    2003-01-01

    Avasimibe is a novel orally bioavailable ACAT inhibitor, currently under clinical development (phase III trials). It was safe when administered to rats, dogs, and humans. In vitro studies in human macrophages demonstrated that avasimibe reduces foam cell formation not only by enhancing free cholesterol efflux, but also by inhibiting the uptake of modified LDL. The concentration-dependent reduction in cellular cholesteryl ester content in these cells was not accompanied by an increase in intracellular free cholesterol, which is in agreement with a good safety profile for avasimibe. In the liver, avasimibe caused a significant reduction in the secretion of apo B and apo B-containing lipoproteins into plasma. Avasimibe induced cholesterol 7alpha-hydroxylase and increased bile acid synthesis in cultured rat hepatocytes, and its administration to rats did not produce an increase in lithogenicity index of the bile. The hypolipidemic efficacy of the compound was demonstrated in cholesterol-fed as well as in non-cholesterol-fed animals. In these models, plasma cholesterol levels were reduced, mainly due to the decrease in the non-HDL cholesterol fraction. Clinical data are scarce, but in a study performed in 130 men and women with combined hyperlipidemia and hypoalphalipoproteinemia, avasimibe, 50-500 mg/day, significantly reduced plasma total triglyceride and VLDL-cholesterol. Although total cholesterol, LDL-cholesterol, and HDL-cholesterol were unchanged, it must be stressed that animal data suggest that avasimibe may have direct antiatherosclerotic activity in addition to its cholesterol-lowering effect. Avasimibe treatment can also contribute to increase plaque stability, as it reduces the accumulation of lipids in the arterial wall, inhibits macrophage infiltration into the media and reduces matrix metalloproteinase expression and activity. Moreover, avasimibe and statins have been shown to have synergistic effects, and the combination therapy may not only inhibit

  6. Synergetic cholesterol-lowering effects of main alkaloids from Rhizoma Coptidis in HepG2 cells and hypercholesterolemia hamsters.

    PubMed

    Kou, Shuming; Han, Bing; Wang, Yue; Huang, Tao; He, Kai; Han, Yulong; Zhou, Xia; Ye, Xiaoli; Li, Xuegang

    2016-04-15

    Hyperlipidemia contributes to the progression of cardiovascular diseases. Main alkaloids from Rhizoma Coptidis including berberine (BBR), coptisine (COP), palmatine (PAL), epiberberine (EPI) and jatrorrhizine (JAT), improved dyslipidemia in hypercholesterolemic hamsters to a different degree. In this study, HepG2 cells and hypercholesterolemic hamsters were used to investigate the synergetic cholesterol-lowering efficacy of these five main alkaloids. The cellular lipid and cholesterol accumulation and in HepG2 cells were evaluated by Oil Red O staining and HPLC analysis. LDL receptor, 3-Hydroxy-3-methylglutaryl CoA reductase (HMGCR) and cholesterol 7-alpha-hydroxylase (CYP7A1) that involving cholesterol metabolism in HepG2 cells were measured by qRT-PCR, western blot and immunofluorescence analysis. The serum profiles including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c), as well as TC and total bile acids (TBA) of feces in hypercholesterolemic hamsters were also measured. As compared to single alkaloids, the combination of five main alkaloids (COM) reduced the lipid and cholesterol accumulation in HepG2 cells more effectively and performed an advantageous effect on controlling TC, TG, LDL-c and HDL-c in hypercholesterolemic hamsters. More effective reduction of TBA and TC levels in feces of hamsters were achieved after the administration of COM. These effects were derived from the up-regulation of LDL receptor and CYP7A1, as well as HMGCR downregulation. Our results demonstrated that COM showed a synergetic cholesterol-lowering efficacy, which was better than single alkaloids and it might be considered as a potential therapy for hypercholesterolemia. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Induction of rat hepatic cytochromes P450 by toxic ingredients in plants: lack of correlation between toxicity and inductive activity.

    PubMed

    Yamada, H; Nakamura, T; Oguri, K

    1998-12-01

    "Animal-Plant Warfare" is one of the hypotheses for the evolution of drug-metabolizing P450s. To address the validity of this hypothesis, we examined the induction of xenobiotic-metabolizing P450s by 12 plant toxins in rats, using hepatic activity for testosterone metabolism as the index. The compounds tested were aconitine, morphine, tubocurarine, physostigmine, pilocarpine, muscarine, cocaine, atropine, amygdalin, digitonin, nicotine and solanine. Drinking water containing a test compound was given to rats for 4 days, and the hepatic activity of testosterone metabolism was determined together with monitoring body weight gain and liver weight as the indices of toxicity. The results showed that while cocaine and nicotine have a minor ability to increase testosterone 16 beta-hydroxylase activity, a marker activity for the CYP2B1 and 2, all other compounds did not have any such effect. No correlation was observed between a change in 16 beta-hydroxylase and toxicity caused by toxins. Therefore, these results did not support the idea that the inducibility of the CYP2B subfamily in animals is acquired through "Animal-Plant Warfare". Several compounds examined here increased or decreased hepatic activities of testosterone 2 alpha-, 6 beta-, 7 alpha- and 16 alpha-hydroxylation and 17-oxidation, indicating a possible effect on the CYP2A, 2C and 3A subfamily. Of these effects, a moderate correlation (r < 0.49) was observed in the changes in the activities of 2 alpha-/16 alpha-hydroxylation and 17-oxidation vs. that in toxicity. It is therefore suggested that inhibition or suppression of the expression of CYP2C11 is one of the mechanisms in the toxicity of plant toxins for rats, although it comes from an examination using limited numbers of compounds.

  8. Digital Gene-Expression Profiling Analysis of the Cholesterol-Lowering Effects of Alfalfa Saponin Extract on Laying Hens

    PubMed Central

    Guo, Rui; Liang, Minggen; Zhu, Xiaoyan; Wang, Chengzhang

    2014-01-01

    Background To prevent cardiovascular disease, people are advised to limit their intake of dietary cholesterol to less than 300 mg/day. Egg consumption has been seriously reduced because of the high levels of cholesterol. The purpose of the present study was to evaluate the cholesterol-lowering effects of alfalfa saponin extract (ASE) in yolk and the molecular mechanisms underlying these effects using digital gene-expression profiling analysis. Liver and ovary tissues were isolated from laying hens fed with ASE for RNA sequencing. Results The cholesterol content of the yolks of eggs from hens fed 120 mg/kg ASE declined considerably on day 60. Other groups (60, 240, 480 mg/kg ASE group) also showed decreases, but they were not significant. Digital gene expression generated over nine million reads per sample, producing expression data for least 12,384 genes. Among these genes, 110 genes showed greater than normal expression in the liver and 107 genes showed greater than normal expression in the ovary. Cholesterol 7 alpha-hydroxylase (Cyp7a1) and apolipoprotein H (Apoh), which act in the synthesis of bile acid and cholesterol efflux, showed more expression in the livers of hens given dietary ASE supplementation. In the ovary, levels of very low density lipoprotein receptor (Vldlr), apolipoprotein B (Apob), apovitellenin 1 (ApovldlII) and vitellogenin (VtgI, VtgII and VtgIII) in ovary decreased with dietary ASE supplementation. Conclusion Transcriptome analysis revealed that the molecular mechanisms underlying the cholesterol-lowering effects of ASE were partially mediated by enhancement of cholesterol efflux in the liver and this reduced of cholesterol deposition in the ovary. PMID:24886784

  9. Validation of a reverse-hybridization StripAssay for the simultaneous analysis of common alpha-thalassemia point mutations and deletions.

    PubMed

    Puehringer, Helene; Najmabadi, Hossein; Law, Hai-Yang; Krugluger, Walter; Viprakasit, Vip; Pissard, Serge; Baysal, Erol; Taher, Ali; Farra, Chantal; Al-Ali, Amein; Al-Ateeq, Suad; Oberkanins, Christian

    2007-01-01

    alpha-Thalassemia is a worldwide disease and considered to be a major public health problem in countries within the so-called thalassemia belt. The complex genetics of alpha-thalassemias requires diagnostic methods with the capacity to screen rapidly and accurately for common causative mutations. We developed and validated a reverse-hybridization assay (Alpha-Globin StripAssay) for the rapid and simultaneous detection of 21 alpha-globin mutations: two single gene deletions (-alpha(3.7); -alpha(4.2)), five double gene deletions [--(MED); --(SEA); --(THAI); --(FIL); -(alpha)(20.5)], alpha alpha alpha(anti-3.7) gene triplication, two point mutations in the alpha1 gene (cd 14 G>A; Hb Adana) and 11 point mutations in the alpha2 gene (initiation cd T>C; cd 19 -G; IVS1 -5nt; cd 59 G>A; Hb Quong Sze; Hb Constant Spring; Hb Icaria; Hb Pakse; Hb Koya Dora; polyA-1; polyA-2). Reliable genotyping of recombinant mutant clones and reference DNA samples was achieved by means of two corresponding test strips presenting parallel arrays of allele-specific oligonucleotides. The entire procedure from blood sampling to the identification of mutations required less than 6 h, and hybridization/detection was manual or automated. The diagnostic potential of this Alpha-Globin StripAssay was carefully evaluated on 272 pre-typed samples in a multicenter validation study. In 96.14% of the cases, StripAssay typing was completely concordant with the reference methods. The Alpha-Globin StripAssay proved to be a fast, easy-to-perform and reliable screening method to identify >90% of alpha-globin mutations in endemic areas worldwide.

  10. Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors.

    PubMed

    Sharma, S D; Jiang, J; Hadley, M E; Bentley, D L; Hruby, V J

    1996-11-26

    We developed two solid-phase reagent systems for microscopic visualization and characterization of melanocyte-stimulating hormone (MSH) receptors of melanoma cells. Multiple copies of [Nle-4,D-Phe-7]-alpha-MSH, a potent analog of alpha-MSH, were conjugated to microspheres (latex beads) or macrospheres (polyamide beads) through a thioether or disulfide bond. Binding between the beads and mouse and human melanoma cells was examined by scanning electron microscopy and by light microscopy. Each mouse and human melanoma cell (of all cell lines) evinced binding to the beads. Binding of the melanotropin conjugates was not restricted to any one phase of the cell cycle. Specificity of binding was demonstrated by several studies. Negative controls included cell types of nonmelanocyte origin (e.g., mammary cancer cells) and beads that lacked the melanotropic ligand or had other attached ligands. Beads with a disulfide-linked melanotropin analog served as a direct control. Treatment of these beads with DTT during or before incubation of the beads with melanoma cells (resulting in release of the MSH analog from the beads) eliminated binding of the beads to melanoma cells. Binding interactions between melanoma cells and melanotropin-bound beads also could be abolished by prior incubation with unconjugated MSH analog. During these experiments, certain membrane receptor-hormone associated phenomena, such as capping (aggregation) of the receptor-ligand complex, also were observed. These results provide visual evidence that MSH receptors are a property common to melanoma cells. Normal human epidermal melanocytes and keratinocytes were also shown to express melanotropin receptors by the same criteria established for melanoma cells.

  11. FXR-mediated down-regulation of CYP7A1 dominates LXRalpha in long-term cholesterol-fed NZW rabbits.

    PubMed

    Xu, Guorong; Li, Hai; Pan, Lu-Xing; Shang, Quan; Honda, Akira; Ananthanarayanan, M; Erickson, Sandra K; Shneider, Benjamin L; Shefer, Sarah; Bollineni, Jaya; Forman, Barry M; Matsuzaki, Yasushi; Suchy, Frederick J; Tint, G Stephen; Salen, Gerald

    2003-10-01

    We investigated how cholesterol feeding regulates cholesterol 7alpha-hydroxylase (CYP7A1) via the nuclear receptors farnesoid X receptor (FXR) and liver X receptor alpha (LXRalpha) in New Zealand white rabbits. After 1 day of 2% cholesterol feeding, when the bile acid pool size had not expanded, mRNA levels of the FXR target genes short-heterodimer partner (SHP) and sterol 12alpha-hydroxylase (CYP8B) were unchanged, indicating that FXR activation remained constant. In contrast, the mRNA levels of the LXRalpha target genes ATP binding cassette transporter A1 (ABCA1) and cholesteryl ester transfer protein (CETP) increased 5-fold and 2.3-fold, respectively, associated with significant increases in hepatic concentrations of oxysterols. Activity and mRNA levels of CYP7A1 increased 2.4 times and 2.2 times, respectively. After 10 days of cholesterol feeding, the bile acid pool size increased nearly 2-fold. SHP mRNA levels increased 4.1-fold while CYP8B declined 64%. ABCA1 mRNA rose 8-fold and CETP mRNA remained elevated. Activity and mRNA of CYP7A1 decreased 60% and 90%, respectively. Feeding cholesterol for 1 day did not enlarge the ligand pool size or change FXR activation, while LXRalpha was activated highly secondary to increased hepatic oxysterols. As a result, CYP7A1 was up-regulated. After 10 days of cholesterol feeding, the bile acid (FXR ligand) pool size increased, which activated FXR and inhibited CYP7A1 despite continued activation of LXRalpha. Thus, in rabbits, when FXR and LXRalpha are activated simultaneously, the inhibitory effect of FXR overrides the stimulatory effect of LXRalpha to suppress CYP7A1 mRNA expression.

  12. Interactions between common genetic polymorphisms in ABCG5/G8 and CYP7A1 on LDL cholesterol-lowering response to atorvastatin.

    PubMed

    Kajinami, Kouji; Brousseau, Margaret E; Ordovas, Jose M; Schaefer, Ernst J

    2004-08-01

    Cholesterol excretion by ATP binding cassette transporters G5 and G8 (ABCG5/G8) and bile acid biosynthesis by cholesterol 7alpha-hydroxylase (CYP7A1) are major pathways for the removal of cholesterol into bile. To investigate the interactions between common polymorphisms in ABCG5/G8 and CYP7A1 and statin response, we examined the relationships between five non-synonymous polymorphisms in ABCG5/G8 (Q604E, D19H, Y54C, T400K, and A632V) and a promoter variant in CYP7A1 (A-204C) in 337 hypercholesterolemic patients treated with atorvastatin 10mg. The ABCG8 H19 allele was significantly associated with a greater LDL cholesterol reduction relative to the wild type D19 allele (39.6% versus 36.6%, P = 0.043). This difference was enhanced in non-carriers of the CYP7A1 promoter polymorphism (42.7% versus 38.2%, P = 0.048), and was diminished in accordance with the number of CYP7A1 variant alleles (1.8% in heterozygotes and 0.2% in homozygotes). Combination analysis of these polymorphisms explained a greater percentage of LDL cholesterol response variation (8.5% difference across subgroups) than did single polymorphism analysis (4.2% in CYP7A1 and 3.0% in ABCG8 D19H). The other ABCG5/G8 polymorphisms did not show any significant interactions with the CYP7A1 polymorphism. We conclude that the ABCG8 H19 and CYP7A1 C-204 alleles appear to interact in a dose-dependent manner on atorvastatin response.

  13. Enhancing effect of taurine on CYP7A1 mRNA expression in Hep G2 cells.

    PubMed

    Lam, N V; Chen, W; Suruga, K; Nishimura, N; Goda, T; Yokogoshi, H

    2006-02-01

    Taurine has been reported to enhance cholesterol 7alpha-hydroxylase (CYP7A1) mRNA expression in animal models. However, no in vitro studies of this effect have been reported. The Hep G2 human hepatoma cell line has been recognized as a good model for studying the regulation of human CYP7A1. This work characterizes the effects of taurine on CYP7A1 mRNA levels of Hep G2 cells in a dose- and time-dependent manner. In the dose-dependent experiment, Hep G2 cells were treated with 0, 2, 10 or 20 mM taurine in the presence or absence of cholesterol 0.2 mM for 48 h. In the time-dependent experiment, Hep G2 cells were treated with 0 or 20 mM taurine for 4, 24 and 48 h with and without cholesterol 0.2 mM. Our data revealed that taurine showed time- and dose-response effects on CYP7A1 mRNA levels in Hep G2 cells. However, glycine - a structural analogue of taurine - did not have an effect on CYP7A1 gene expression. These results show that, in agreement to previous studies on animal models, taurine induces the mRNA levels of CYP7A1 in Hep G2 cells, which could enhance cholesterol conversion into bile acids. Also, Hep G2 cell line may be an appropriate model to study the effects of taurine on human cholesterol metabolism.

  14. Significance and mechanism of CYP7a1 gene regulation during the acute phase of liver regeneration.

    PubMed

    Zhang, Lisheng; Huang, Xiongfei; Meng, Zhipeng; Dong, Bingning; Shiah, Steven; Moore, David D; Huang, Wendong

    2009-02-01

    Cholesterol 7alpha-hydroxylase (CYP7a1) is the rate-limiting enzyme in the classic pathway of bile acid synthesis. Expression of CYP7a1 is regulated by a negative feedback pathway of bile acid signaling. Previous studies have suggested that bile acid signaling is also required for normal liver regeneration, and CYP7a1 expression is strongly repressed after 70% partial hepatectomy (PH). Both the effect of CYP7a1 suppression on liver regrowth and the mechanism by which 70% PH suppresses CYP7a1 expression are unknown. Here we show that liver-specific overexpression of an exogenous CYP7a1 gene impaired liver regeneration after 70% PH, which was accompanied by increased hepatocyte apoptosis and liver injury. CYP7a1 expression was initially suppressed after 70% PH in an farnesoid X receptor/ small heterodimer partner-independent manner; however, both farnesoid X receptor and small heterodimer partner were required to regulate CYP7a1 expression at the later stage of liver regeneration. c-Jun N-terminus kinase and hepatocyte growth factor signaling pathways are activated during the acute phase of liver regeneration. We determined that hepatocyte growth factor and c-Jun N-terminus kinase pathways were involved in the suppressing of the CYP7a1 expression in the acute phase of live regeneration. Taken together, our results provide the significance that CYP7a1 suppression is required for liver protection after 70% PH and there are two distinct phases of CYP7a1 gene regulation during liver regeneration.

  15. In vivo regulation of murine CYP7A1 by HNF-6: a novel mechanism for diminished CYP7A1 expression in biliary obstruction.

    PubMed

    Wang, Minhua; Tan, Yongjun; Costa, Robert H; Holterman, Ai-Xuan L

    2004-09-01

    Disruption of the enterohepatic bile acid circulation during biliary tract obstruction leads to profound perturbation of the cholesterol and bile acid metabolic pathways. Several families of nuclear receptor proteins have been shown to modulate this critical process by regulating hepatic cholesterol catabolism and bile acid synthesis through the transcriptional control of cholesterol 7-alpha hydroxylase (CYP7A1). Hepatocyte nuclear factor (HNF) 6 (also known as OC-1) is a member of the ONECUT family of transcription factors that activate numerous hepatic target genes essential to liver function. We have previously shown that hepatic expression of mouse HNF-6 messenger RNA (mRNA) and protein significantly decrease following bile duct ligation. Because CYP7A1 contains potential HNF-6 binding sites in its promoter region, we tested the hypothesis that HNF-6 transcriptionally regulates CYP7A1. Following bile duct ligation, we demonstrated that diminished HNF-6 mRNA levels correlate with a reduction in CYP7A1 mRNA expression. Increasing hepatic levels of HNF-6 either by infection with recombinant adenovirus vector expressing HNF-6 cDNA by growth hormone treatment leads to an induction of CYP7A1 mRNA. To directly evaluate if HNF-6 is a transcriptional activator for CYP7A1, we used deletional and mutational analyses of CYP7A1 promoter sequences and defined sequences -206/-194 to be critical for CYP7A1 transcriptional stimulation by HNF-6 in cotransfection assays. In conclusion, the HNF-6 protein is a component of the complex network of hepatic transcription factors that regulates the expression of hepatic genes essential for bile acid homeostasis and cholesterol/lipid metabolism in normal and pathological conditions.

  16. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha activation of CYP7A1 during food restriction and diabetes is still inhibited by small heterodimer partner.

    PubMed

    Shin, Dong-Ju; Osborne, Timothy F

    2008-05-30

    Cholesterol 7alpha-hydroxylase (CYP7A1) catalyzes the rate-limiting step in the classic pathway of hepatic bile acid biosynthesis from cholesterol. During fasting and in type I diabetes, elevated levels of peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1alpha) induce expression of the Cyp7A1 gene and overexpression of PGC-1alpha in hepatoma cells stimulates bile acid synthesis. Using Ad-PGC-1alpha-RNA interference to induce acute disruption of PGC-1alpha in mice, here we show that PGC-1alpha is necessary for fasting-mediated induction of CYP7A1. Co-immunoprecipitation and promoter activation studies reveal that the induction of CYP7A1 is mediated by direct interaction between PGC-1alpha and the AF2 domain of liver receptor homolog-1 (LRH-1). In contrast, the very similar PGC-1beta could not substitute for PGC-1alpha. We also show that transactivation of PGC-1alpha and LRH-1 is repressed by the small heterodimer partner (SHP). Treatment of mice with GW4064, a synthetic agonist for farnesoid X receptor, induced SHP expression and decreased both the recruitment of PGC-1alpha to the Cyp7A1 promoter and the fasting-induced expression of CYP7A1 mRNA. These data suggest that PGC-1alpha is an important co-activator for LRH-1 and that SHP targets the interaction between LRH-1 and PGC-1alpha to inhibit CYP7A1 expression. Overall, these studies provide further evidence for the important role of PGC-1alpha in bile acid homeostasis and suggest that pharmacological targeting of farnesoid X receptor in vivo can be used to reverse the increase in CYP7A1 associated with adverse metabolic conditions.

  17. Transgenic expression of CYP7A1 in LDL receptor-deficient mice blocks diet-induced hypercholesterolemia.

    PubMed

    Ratliff, Eric P; Gutierrez, Alejandra; Davis, Roger A

    2006-07-01

    Constitutive expression of a cholesterol-7alpha-hydroxylase (CYP7A1) transgene in LDL receptor-deficient mice blocked the ability of a cholesterol-enriched diet to increase plasma levels of apolipoprotein B-containing lipoproteins. LDL receptor-deficient mice expressing the CYP7A1 transgene exhibited complete resistance to diet-induced hypercholesterolemia and to the accumulation of cholesterol in the liver. Hepatic mRNA expression of liver X receptor-inducible ABCG5 and ABCG8 was decreased in CYP7A1 transgenic, LDL receptor-deficient mice fed a cholesterol-enriched diet. Thus, increased biliary cholesterol excretion could not account for the maintenance of cholesterol homeostasis. CYP7A1 transgenic, LDL receptor-deficient mice fed the cholesterol-enriched diet exhibited decreased jejunal Niemann-Pick C1-Like 1 protein (NPC1L1) mRNA expression, an important mediator of intestinal cholesterol absorption. A taurocholate-enriched diet also decreased NPC1L1 mRNA expression in a farnesoid X receptor-independent manner. Reduced expression of NPC1L1 mRNA was associated with decreased cholesterol absorption ( approximately 20%; P < 0.05) exhibited by CYP7A1 transgenic LDL receptor-deficient mice fed the cholesterol-enriched diet. The combined data show that enhanced expression of CYP7A1 is an effective means to prevent the accumulation of cholesterol in the liver and of atherogenic apolipoprotein B-containing lipoproteins in plasma.

  18. NO-1886 suppresses diet-induced insulin resistance and cholesterol accumulation through STAT5-dependent upregulation of IGF1 and CYP7A1.

    PubMed

    Li, Qinkai; Yin, Weidong; Cai, Manbo; Liu, Yi; Hou, Hongjie; Shen, Qingyun; Zhang, Chi; Xiao, Junxia; Hu, Xiaobo; Wu, Qishisan; Funaki, Makoto; Nakaya, Yutaka

    2010-01-01

    Insulin resistance and dyslipidemia are both considered to be risk factors for metabolic syndrome. Low levels of IGF1 are associated with insulin resistance. Elevation of low-density lipoprotein cholesterol (LDL-C) concomitant with depression of high-density lipoprotein cholesterol (HDL-C) increase the risk of obesity and type 2 diabetes mellitus (T2DM). Liver secretes IGF1 and catabolizes cholesterol regulated by the rate-limiting enzyme of bile acid synthesis from cholesterol 7alpha-hydroxylase (CYP7A1). NO-1886, a chemically synthesized lipoprotein lipase activator, suppresses diet-induced insulin resistance with the improvement of HDL-C. The goal of the present study is to evaluate whether NO-1886 upregulates IGF1 and CYP7A1 to benefit glucose and cholesterol metabolism. By using human hepatoma cell lines (HepG2 cells) as an in vitro model, we found that NO-1886 promoted IGF1 secretion and CYP7A1 expression through the activation of signal transducer and activator of transcription 5 (STAT5). Pretreatment of cells with AG 490, the inhibitor of STAT pathway, completely abolished NO-1886-induced IGF1 secretion and CYP7A1 expression. Studies performed in Chinese Bama minipigs pointed out an augmentation of plasma IGF1 elicited by a single dose administration of NO-1886. Long-term supplementation with NO-1886 recovered hyperinsulinemia and low plasma levels of IGF1 suppressed LDL-C and facilitated reverse cholesterol transport by decreasing hepatic cholesterol accumulation through increasing CYP7A1 expression in high-fat/high-sucrose/high-cholesterol diet minipigs. These findings indicate that NO-1886 upregulates IGF1 secretion and CYP7A1 expression to improve insulin resistance and hepatic cholesterol accumulation, which may represent an alternative therapeutic avenue of NO-1886 for T2DM and metabolic syndrome.

  19. PGC-1alpha activates CYP7A1 and bile acid biosynthesis.

    PubMed

    Shin, Dong-Ju; Campos, Jose A; Gil, Gregorio; Osborne, Timothy F

    2003-12-12

    Cholesterol 7-alpha-hydroxylase (CYP7A1) is the key enzyme that commits cholesterol to the neutral bile acid biosynthesis pathway and is highly regulated. In the current studies, we have uncovered a role for the transcriptional co-activator PGC-1alpha in CYP7A1 gene transcription. PGC-1alpha plays a vital role in adaptive thermogenesis in brown adipose tissue and stimulates genes important to mitochondrial function and oxidative metabolism. It is also involved in the activation of hepatic gluconeogenesic gene expression during fasting. Because the mRNA for CYP7A1 was also induced in mouse liver by fasting, we reasoned that PGC-1alpha might be an important co-activator for CYP7A1. Here we show that PGC-1alpha and CYP7A1 are also co-induced in livers of mice in response to streptozotocin induced diabetes. Additionally, infection of cultured HepG2 cells with a recombinant adenovirus expressing PGC-1alpha directly activates CYP7A1 gene expression and increases bile acid biosynthesis as well. Furthermore, we show that PGC-1alpha activates the CYP7A1 promoter directly in transient transfection assays in cultured cells. Thus, PGC-1alpha is a key activator of CYP7A1 and bile acid biosynthesis and is likely responsible for the fasting and diabetes dependent induction of CYP7A1. PGC-1alpha has already been shown to be a critical activator of several other oxidative processes including adaptive thermogenesis and fatty acid oxidation. Our studies provide further evidence of the fundamental role played by PGC-1alpha in oxidative metabolism and define PGC-1alpha as a link between diabetes and bile acid metabolism.

  20. Preparation of (3H)collagen for studies of the biologic fate of xenogenic collagen implants in vivo

    SciTech Connect

    McPherson, J.M.; Sawamura, S.J.; Conti, A.

    1986-06-01

    Reduction of a commercially available, pepsin-solubilized, bovine dermal collagen (Vitrogen 100) with sodium (3H)borohydride provided radiolabeled collagen preparations with specific activities ranging from 7.1-12.0 muCi/mg collagen. These specific activities were 2-3 times greater than those obtained by reduction of intact rat tail tendon collagen under similar conditions. The alpha, beta, and higher aggregate components of type I collagen were radiolabeled as well as the alpha component of a small amount of type III collagen present in the samples. Fractionation of cyanogen bromide peptides showed that alpha 1(I)CB7, alpha 1(I)CB8, and alpha 2(I)CB3,5 were the predominant peptides labeled by this procedure. Amino acid analysis indicated that the majority of the radioactivity was in reducible cross-links, precursors of these cross-links, and in hexosyllysine residues. Reconstitution experiments comparing this radiolabeled collagen with nonlabeled collagen showed them to be indistinguishable. Bacterial collagenase digestion of this reconstituted fibrillar collagen in both a lightly cross-linked (glutaraldehyde 0.0075%) and noncross-linked form provided evidence that digestion of labeled and nonlabeled collagens proceeded at similar rates. Thus, labeling did not change the properties of the collagen. Cross-linking made the preparation refractory to proteolytic degradation. Injection of fibrillar collagen preparations, spiked with radiolabeled collagen, into the guinea pig dermis followed by quantitation of the amount of radioactivity recovered from implant sites as a function of time, indicated that the lightly cross-linked samples also were more resistant to degradation in vivo than the noncross-linked preparation. The half-life of noncross-linked collagen was about 4 days while that of the cross-linked collagen was about 25 days.

  1. Integrin ligands mobilize Ca2+ from ryanodine receptor-gated stores and lysosome-related acidic organelles in pulmonary arterial smooth muscle cells.

    PubMed

    Umesh, Anita; Thompson, Michael A; Chini, Eduardo N; Yip, Kay-Pong; Sham, James S K

    2006-11-10

    Extracellular matrix (ECM) protein receptors, or integrins, participate in vascular remodeling and the systemic myogenic response. Synthetic ligands and ECM fragments regulate the vascular smooth muscle cell contractile state by altering intracellular Ca2+ levels ([Ca2+]i). Information on the Ca2+ effect of integrins in vascular smooth muscle cells is limited, but nonexistent in pulmonary arterial smooth muscle cells (PASMCs). We therefore characterized integrin expression in endothelium-denuded pulmonary arteries, and explored [Ca2+]i mobilization pathways induced by soluble ligands in rat PASMCs. Reverse transcriptase-PCR showed mRNA expression of integrins alpha1, alpha2, alpha3, alpha4, alpha5, alpha7, alpha8, alpha(v), beta1, beta3, and beta4, and immunoblots of alpha5, alpha(v), beta1, and beta3 confirmed protein expression. Exposure of PASMCs to integrin-binding peptides (0.5 mM) containing the arginine-glycine-aspartate (RGD) motif elicited [Ca2+]i responses with an order of potency of GRGDNP > GRGDSP > GRGDTP = cyclo-RGD. Pharmacological analysis revealed that the GRGDSP-induced Ca2+ response was unrelated to Ca2+ influx and the inositol triphosphate receptor-gated Ca2+ store, but partially blocked by ryanodine or inhibition of lysosome-related acidic organelles with bafilomycin A1. Simultaneous inhibition of both pathways was necessary to abolish the response. GRGDSP treatment increased cyclic ADP-ribose, the endogenous activator of ryanodine receptors, by 70%. GRGDSP also rapidly reduced Lysotracker Red accumulation, confirming direct modulation of acidic organelles. These data are the first demonstration of integrin-mediated Ca2+ regulation in PASMCs. The presence of an array of integrins, and activation of ryanodine-sensitive Ca2+ stores and lysosome-like organelles by GRGDSP suggest important roles for integrin-dependent Ca2+ signaling in regulating PASMC function.

  2. Presence of phytosterol oxides in crude vegetable oils and their fate during refining.

    PubMed

    Bortolomeazzi, Renzo; Cordaro, Francesca; Pizzale, Lorena; Conte, Lanfranco S

    2003-04-09

    The content of phytosterol oxidation products was determined in samples of crude vegetable oils: peanut, sunflower, maize, palm nut, and lampante olive oils that were intended for refining and not for direct consumption. The 7 alpha- and 7 beta-hydroxy derivatives of beta-sitosterol, stigmasterol, and campesterol and the 7-keto-beta-sitosterol were the principal phytosterol oxides found in almost all of the oils analyzed. In some oils, the epoxy and dihydroxy derivatives of beta-sitosterol were also found at very low levels. The highest total concentrations of phytosterol oxides, ranging from 4.5 to 67.5 and from 4.1 to 60.1 ppm, were found in sunflower and maize oils, respectively. Lower concentrations were present in the peanut oils, 2.7-9.6 ppm, and in the palm nut oil, 5.5 ppm, whereas in the lampante olive oils, only three samples of the six analyzed contained a low concentration (1.5-2.5 ppm) of oxyphytosterols. No detectable levels of phytosterol oxides were found in the samples of palm and coconut oils. Bleaching experiments were carried out on a sample of sunflower oil at 80 degrees C for 1 h with 1 and 2% of both acidic and neutral earths. The bleaching caused a reduction of the hydroxyphytosterol with partial formation of steroidal hydrocarbons with three double bonds in the ring system at the 2-, 4-, and 6-positions (steratrienes). The same sunflower oil was deodorized at 180 degrees C under vacuum for 1 h, and no dehydration products were formed with a complete recovery of the hydroxyphytosterols. A bleaching test with acidic earths was carried out also with an extra virgin olive oil fortified with 7-keto-cholesterol, dihydroxycholesterol, and alpha-epoxy-cholesterol. There was no formation of steratrienes from these compounds, but dihydroxycholesterol underwent considerable decomposition and alpha-epoxycholesterol underwent ring opening with formation of the dihydroxy derivative, whereas 7-ketocholesterol was rather stable

  3. Dietary fish protein alters blood lipid concentrations and hepatic genes involved in cholesterol homeostasis in the rat model.

    PubMed

    Shukla, Anjali; Bettzieche, Anja; Hirche, Frank; Brandsch, Corinna; Stangl, Gabriele I; Eder, Klaus

    2006-10-01

    It is known that various dietary plant proteins are capable of influencing the lipid metabolism of human subjects and animals when compared with casein. Less, however, is known about the effects of fish protein on the cholesterol and triacylglycerol metabolism. Therefore, two experiments were conducted in which rats were fed diets containing 200 g of either fish protein, prepared from Alaska pollack fillets, or casein, which served as control, per kilogram, over 20 and 22 d, respectively. As parameters of lipid metabolism, the concentrations of cholesterol and triacylglycerols in the plasma and liver, the faecal excretion of bile acids and the hepatic expression of genes encoding proteins involved in lipid homeostasis were determined. In both experiments, rats fed fish protein had higher concentrations of cholesteryl esters in the liver, a lower concentration of cholesterol in the HDL fraction (rho > 1.063 kg/l) and lower plasma triacylglycerol concentrations than rats fed casein (P < 0.05). The gene expression analysis performed in experiment 2 showed that rats fed fish protein had higher relative mRNA concentrations of sterol regulatory element-binding protein (SREBP)-2, 3-hydroxy-3-methylglutaryl coenzyme A reductase, LDL receptor, apo AI, scavenger receptor B1 and lecithin-cholesterol-acyltransferase in their liver than did rats fed casein (P < 0.05). The faecal excretion of bile acids and the mRNA concentrations of cholesterol 7alpha-hydroxylase, SREBP-1c and corresponding target genes were not altered. These findings show that fish protein had multiple effects on plasma and liver lipids that were at least in part caused by an altered expression of the hepatic genes involved in lipid homeostasis.

  4. Candidate genes involved in cardiovascular risk factors by a family-based association study on the island of Kosrae, Federated States of Micronesia.

    PubMed

    Han, Zhihua; Heath, Simon C; Shmulewitz, Dvora; Li, Wentian; Auerbach, Steve B; Blundell, Maude L; Lehner, Thomas; Ott, Jurg; Stoffel, Markus; Friedman, Jeffrey M; Breslow, Jan L

    2002-07-01

    Altered plasma levels of lipids and lipoproteins, obesity, hypertension, and diabetes are major risk factors for atherosclerotic cardiovascular disease. To identify genes that affect these traits and disorders, we looked for association between markers in candidate genes (apolipoprotein AII (apo AII), apolipoprotein AI-CIII-AIV gene cluster (apo AI-CIII-AIV), apolipoprotein E (apo E), cholesteryl ester transfer protein (CETP), cholesterol 7alpha-hydroxylase (CYP7a), hepatic lipase (HL), and microsomal triglyceride transfer protein (MTP)) and known risk factors (triglycerides (Tg), total cholesterol (TC), apolipoprotein AI (apo AI), apolipoprotein AII (apo AII), apolipoprotein B (apo B), body mass index (BMI), blood pressure (BP), leptin, and fasting blood sugar (FBS) levels.) A total of 1,102 individuals from the Pacific island of Kosrae were genotyped for the following markers: Apo AII/MspI, Apo CIII/SstI, Apo AI/XmnI, Apo E/HhaI, CETP/TaqIB, CYP7a/BsaI, HL/DraI, and MTP/HhpI. After testing for population stratification, family-based association analysis was carried out. Novel associations found were: 1) the apo AII/MspI with apo AI and BP levels, 2) the CYP7a/BsaI with apo AI and BMI levels. We also confirmed the following associations: 1) the apo AII/MspI with Tg level; 2) the apo CIII/SstI with Tg, TC, and apo B levels; 3) the Apo E/HhaI E2, E3, and E4 alleles with TC, apo AI, and apo B levels; and 4) the CETP/TaqIB with apo AI level. We further confirmed the connection between the apo AII gene and Tg level by a nonparametric linkage analysis. We therefore conclude that many of these candidate genes may play a significant role in susceptibility to heart disease.

  5. The Mechanisms Underlying the Hypolipidaemic Effects of Grifola frondosa in the Liver of Rats

    PubMed Central

    Ding, Yinrun; Xiao, Chun; Wu, Qingping; Xie, Yizhen; Li, Xiangmin; Hu, Huiping; Li, Liangqiu

    2016-01-01

    The present study investigated the hypolipidaemic effects of Grifola frondosa and its regulation mechanism involved in lipid metabolism in liver of rats fed a high-cholesterol diet. The body weights and serum lipid levels of control rats, of hyperlipidaemic rats, and of hyperlipidaemic rats treated with oral G. frondosa were determined. mRNA expression and concentration of key lipid metabolism enzymes were investigated. Serum cholesterol, triacylglycerol, and low-density lipoprotein cholesterol levels were markedly decreased in hyperlipidaemic rats treated with G. frondosa compared with untreated hyperlipidaemic rats. mRNA expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), acyl-coenzyme A: cholesterol acyltransferase (ACAT2), apolipoprotein B (ApoB), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC1) were significantly down-regulated, while expression of cholesterol 7-alpha-hydroxylase (CYP7A1) was significantly up-regulated in the livers of treated rats compared with untreated hyperlipidaemic rats. The concentrations of these enzymes also paralleled the observed changes in mRNA expression. Two-dimensional polyacrylamide gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) were used to identify 20 proteins differentially expressed in livers of rats treated with G. frondosa compared with untreated hyperlipidemic rats. Of these 20 proteins, seven proteins were down-regulated, and 13 proteins were up-regulated. These findings indicate that the hypolipidaemic effects of G. frondosa reflected its modulation of key enzymes involved in cholesterol and triacylglycerol biosynthesis, absorption, and catabolic pathways. G. frondosa may exert anti-atherosclerotic effects by inhibiting LDL oxidation through down-regulation and up-regulating proteins expression in the liver of rats. Therefore, G. frondosa may produce both hypolipidaemic and anti-atherosclerotic effects, and potentially

  6. Regulation of prolactin synthesis in vitro by estrogenic and antiestrogenic derivatives of estradiol and estrone.

    PubMed

    Jordan, V C; Koch, R

    1989-04-01

    The estrogenic and antiestrogenic activities of derivatives of estradiol and estrone were determined in vitro using the ability of primary cultures of immature rat pituitary cells to synthesize PRL. Estradiol derivatives were the most potent estrogens in the assay. Large ethinyl substitutions in the 17 alpha position generally caused a decrease in estrogenic potency (up to 1000-fold). The 3 phenolic hydroxyl was important, but not essential, for the estrogenic activity of the estradiol molecule. Estratriene was approximately 1000 times less potent than estradiol. However, significant estrogenic activity was observed with the compound anordin (EC50, 8 x 10(-9) M), which could potentially be converted to a dihydroxylated derivative but without an aromatic A ring. Similarly, the steroid androst-5-ene-3,17-diol was weakly estrogenic (EC50, 3 x 10(-8) M). Steriods with a ketone in the A and D rings were generally inactive as estrogens and antiestrogens. Estradiol derivatives with 17 beta amines were only weak estrogens. Estrone derivatives were less active than the corresponding estradiol derivatives. 4-Nitromethoxyestrone exhibited weak antiestrogenic properties; however, 4-nitroestrone and methoxyestrone were both estrogens. The reason for the antiestrogenic properties of 4-nitromethoxyestrone is obscure, as the compound does not have structural features similar to those of known nonsteroidal antiestrogens. Minor alterations to the estradiol molecule at the 11 beta (OH) or 6 (ketone) position had little effect on estrogenic potency; however, large substitutions at the 11 beta (RU 39,411) or 7 alpha (ICI 164384) position produced antiestrogenic compounds. RU 39,411 was approximately 10 times more active as an antiestrogen than 4-hydroxytamoxifen, whereas ICI 164,384 was approximately 10 times less active than 4-hydroxytamoxifen. A series of hypothetical models is proposed that could explain the antiestrogenic properties of RU 39,411 and ICI 164,384 by an interaction

  7. Chemical and functional identification and characterization of novel sulfated alpha-conotoxins from the cone snail Conus anemone.

    PubMed

    Loughnan, Marion L; Nicke, Annette; Jones, Alun; Adams, David J; Alewood, Paul F; Lewis, Richard J

    2004-02-26

    An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the alpha-conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3beta2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr16]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC50 0.6 nM) and alpha7 (IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC50 367 nM) than at the alpha3beta2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.

  8. Melanogenesis inhibitory, anti-inflammatory, and chemopreventive effects of limonoids from the seeds of Azadirachta indicia A. Juss. (neem).

    PubMed

    Akihisa, Toshihiro; Noto, Taisuke; Takahashi, Akitomo; Fujita, Yukiko; Banno, Norihiro; Tokuda, Harukuni; Koike, Kazuo; Suzuki, Takashi; Yasukawa, Ken; Kimura, Yumiko

    2009-01-01

    Thirty-one nortriterpenoids, including 28 limonoids (1-28) and 3 degraded limonoids (29-31), and one diterpenoid (32), were isolated from the seed extract of Azadirachta indica (neem). Among these, six were new compounds and their structures were established to be 15-hydroxyazadiradione (3), 7-benzoyl-17-hydroxynimbocinol (5), 23-deoxyazadironolide (12), limocin E (13), 23-epilimocin E (14), and 7alpha-acetoxy-3-oxoisocopala-1,13-dien-15-oic acid (32). Upon evaluation of compounds 1-32 on the melanogenesis in the B16 melanoma cells, five compounds, 20, 26, 27, 29, and 31, exhibited marked inhibitory effect (74-91% reduction of melanin content at 25 microg/mL) with no or almost no toxicity to the cells. Seven compounds, 1, 6, 9, 10, 18, 20, and 26, on evaluation for their inhibitory effect against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation (1 microg/ear) in mice, exhibited, except for compound 26, marked anti-inflammatory activity (ID(50) values 0.09-0.26 mg/ear). In addition, all of the 32 compounds exhibited moderate or potent inhibitory effects (IC(50) values of 230-501 mol ratio/32 pmol TPA) against the Epstein-Barr virus early antigen (EBV-EA) activation induced by TPA. Furthermore, on evaluation of azadirachtin B (21) for its anti-tumor-initiating activity on the two-stage carcinogenesis of mouse skin tumor induced by peroxynitrite (ONOO-; PN) as an initiator and TPA as a promoter, this exhibited marked inhibitory activity.

  9. Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment

    PubMed Central

    1996-01-01

    Stable attachment of external epithelia to the basement membrane and underlying stroma is mediated by transmembrane proteins such as the integrin alpha6beta4 and bullous pemphigoid antigen 2 within the hemidesmosomes along the basolateral surface of the epithelial cell and their ligands that include a specialized subfamily of laminins. The laminin 5 molecule (previously termed kalinin/nicein/epiligrin) is a member of this epithelial-specific subfamily. Laminin 5 chains are not only considerably truncated within domains III-VI, but are also extensively proteolytically processed in vitro and in vivo. As a result, the domains expected to be required for the association of laminins with other basement membrane components are lacking in the mature laminin 5 molecule. Therefore, the tight binding of laminin 5 to the basement membrane may occur by a unique mechanism. To examine laminin 5 in tissue, we chose human amnion as the source, because of its availability and the similarity of the amniotic epithelial basement membrane with that of skin. We isolated the laminin 5 contained within the basement membrane of human amnion. In addition to monomeric laminin 5, we find that much of the laminin 5 isolated is covalently adducted with laminin 6 (alpha3beta1gamma1) and a novel laminin isotype we have termed laminin 7 (alpha3beta2gamma1). We propose that the association between laminin 5 and laminins 6 and 7 is a mechanism used in amnion to allow stable association of laminin 5 with the basement membrane. The beta2 chain is seen at the human amniotic epithelial-stromal interface and at the dermal-epidermal junction of fetal and adult bovine skin by immunofluorescence, but is not present, or only weakly present, in neonatal human skin. PMID:8601594

  10. Down-regulation of bile acid synthesis and a metabolic co-activator under hypoxic conditions - implications in obstructive sleep apnea.

    PubMed

    Johnson, Paul R

    2008-10-01

    Obstructive sleep apnea is characterized by episodes of intermittent hypoxia that occur while the afflicted person sleeps, and is believed to affect and have a negative impact on many people worldwide. Both observational evidence in obstructive sleep apnea patients and direct studies in mice mimicking the disease strongly support an increased risk for atherosclerosis in these groups. Studies to date on atherosclerosis in obstructive sleep apnea have been focused on cholesterol synthesis. However, dysregulation of bile acid synthesis, which is an important means of cholesterol removal, has not been considered in any sleep disorder model to date. While the relationship between hypoxia and bile acid regulation has been explored in a variety of models, no unifying theory currently ties in the pathologic effect of intermittent hypoxia on bile acid regulation in humans or animals. The goal of this article is to propose the role intermittent hypoxia plays in disrupting bile acid synthesis in patients with obstructive sleep apnea, and its metabolic consequences. Reviewed and discussed are the complex interactions of several key molecular players known to be involved in metabolism with emphasis on the principle bile acid synthesis enzyme, cholesterol 7-alpha-hydroxylase, which is proposed to have reduced activity under hypoxic conditions. Furthermore, the metabolic consequences of reduced levels of active peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1alpha) under hypoxia, and hypothetically in obstructive sleep apnea, are explored. A better molecular understanding of bile acid synthesis and metabolic dysregulation in this context will hopefully promote the study of new targets in human sleep apneas, and encourage clinical trials using existing therapeutic and dietary interventions in patients afflicted with these conditions.

  11. Behavioral and toxicological responses of Rhodnius prolixus and Triatoma infestans (Hemiptera: Reduviidae) to 10 monoterpene alcohols.

    PubMed

    Moretti, A N; Zerba, E N; Alzogaray, Raúl A

    2013-09-01

    The effect on locomotor activity, the repellency, and the knock-down produced by 10 monoterpene alcohols were evaluated on first-instar nymphs of Rhodnius prolixus and Triatoma infestans, vectors of Chagas disease. A video tracking technique was used to evaluate locomotor activity and repellency by exposure to papers impregnated with monoterpenes. Eugenol on R. prolixus and (S)-cis-verbenol on T. infestans did not modify the locomotor activity. The remaining monoterpenes produced hyperactivity on both species, although the concentration required was at least a 1,000 times higher than that of deltamethrin (positive control). Carvacrol, eugenol, and geraniol resulted as repellent as N,N-diethyl-m-toluamide (positive control) for both species. A similar result was observed for almost every monoterpene on T. infestans. Knock-down effect was evaluated by exposing the nymphs in closed recipients. The order of increasing toxicity on R. prolixus was (KT50 values in min): geraniol (213.7) < alpha-terpineol (164.5) < linalool (124.2) < carvacrol (111.6) < eugenol (89.8) < thymol (78.9), and on T. infestans: alpha-terpineol (289.8) < eugenol (221.3) < carvacrol (164.2) < linalool (154.9) < thymol (96.7). All monoterpenes were less toxic than the positive control, dichlorvos (3.6 min for R. prolixus and 3.9 min for T. infestans). After 7 h of exposure, (-)-carveol, citronellol, and menthol (on both species) and geraniol (on T. infestans) produced < 50% of knock-down. After these results, it is worthwhile to explore more deeply the potential of these compounds as tools for controlling Chagas disease vectors.

  12. Anti-inflammatory ergostanes from the basidiomata of Antrodia salmonea.

    PubMed

    Shen, Chien-Chang; Wang, Yea-Hwey; Chang, Tun-Tschu; Lin, Lie-Chwen; Don, Ming-Jaw; Hou, Yu-Chang; Liou, Kuo-Tong; Chang, Shiou; Wang, Wen-Yen; Ko, Han-Chieh; Shen, Yuh-Chiang

    2007-09-01

    Three new anti-oxidative ergostanes, methyl antcinate L (1), antcin M (2), and methyl antcinate K (3), together with nine additional known compounds, 3-ketodehydrosulphurenic acid, sulphurenic acid, dehydrosulphurenic acid, 3beta,15alpha-dihydroxylanosta-7,9(11),24-trien-21-oic acid, zhankuic acid A, zhankuic acid B, zhankuic acid C, antcin C, and antcin K were isolated from the basidiomata of Antrodia salmonea, a newly identified species of Antrodia (Polyporaceae) in Taiwan. These three new compounds were identified as methyl 3alpha,7alpha,12alpha-trihydroxy-4alpha-methylergosta-8,24(29)-dien-11-on-26-oate (1), 3alpha,12alpha-dihydroxy-4alpha-methylergosta-8,24(29)-dien-11-on-26-oic acid (2), and methyl 3alpha,4beta,7beta-trihydroxy-4alpha-methylergosta-8,24(29)-dien-11-on-26-oate (3) by spectroscopic analysis. We studied their antioxidative potential on the production of reactive oxygen species and nitric oxide (NO) in neutrophils and microglial cells, respectively. Compounds 1-3 displayed potent antioxidative activity with IC50 values of around 2.0-8.8 microM that was partially due to inhibition (6-67%) of NADPH oxidase activity but not through direct radical-scavenging properties. Compounds 1-3 also inhibited NO production with IC50 values of around 1.7-16.5 microM and were more potent than a non-specific NOS inhibitor. We conclude that these three new compounds 1, 2, and 3 exhibit anti-inflammatory activities in activated inflammatory cells.

  13. Essential oil of Nepeta x faassenii Bergmans ex Stearn (N. mussinii Spreng. x N. nepetella L.): a comparison study.

    PubMed

    Radulović, Niko; Blagojević, Polina D; Rabbitt, Kevin; Menezes, Fabio de Sousa

    2011-07-01

    Analysis (GC and GC/MS) of an essential oil sample obtained from dry leaves of Nepeta x faassenii Bergmans ex Steam, a hybrid species produced by crossbreeding N. mussinii Spreng. with N. nepetella L., led to the identification of 109 constituents that represented 95.9% of the oil. The major constituents were 4aalpha,7alpha,7aalpha-nepetalactone (67.8%), 1,8-cineole (6.6%), germacrene D (4.8%), beta-pinene (2.7%), (E)-beta-ocimene (2.6%), 4aalpha,7beta,7aalpha-nepetalactone (2.3%) and (E)-beta-farnesene (1.0%). Chemical composition of the oil was compared, using multivariate statistical analyses (MVA) with those of the oils of other Nepeta taxa, in particular N. mussinii and N. nepetella. This was done in order to explore the mode of inheritance of the monoterpene biosynthetic apparatus of N. faassenii. Chemical composition of the volatiles of a Nepeta taxon (different populations) can be subject to variation due to environmental and geographical factors. To accommodate this fact in the MVAs, along side with N. faassenii essential oil, additional 6 oils (3 different populations of N. nuda L. and N. cataria L. from Serbia) were included in this study (isolated and analyzed (chemically and statistically)). The MVA analyses recognized N. faassenii as being closely related to both N. mussinii and N. nepetella. If the relative content of oil constituents per plant and not per chromatogram were used as variables in the MVA (this was done by simple multiplication of the yields and relative percentages of components) a higher degree of mutual similarity (in respect to the monoterpene biosynthesis) of N. faassenii to N. mussinii, than to the other parent species, was observed.

  14. Recent advances in the development of farnesoid X receptor agonists

    PubMed Central

    Carey, Elizabeth J.; Lindor, Keith D.

    2015-01-01

    Farnesoid X receptors (FXRs) are nuclear hormone receptors expressed in high amounts in body tissues that participate in bilirubin metabolism including the liver, intestines, and kidneys. Bile acids (BAs) are the natural ligands of the FXRs. FXRs regulate the expression of the gene encoding for cholesterol 7 alpha-hydroxylase, which is the rate-limiting enzyme in BA synthesis. In addition, FXRs play a critical role in carbohydrate and lipid metabolism and regulation of insulin sensitivity. FXRs also modulate live growth and regeneration during liver injury. Preclinical studies have shown that FXR activation protects against cholestasis-induced liver injury. Moreover, FXR activation protects against fatty liver injury in animal models of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), and improved hyperlipidemia, glucose intolerance, and insulin sensitivity. Obeticholic acid (OCA), a 6α-ethyl derivative of the natural human BA chenodeoxycholic acid (CDCA) is the first-in-class selective FXR agonist that is ~100-fold more potent than CDCA. Preliminary human clinical trials have shown that OCA is safe and effective. In a phase II clinical trial, administration of OCA was well-tolerated, increased insulin sensitivity and reduced markers of liver inflammation and fibrosis in patients with type II diabetes mellitus and NAFLD. In two clinical trials of OCA in patients with primary biliary cirrhosis (PBC), a progressive cholestatic liver disease, OCA significantly reduced serum alkaline phosphatase (ALP) levels, an important disease marker that correlates well with clinical outcomes of patients with PBC. Together, these studies suggest that FXR agonists could potentially be used as therapeutic tools in patients suffering from nonalcoholic fatty and cholestatic liver diseases. Larger and Longer-term studies are currently ongoing. PMID:25705637

  15. Modulation of cholesterol levels in broiler meat by dietary garlic and copper.

    PubMed

    Konjufca, V H; Pesti, G M; Bakalli, R I

    1997-09-01

    Male Ross x Ross 208 chickens were fed from hatching to 21 d of age either a control diet (based on corn and soybean meal) or the control diet supplemented with 0, 1.5, 3.0, and 4.5% of a commercial garlic powder in Experiments 1 and 2. Once the dose-response relationship was established, 3% garlic powder or 63 or 180 mg/kg copper as cupric citrate or cupric sulfate pentahydrate were supplemented to the diet (Experiments 3, 4, 5, and 6). In the first two experiments, reductions of plasma cholesterol (P = 0.006) and triacylglycerols (P = 0.013) and liver (P = 0.012) and breast muscle (P = 0.165) cholesterol were observed in garlic-supplemented birds. Feeding either garlic powder or copper (63 and 180 mg/kg) resulted in reduced levels of plasma cholesterol, liver cholesterol, blood reduced glutathione, and breast and thigh muscle cholesterol. Differences were significant at P < 0.05 in at least one experiment. 3-Hydroxy-3-methylglutaryl reductase activity was decreased due to dietary garlic (P = 0.0369), but not by pharmacological levels of dietary copper (P = 0.982). The activity of fatty acid synthetase was decreased in birds fed copper (P = 0.035). Both garlic and copper supplements decreased cholesterol 7 alpha-hydroxylase activity (P = 0.024 and P = 0.022, respectively). The results of these trials confirm the findings that garlic and copper alter lipid and cholesterol metabolism. However, they do not work by the same mechanism. Feeding dietary garlic or copper for 21 d reduced cholesterol levels of broiler meat without altering growth of the chickens or feed efficiency.

  16. Bile Acids and Dysbiosis in Non-Alcoholic Fatty Liver Disease

    PubMed Central

    Bandsma, Robert; Comelli, Elena M.; Arendt, Bianca M.; Zhang, Ling; Fung, Scott; Fischer, Sandra E.; McGilvray, Ian G.; Allard, Johane P.

    2016-01-01

    Background & Aims Non-alcoholic fatty liver disease (NAFLD) is characterized by dysbiosis. The bidirectional effects between intestinal microbiota (IM) and bile acids (BA) suggest that dysbiosis may be accompanied by an altered bile acid (BA) homeostasis, which in turn can contribute to the metabolic dysregulation seen in NAFLD. This study sought to examine BA homeostasis in patients with NAFLD and to relate that with IM data. Methods This was a prospective, cross-sectional study of adults with biopsy-confirmed NAFLD (non-alcoholic fatty liver: NAFL or non-alcoholic steatohepatitis: NASH) and healthy controls (HC). Clinical and laboratory data, stool samples and 7-day food records were collected. Fecal BA profiles, serum markers of BA synthesis 7-alpha-hydroxy-4-cholesten-3-one (C4) and intestinal BA signalling, as well as IM composition were assessed. Results 53 subjects were included: 25 HC, 12 NAFL and 16 NASH. Levels of total fecal BA, cholic acid (CA), chenodeoxycholic acid (CDCA) and BA synthesis were higher in patients with NASH compared to HC (p<0.05 for all comparisons). The primary to secondary BA ratio was higher in NASH compared to HC (p = 0.004), but ratio of conjugated to unconjugated BAs was not different between the groups. Bacteroidetes and Clostridium leptum counts were decreased in in a subset of 16 patients with NASH compared to 25 HC, after adjusting for body mass index and weight-adjusted calorie intake (p = 0.028 and p = 0.030, respectively). C. leptum was positively correlated with fecal unconjugated lithocholic acid (LCA) (r = 0.526, p = 0.003) and inversely with unconjugated CA (r = -0.669, p<0.0001) and unconjugated CDCA (r = - 0.630, p<0.0001). FGF19 levels were not different between the groups (p = 0.114). Conclusions In adults with NAFLD, dysbiosis is associated with altered BA homeostasis, which renders them at increased risk of hepatic injury. PMID:27203081

  17. Elevated homocysteine reduces apolipoprotein A-I expression in hyperhomocysteinemic mice and in males with coronary artery disease.

    PubMed

    Mikael, Leonie G; Genest, Jacques; Rozen, Rima

    2006-03-03

    Hyperhomocysteinemia, a risk factor for cardiovascular disease, is caused by nutritional or genetic disturbances in homocysteine metabolism. A polymorphism in methylenetetrahydrofolate reductase (MTHFR) is the most common genetic cause of mild hyperhomocysteinemia. To examine mechanisms by which an elevation in plasma homocysteine leads to vascular disease, we first performed microarray analyses in livers of Mthfr-deficient mice and identified differentially expressed genes that are involved in lipid and cholesterol metabolism. Microarrays and RT-PCR showed decreased mRNA for apolipoprotein A (ApoA)-IV and for ApoA-I and increased mRNA for cholesterol 7alpha hydroxylase (Cyp7A1) in Mthfr(+/-) mice compared with Mthfr(+/+) mice. Western blotting revealed that ApoA-I protein levels in liver and plasma of Mthfr(+/-) mice were 52% and 62% of levels in the respective tissues of Mthfr(+/+) mice. We also performed Western analysis for plasma ApoA-I protein levels in 60 males with coronary artery disease and identified a significant (P<0.01) negative correlation (-0.33) between ApoA-I and plasma homocysteine levels. This cohort also displayed a negative correlation (-0.24, P=0.06) between high-density lipoprotein cholesterol and plasma homocysteine. Treatment of HepG2 cells with supraphysiological levels of 5 mmol/L homocysteine reduced peroxisome proliferator-activated receptor (PPAR) alpha and ApoA-I protein levels and decreased ApoA-I promoter activity. Transfection with a PPARalpha construct upregulated ApoA-I and MTHFR. Our results suggest that hyperhomocysteinemia may increase risk of atherosclerosis by decreasing expression of ApoA-I and increasing expression of CYP7A1.

  18. Ergosteroids. II: Biologically active metabolites and synthetic derivatives of dehydroepiandrosterone.

    PubMed

    Lardy, H; Kneer, N; Wei, Y; Partridge, B; Marwah, P

    1998-03-01

    An improved procedure for the synthesis of 3 beta-hydroxyandrost-5-ene-7,17-dione, a natural metabolite of dehydroepiandrosterone (DHEA) is described. The synthesis and magnetic resonance spectra of several other related steroids are presented. Feeding dehydroepiandrosterone to rats induces enhanced formation of several liver enzymes among which are mitochondrial sn-glycerol 3-phosphate dehydrogenase (GPDH) and cytosolic malic enzyme. The induction of these two enzymes, that complete a thermogenic system in rat liver, was used as an assay to search for derivatives of DHEA that might be more active than the parent steroid. Activity is retained in steroids that are reduced to the corresponding 17 beta-hydroxy derivative, or hydroxylated at 7 alpha or 7 beta, and is considerably enhanced when the 17-hydroxy or 17-carbonyl steroid is converted to the 7-oxo derivative. Several derivatives of DHEA did not induce the thermogenic enzymes whereas the corresponding 7-oxo compounds did. Both short and long chain acyl esters of DHEA and of 7-oxo-DHEA are active inducers of the liver enzymes when fed to rats. 7-Oxo-DHEA-3-sulfate is as active as 7-oxo-DHEA or its 3-acetyl ester, whereas DHEA-3-sulfate is much less active than DHEA. Among many steroids tested, those possessing a carbonyl group at position 3, a methyl group at 7, a hydroxyl group at positions 1, 2, 4, 11, or 19, or a saturated B ring, with or without a 4-5 double bond, were inactive.

  19. ACAT inhibition reverses LCAT deficiency and improves plasma HDL in chronic renal failure.

    PubMed

    Vaziri, N D; Liang, K

    2004-11-01

    Chronic renal failure (CRF) is associated with increased risk of arteriosclerotic cardiovascular disease and profound alteration of plasma lipid profile. Uremic dyslipidemia is marked by increased plasma concentration of ApoB-containing lipoproteins and impaired high-density lipoprotein (HDL)-mediated reverse cholesterol transport. These abnormalities are, in part, due to acquired LCAT deficiency and upregulation of hepatic acyl-CoA:cholesterol acyltransferase (ACAT). ACAT catalyzes intracellular esterification of cholesterol, thereby promoting hepatic production of ApoB-containing lipoproteins and constraining HDL-mediated cholesterol uptake in the peripheral tissues. In view of the above considerations, we tested the hypothesis that pharmacological inhibition of ACAT may ameliorate CRF-induced dyslipidemia. 5/6 Nephrectomized rats were treated with either ACAT inhibitor IC-976 (30 mg.kg(-1).day(-1)) or placebo for 6 wk. Sham-operated rats served as controls. Key cholesterol-regulating enzymes, plasma lipids, and creatinine clearance were measured. The untreated CRF rats exhibited increased plasma low-density lipoprotein (LDL) and very LDL (VLDL) cholesterol, unchanged plasma HDL cholesterol, elevated total cholesterol-to-HDL cholesterol ratio, reduced liver microsomal free cholesterol, and diminished creatinine clearance. This was accompanied by reduced plasma LCAT, increased hepatic ACAT-2 mRNA, ACAT-2 protein and ACAT activity, and unchanged hepatic HMG-CoA reductase and cholesterol 7alpha-hydroxylase. ACAT inhibitor raised plasma HDL cholesterol, lowered LDL and VLDL cholesterol, and normalized total cholesterol-to-HDL cholesterol ratio without changing total cholesterol concentration (hence, a shift from ApoB-containing lipoproteins to HDL). This was accompanied by normalizations of hepatic ACAT activity and plasma LCAT. In conclusion, inhibition of ACAT reversed LCAT deficiency and improved plasma HDL level in CRF rats. Future studies are needed to explore

  20. Corn silk extract improves cholesterol metabolism in C57BL/6J mouse fed high-fat diets

    PubMed Central

    Cha, Jae Hoon; Kim, Sun Rim; Kang, Hyun Joong; Kim, Myung Hwan; Ha, Ae Wha

    2016-01-01

    BACKGROUND/OBJECTIVES Corn silk (CS) extract contains large amounts of maysin, which is a major flavonoid in CS. However, studies regarding the effect of CS extract on cholesterol metabolism is limited. Therefore, the purpose of this study was to determine the effect of CS extract on cholesterol metabolism in C57BL/6J mouse fed high-fat diets. MATERIALS/METHODS Normal-fat group fed 7% fat diet, high-fat (HF) group fed 25% fat diet, and high-fat with corn silk (HFCS) group were orally administered CS extract (100 mg/kg body weight) daily. Serum and hepatic levels of total lipids, triglycerides, and total cholesterol as well as serum free fatty acid, glucose, and insulin levels were determined. The mRNA expression levels of acyl-CoA: cholesterol acyltransferase (ACAT), cholesterol 7-alpha hydroxylase (CYP7A1), farnesoid X receptor (FXR), lecithin cholesterol acyltransferase (LCAT), low-density lipoprotein receptor, 3-hyroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), adiponectin, leptin, and tumor necrosis factor α were determined. RESULTS Oral administration of CS extract with HF improved serum glucose and insulin levels as well as attenuated HF-induced fatty liver. CS extracts significantly elevated mRNA expression levels of adipocytokines and reduced mRNA expression levels of HMG-CoA reductase, ACAT, and FXR. The mRNA expression levels of CYP7A1 and LCAT between the HF group and HFCS group were not statistically different. CONCLUSIONS CS extract supplementation with a high-fat diet improves levels of adipocytokine secretion and glucose homeostasis. CS extract is also effective in decreasing the regulatory pool of hepatic cholesterol, in line with decreased blood and hepatic levels of cholesterol though modulation of mRNA expression levels of HMG-CoA reductase, ACAT, and FXR. PMID:27698957

  1. Binding of collagens to an enterotoxigenic strain of Escherichia coli

    SciTech Connect

    Visai, L.; Speziale, P.; Bozzini, S. )

    1990-02-01

    An enterotoxigenic strain of Escherichia coli, B34289c, has been shown to bind the N-terminal region of fibronectin with high affinity. We now report that this strain also binds collagen. The binding of 125I-labeled type II collagen to bacteria was time dependent and reversible. Bacteria expressed a limited number of collagen receptors (2.2 x 10(4) per cell) and bound collagen with a Kd of 20 nM. All collagen types tested (I to V) as well as all tested cyanogen bromide-generated peptides (alpha 1(I)CB2, alpha 1(I)CB3, alpha 1(I)CB7, alpha 1(I)CB8, and alpha 2(I)CB4) were recognized by bacterial receptors, as demonstrated by the ability of these proteins to inhibit the binding of 125I-labeled collagen to bacteria. Of several unlabeled proteins tested in competition experiments, fibronectin and its N-terminal region strongly inhibited binding of the radiolabeled collagen to E. coli cells. Conversely, collagen competed with an 125I-labeled 28-kilodalton fibronectin fragment for bacterial binding. Collagen bound to bacteria could be displaced by excess amounts of either unlabeled fibronectin or its N-terminal fragment. Similarly, collagen could displace 125I-labeled N-terminal peptide of fibronectin bound to the bacterial cell surface. Bacteria grown at 41 degrees C or in the presence of glucose did not express collagen or fibronectin receptors. These results indicate the presence of specific binding sites for collagen on the surface of E. coli cells and furthermore that the collagen and fibronectin binding sites are located in close proximity, possibly on the same structure.

  2. Epiberberine reduces serum cholesterol in diet-induced dyslipidemia Syrian golden hamsters via network pathways involving cholesterol metabolism.

    PubMed

    Zou, Zong-Yao; Hu, Yin-Ran; Ma, Hang; Feng, Min; Li, Xue-Gang; Ye, Xiao-Li

    2016-03-05

    This study aimed to evaluate the cholesterol-lowering effect of epiberberine in dyslipidemia Syrian golden hamsters induced by high fat and high cholesterol (HFHC) diet and its regulation mechanism on some key genes involved in cholesterol metabolism. Hamsters were divided into six groups: normal control group (NC), HFHC group, simvastatin (Sim) and three doses of epiberberine group. The body weight, organs weight and serum lipid levels, as well as total cholesterol (TC) and total bile acids (TBA) levels in liver and feces were determined. Furthermore, the antidyslipidemia effect of epiberberine on key genes involved in cholesterol biosynthesis, uptake, conversion and elimination such as 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), low density lipoprotein receptor (LDL receptor), 7-alpha-hydroxylase (CYP7A1) and apical sodium dependent bile acid transporter (ASBT) were investigated. The results showed that epiberberine at high dosage significantly reduced serum TC, low density lipoprotein cholesterol (LDL-c) and TBA levels by 20.2%, 22.3% and 43.8%, respectively, and increased TBA and TC levels in feces. Epiberberine inhibited HMGCR mRNA and protein expressions and slightly reduced the protein level of ASBT, as well as dramatically up-regulated mRNA and protein expressions of CYP7A1 and LDL receptor. These findings suggested that the antidyslipidemia effects of epiberberine can be achieved via inhibiting the synthesis of cholesterol, promoting the uptake and conversion of TC in liver and increasing the excretion of TC and TBA in feces. Thus, epiberberine should be considered as one of the promising natural drugs for the treatment of dyslipidemia.

  3. Reversed-phase high-performance liquid chromatography studies of alpha-MSH fragments.

    PubMed

    Cody, W L; Wilkes, B C; Hruby, V J

    1984-11-30

    alpha-Melanotropin (alpha-MSH) is a linear tridecapeptide (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2), that is primarily known for its ability to stimulate melanosome dispersion within integumental melanocytes (F. J. H. Tilders, D. F. Swaab and T. B. van Wimersma Greidanus (Editors), Frontiers of Hormone Research, Vol. 4, Karger, Basel, 1977; J. Ramachandran, S. W. Farmer, S. Liles and C. H. Li, Biochim. Biophys. Acta, 428 (1976) 347). In our efforts to understand the relationships of structure and conformation to the biological activities of alpha-MSH, we have prepared a series of diastereoisomeric analogues based on the highly potent analogue Ac-[Nle4, D-Phe7]-alpha-MSH4-11-NH2 (T. K. Sawyer, V. J. Hruby, B. C. Wilkes, M. T. Draelos, M. E. Hadley and J. Bergsneider, J. Med. Chem., 25 (1982) 1022). These analogues differed only in the amino acid substituted in the seven position, which was thought to be a critical residue for the biological activity of alpha-MSH. The chromatographic behavior of these analogues was examined on a C18 Vydac (16-micron) reversed-phase column with five different mobile phases. The selectivity (alpha) for the analogues was compared in 0.10% trifluoroacetic acid (TFA), 0.10% heptafluorobutyric acid (HFBA) and 0.25 M triethylammonium phosphate (TEAP) using either acetonitrile or methanol as the organic modifier. With only one exception all analogues substituted with a D-amino acid in the seven position were eluted prior to their L-amino acid counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Putative neurotrophic factors and functional recovery from peripheral nerve damage in the rat.

    PubMed Central

    Van der Zee, C. E.; Brakkee, J. H.; Gispen, W. H.

    1991-01-01

    1. In rats, recovery of sensory-motor function following a crush lesion of the sciatic or tibial nerve was monitored by measuring foot reflex withdrawal from a local noxious stimulation of the foot sole. 2. Putative neurotrophic compounds were tested on this functional recovery model: melanocortins (peptides derived from ACTH (corticotropin) and alpha-MSH (melanotropin], gangliosides and nimodipine were effective whereas isaxonine and TRH (thyrotropin releasing hormone) were not. 3. Structure-activity studies with melanocortins revealed a similar effectiveness of alpha-MSH, [N-Leu4, D-Phe7]-alpha-MSH, desacetyl-alpha-MSH and the ACTH analogue ORG 2766, questioning the validity of the previously suggested notion that the melanotrophic properties of these peptides are responsible for their neurotrophic effect. 4. As recovery of function after peripheral nerve damage follows a similar time course in hypophysectomized (five days post operation) and sham-operated rats, effective melanocortin therapy does not mimic an endogenous peptide signal in the repair process from pituitary origin. 5. Subcutaneous treatment with ORG 2766 (7.5 micrograms kg-1 48 h-1) facilitates recovery of function following peripheral nerve damage in young (6-7 weeks old), mature (5 month old) and old (20 month old) rats. 6. In view of the diversity in structure of the effective neurotrophic factors and the complexity of nerve repair, the present data support the notion that peripheral nerve repair may be facilitated by different humoral factors likely to be active on different aspects of the recovery process. PMID:1678980

  5. Auto/paracrine control of inflammatory cytokines by acetylcholine in macrophage-like U937 cells through nicotinic receptors.

    PubMed

    Chernyavsky, Alexander I; Arredondo, Juan; Skok, Maryna; Grando, Sergei A

    2010-03-01

    Although acetylcholine (ACh) is well known for its neurotransmitter function, recent studies have indicated that it also functions as an immune cytokine that prevents macrophage activation through a 'cholinergic (nicotinic) anti-inflammatory pathway'. In this study, we used the macrophage-like U937 cells to elucidate the mechanisms of the physiologic control of cytokine production by auto/paracrine ACh through the nicotinic class of ACh receptors (nAChRs) expressed in these cells. Stimulation of cells with lipopolysaccharide up-regulated expression of alpha1, alpha4, alpha5, alpha7, alpha10, beta1 and beta3 subunits, down-regulated alpha6 and beta2 subunits, and did not alter the relative quantity of alpha9 and beta4 mRNAs. Distinct nAChR subtypes showed differential regulation of the production of pro- and anti-inflammatory cytokines. While inhibition of the expression of the TNF-alpha gene was mediated predominantly by the alpha-bungarotoxin sensitive nAChRs, that of the IL-6 and IL-18 genes-by the mecamylamine-sensitive nAChRs. Both the Mec- and alphaBtx-sensitive nAChRs regulated expression of the IL-1beta gene equally efficiently. Upregulation of IL-10 production by auto/paracrine ACh was mediated predominantly through alpha7 nAChR. These findings offer a new insight on how nicotinic agonists control inflammation, thus laying a groundwork for the development of novel immunomodulatory therapies based on the nAChR subtype selectivity of nicotinic agonists.

  6. 7-Alkylguanine adduct levels in urine, lungs and liver of mice exposed to styrene by inhalation

    SciTech Connect

    Vodicka, Pavel Erik . E-mail: pvodicka@biomed.cas.cz; Linhart, Igor; Novak, Jan; Koskinen, Mikko; Vodickova, Ludmila; Hemminki, Kari

    2006-01-15

    This study describes urinary excretion of two nucleobase adducts derived from styrene 7,8-oxide (SO), i.e., 7-(2-hydroxy-1-phenylethyl)guanine (N7{alpha}G) and 7-(2-hydroxy-2-phenylethyl)guanine (N7{beta}G), as well as a formation of N7-SO-guanine adducts in lungs and liver of two month old male NMRI mice exposed to styrene by inhalation in a 3-week subacute study. Strikingly higher excretion of both isomeric nucleobase adducts in the first day of exposure was recorded, while the daily excretion of nucleobase adducts in following time intervals reached the steady-state level at 4.32 + 1.14 and 6.91 + 1.17 pmol/animal for lower and higher styrene exposure, respectively. {beta}-SO-guanine DNA adducts in lungs increased with exposure in a linear way (F = 13.7 for linearity and 0.17 for non-linearity, respectively), reaching at the 21st day the level of 23.0 adducts/10{sup 8} normal nucleotides, i.e., 0.74 fmol/{mu}g DNA of 7-alkylguanine DNA adducts for the concentration of 1500 mg/m{sup 3}, while no 7-SO-guanine DNA adducts were detected in the liver after 21 days of inhalation exposure to both of styrene concentrations. A comparison of 7-alkylguanines excreted in urine with 7-SO-guanines in lungs (after correction for depurination and for missing {alpha}-isomers) revealed that persisting 7-SO-guanine DNA adducts in lungs account for about 0.5% of the total alkylation at N7 of guanine. The total styrene-specific 7-guanine alkylation accounts for about 1.0 x 10{sup -5}% of the total styrene uptake, while N1-adenine alkylation contributes to this percentage only negligibly.

  7. Genistein modulates the effects of parathyroid hormone in human osteoblastic SaOS-2 cells.

    PubMed

    Chen, Wen-Fang; Wong, Man-Sau

    2006-06-01

    Genistein and parathyroid hormone (PTH) are anabolic agents that stimulate bone formation through their direct actions in osteoblastic cells. In the present study, we aimed to determine whether genistein modulates the actions of PTH in human osteoblastic SaOS-2 cells in an oestrogen-depleted condition. The present results showed that genistein (10(-8) to 10(-6) m) induced alkaline phosphatase (ALP) activity and osteoprotegrin (OPG) expression in SaOS-2 cells in a dose-dependent manner. These effects could be completely abolished by co-treatment with oestrogen antagonist ICI 182780 (7alpha-[9-[(4,4,5,5,5-pentafluoropentyl)sulfonyl]nonyl]-estra-1,3,5(10)-triene-3,17beta-diol). Genistein (at 1 microM) could stimulate the mRNA expression of receptor activator of NF-kappaB ligand (RANKL). As OPG and RANKL are known to modulate osteoclastogenesis, the ability of genistein to modulate OPG and RANKL expression in SaOS-2 cells suggested that it might modulate osteoclastogenesis through its direct actions on osteoblastic cells. PTH (at 10 nM) stimulated ALP activity, induced RANKL mRNA expression and suppressed OPG mRNA expression in SaOS-2 cells, confirming its bi-directional effects on osteoblastic cells. Pre-treatment of SaOS-2 cells with genistein and oestrogen not only enhanced PTH-induced ALP activity, but also attenuated PTH up regulation of RANKL mRNA expression and PTH down regulation of OPG mRNA expression. Taken together, the present study provides the first evidence that genistein could modulate the actions of PTH in human osteoblastic SaOS-2 cells in an oestrogen-depleted condition.

  8. Cefotetan, a new cephamycin: comparison of in vitro antimicrobial activity with other cephems, beta-lactamase stability, and preliminary recommendations for disk diffusion testing.

    PubMed Central

    Ayers, L W; Jones, R N; Barry, A L; Thornsberry, C; Fuchs, P C; Gavan, T L; Gerlach, E H; Sommers, H M

    1982-01-01

    Cefotetan is a new, potent, 7 alpha-methoxy cephalosporin (cephamycin). The in vitro activity of cefotetan tested in a multiphasic, collaborative study against 12,260 consecutive clinical isolates and 448 selected isolates showed 93% of Enterobacteriaceae, 90% of methicillin-susceptible Staphylococcus aureus (broth dilution), 83% of Bacteroides fragilis, and 72% of non-enterococcal streptococci to be inhibited by less than or equal to 8 micrograms/ml. Beta-Lactamase-producing and -nonproducing Haemophilus influenzae strains were inhibited by less than or equal to 1.0 micrograms/ml. Cefotetan's inhibitory spectrum paralleled those of the newest generation of cephems and exceeded those of cefoxitin and cefamandole. No useful activity was present against Streptococcus faecalis or Pseudomonas aeruginosa. Cefotetan was bactericidal without significant inoculum effect and was highly resistant to hydrolysis by Richmond-Sykes types I, III, and IV beta-lactamases. Hydrolysis of the chromogenic cephalosporin PADAC (pyridine-2-azo-p-dimethylaniline cephalosporin) by type I beta-lactamases was markedly inhibited by concentrations of cefotetan similar to those of the potent inhibitor dicloxacillin. Analysis of agar disk diffusion for several disk potencies and broth dilution susceptibility tests by regression and error rate-bounding methods produced preliminary tentative zone standards (30-micrograms disk, using minimal inhibitory concentration breakpoints of less than or equal to 8 micrograms/ml susceptible and greater than 32 micrograms/ml resistant, or 75-micrograms disk, using minimal inhibitory concentration breakpoints of less than or equal to 16 micrograms/ml susceptible and greater than or equal to 64 micrograms/ml resistant) of greater than or equal to 18 mm susceptible, less than or equal to 14 mm resistant, and 15 to 17 mm indeterminate. Staphylococcus aureus testing with the 30-micrograms disk is not recommended. PMID:6983862

  9. Probiotics and the BSH-related cholesterol lowering mechanism: a Jekyll and Hyde scenario.

    PubMed

    Choi, Sy-Bing; Lew, Lee-Ching; Yeo, Siok-Koon; Nair Parvathy, Seema; Liong, Min-Tze

    2015-01-01

    Probiotic microorganisms have been documented over the past two decades to play a role in cholesterol-lowering properties via various clinical trials. Several mechanisms have also been proposed and the ability of these microorganisms to deconjugate bile via production of bile salt hydrolase (BSH) has been widely associated with their cholesterol lowering potentials in prevention of hypercholesterolemia. Deconjugated bile salts are more hydrophobic than their conjugated counterparts, thus are less reabsorbed through the intestines resulting in higher excretion into the feces. Replacement of new bile salts from cholesterol as a precursor subsequently leads to decreased serum cholesterol levels. However, some controversies have risen attributed to the activities of deconjugated bile acids that repress the synthesis of bile acids from cholesterol. Deconjugated bile acids have higher binding affinity towards some orphan nuclear receptors namely the farsenoid X receptor (FXR), leading to a suppressed transcription of the enzyme cholesterol 7-alpha hydroxylase (7AH), which is responsible in bile acid synthesis from cholesterol. This notion was further corroborated by our current docking data, which indicated that deconjugated bile acids have higher propensities to bind with the FXR receptor as compared to conjugated bile acids. Bile acids-activated FXR also induces transcription of the IBABP gene, leading to enhanced recycling of bile acids from the intestine back to the liver, which subsequently reduces the need for new bile formation from cholesterol. Possible detrimental effects due to increased deconjugation of bile salts such as malabsorption of lipids, colon carcinogenesis, gallstones formation and altered gut microbial populations, which contribute to other varying gut diseases, were also included in this review. Our current findings and review substantiate the need to look beyond BSH deconjugation as a single factor/mechanism in strain selection for

  10. Interaction of glucocorticosteroid receptor and wild-type or mutated 90-kDa heat shock protein coexpressed in baculovirus-infected Sf9 cells.

    PubMed

    Cadepond, F; Binart, N; Chambraud, B; Jibard, N; Schweizer-Groyer, G; Segard-Maurel, I; Baulieu, E E

    1993-11-15

    Coexpression of the human glucocorticosteroid receptor (hGR) and chicken 90-kDa heat shock protein alpha (chsp90) in recombinant baculovirus-infected Sf9 cells is a system that provides a large quantity of wild-type chsp90-hGR complexes able to bind hormone ([3H]triamcinolone acetonide; TA), sedimenting at 8 S, and displaceable to 11 S by BF4 and D7 alpha anti-chsp90 monoclonal antibodies. Thus, we were able to examine the effects of selective chsp90 mutations on hetero-oligomeric complex formation. Two deletions involved hydrophilic regions, A between amino acids 221 and 290 and B between amino acids 530 and 581, and the third, Z, removed a central leucine heptad repeat region (amino acids 392-419). When these chsp90 mutants were expressed, the lack of displacement of [3H]TA receptor complexes on sucrose gradient by specific chsp90 antibodies was consistent with the formation of [3H]TA receptor complexes containing only endogenous insect hsp90. By using an immunoadsorption method and sedimentation analysis, we found that the deletion of region A precluded the interaction of chsp90 with the hGR, while B and Z deletions led to formation of abnormal complexes with the hGR, which displayed large forms (> 10 S), were unable to bind hormone, and apparently formed only small amounts of tightly bound nuclei hGR upon in vivo hormone treatment. As a whole, the data are consistent with distinct roles of hsp90 regions in hGR function.

  11. Expression of the VLA beta 1 integrin family in bladder cancer.

    PubMed Central

    Liebert, M.; Washington, R.; Stein, J.; Wedemeyer, G.; Grossman, H. B.

    1994-01-01

    Integrins are a family of transmembrane heterodimers, many of which function as receptors for extracellular matrix molecules and play a role in adherence to and motility on matrix components. Because of these functions, integrins are suspected of participating in metastatic processes. We investigated the expression of beta 1 integrins in human bladder cancer cell lines and tissues. Expression of beta 1 integrins on cultured bladder cancer cell lines was evaluated by flow cytometry, of 8 cell lines tested, alpha 1 was found in 4, alpha 2 and alpha 3 in all 8, alpha 4 in 1, and alpha 5 in 3. These results were in sharp contrast to the expression detected by immunostaining tissues containing normal urothelium and low stage (noninvasive) and high stage (invasive) bladder cancers. All normal urothelial tissues tested expressed alpha 2 and alpha 3 and none expressed alpha 1, alpha 4, or alpha 5. Similarly, a majority (77%) of low stage (noninvasive) bladder cancers stained positively for alpha 3, whereas only 6 of 13 expressed alpha 2 and none expressed alpha 1, alpha 4, or alpha 5. Among invasive bladder cancers, alpha 1 was detected in 7%, alpha 2 in 24%, alpha 3 in 68%, alpha 5 in 10%, and alpha 4 was not found in any samples. These results indicate that integrin expression in cultured human bladder cancer cell lines does not represent expression observed in tissue samples and may reflect adaption to or selection during tissue culture conditions. A progressive loss of alpha 2 expression is seen from normal urothelial cells through invasive bladder cancers. This loss may contribute to an invasive phenotype by a loss of the cell-cell adherence function mediated by the alpha 2 beta 1 and alpha 3 beta 1 integrins. Images Figure 2 Figure 3 PMID:8178925

  12. Alpha-melanocyte-stimulating hormone peptide analogs labeled with technetium-99m and indium-111 for malignant melanoma targeting.

    PubMed

    Chen, JianQing; Cheng, Zhen; Miao, Yubin; Jurisson, Silvia S; Quinn, Thomas P

    2002-02-15

    Previous studies have shown that the compact structure of a rhenium-cyclized alpha--melanocyte-stimulating hormone peptide analog, [Cys3410,D-Phe7]alpha-MSH(3--13), or Re-CCMSH, significantly enhanced its in vivo tumor uptake and retention. In this study, the metal chelate 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was coupled to the N-terminus of Re-CCMSH in order to develop a melanoma-targeting peptide that could be labeled with a wider variety of imaging and therapeutic radionuclides. Biodistribution properties of indium-111 ((111)In)--labeled DOTA-Re-CCMSH were compared with the non-DOTA-containing technetium-99m ((99m)Tc)--CCMSH in murine melanoma--bearing C57 mice to determine the effects of DOTA on tumor uptake and whole-body clearance. The tumor targeting capacity and clearance kinetics of (111)In-DOTA-Re-CCMSH were also compared with other related cyclic and linear (111)In-labeled DOTA-alpha-MSH complexes. The in vivo distribution data showed that the conjugation of DOTA to Re-CCMSH did not reduce its initial tumor uptake kinetics but did enhance its tumor retention and renal clearance properties. The tumor uptake of (111)In-DOTA-Re-CCMSH was significantly higher than the other (111)In-DOTA--coupled cyclic or linear alpha-MSH analogs used in this study. Moreover, (111)In-DOTA-Re-CCMSH displayed lower radioactivity accumulation in normal tissues of interest than its non-Re-cyclized counterpart, (111)In-DOTA-CCMSH; the disulfide bond--cyclized (111)In-DOTA-CMSH; or the linear (111)In-DOTA-NDP. Peptide cyclization via rhenium coordination significantly enhanced the tumor targeting and renal clearance properties of DOTA-Re-CCMSH, making it an excellent candidate for melanoma radiodetection and radiotherapy. Copyright 2002 American Cancer Society.

  13. Design and characterization of alpha-melanotropin peptide analogs cyclized through rhenium and technetium metal coordination.

    PubMed

    Giblin, M F; Wang, N; Hoffman, T J; Jurisson, S S; Quinn, T P

    1998-10-27

    alpha-Melanocyte stimulating hormone (alpha-MSH) analogs, cyclized through site-specific rhenium (Re) and technetium (Tc) metal coordination, were structurally characterized and analyzed for their abilities to bind alpha-MSH receptors present on melanoma cells and in tumor-bearing mice. Results from receptor-binding assays conducted with B16 F1 murine melanoma cells indicated that receptor-binding affinity was reduced to approximately 1% of its original levels after Re incorporation into the cyclic Cys4,10, D-Phe7-alpha-MSH4-13 analog. Structural analysis of the Re-peptide complex showed that the disulfide bond of the original peptide was replaced by thiolate-metal-thiolate cyclization. A comparison of the metal-bound and metal-free structures indicated that metal complexation dramatically altered the structure of the receptor-binding core sequence. Redesign of the metal binding site resulted in a second-generation Re-peptide complex (ReCCMSH) that displayed a receptor-binding affinity of 2.9 nM, 25-fold higher than the initial Re-alpha-MSH analog. Characterization of the second-generation Re-peptide complex indicated that the peptide was still cyclized through Re coordination, but the structure of the receptor-binding sequence was no longer constrained. The corresponding 99mTc- and 188ReCCMSH complexes were synthesized and shown to be stable in phosphate-buffered saline and to challenges from diethylenetriaminepentaacetic acid (DTPA) and free cysteine. In vivo, the 99mTcCCMSH complex exhibited significant tumor uptake and retention and was effective in imaging melanoma in a murine-tumor model system. Cyclization of alpha-MSH analogs via 99mTc and 188Re yields chemically stable and biologically active molecules with potential melanoma-imaging and therapeutic properties.

  14. Evolution and Taxonomic Classification of Alphapapillomavirus 7 Complete Genomes: HPV18, HPV39, HPV45, HPV59, HPV68 and HPV70

    PubMed Central

    Chen, Zigui; Schiffman, Mark; Herrero, Rolando; DeSalle, Rob; Anastos, Kathryn; Segondy, Michel; Sahasrabuddhe, Vikrant V.; Gravitt, Patti E.; Hsing, Ann W.; Burk, Robert D.

    2013-01-01

    Background The species Alphapapillomavirus 7 (alpha-7) contains human papillomavirus genotypes that account for 15% of invasive cervical cancers and are disproportionately associated with adenocarcinoma of the cervix. Complete genome analyses enable identification and nomenclature of variant lineages and sublineages. Methods The URR/E6 region was sequenced to screen for novel variants of HPV18, 39, 45, 59, 68, 70, 85 and 97 from 1147 cervical samples obtained from multiple geographic regions that had previously been shown to contain an alpha-7 HPV isolate. To study viral heterogeneity, the complete 8 kb genome of 128 isolates, including 109 sequenced for this analysis, were annotated and analyzed. Viral evolution was characterized by constructing phylogenic trees using maximum-likelihood and Bayesian algorithms. Global and pairwise alignments were used to calculate total and ORF/region nucleotide differences; lineages and sublineages were assigned using an alphanumeric system. The prototype genome was assigned to the A lineage or A1 sublineage. Results The genomic diversity of alpha-7 HPV types ranged from 1.1% to 6.7% nucleotide sequence differences; the extent of genome-genome pairwise intratype heterogeneity was 1.1% for HPV39, 1.3% for HPV59, 1.5% for HPV45, 1.6% for HPV70, 2.1% for HPV18, and 6.7% for HPV68. ME180 (previously a subtype of HPV68) was designated as the representative genome for HPV68 sublineage C1. Each ORF/region differed in sequence diversity, from most variable to least variable: noncoding region 1 (NCR1) / noncoding region 2 (NCR2) > upstream regulatory region (URR) > E6 / E7 > E2 / L2 > E1 / L1. Conclusions These data provide estimates of the maximum viral genomic heterogeneity of alpha-7 HPV type variants. The proposed taxonomic system facilitates the comparison of variants across epidemiological and molecular studies. Sequence diversity, geographic distribution and phylogenetic topology of this clinically important group of HPVs suggest

  15. 1-[4-[4[(4R,5R)-3,3-Dibutyl-7-(dimethylamino)-2,3,4,5-tetrahydro-4-hydroxy-1,1-dioxido-1-benzothiepin-5-yl]phenoxy]butyl]-4-aza-1-azoniabicyclo[2.2.2]octane methanesulfonate (SC-435), an ileal apical sodium-codependent bile acid transporter inhibitor alters hepatic cholesterol metabolism and lowers plasma low-density lipoprotein-cholesterol concentrations in guinea pigs.

    PubMed

    West, Kristy L; Ramjiganesh, Tripurasundari; Roy, Suheeta; Keller, Bradley T; Fernandez, Maria Luz

    2002-10-01

    Male Hartley guinea pigs (10/group) were assigned either to a control diet (no drug treatment) or to diets containing 0.4, 2.2, or 7.3 mg/day of an ileal apical sodium-codependent bile acid transporter (ASBT) inhibitor, 1-[4-[4[(4R,5R)-3,3-dibutyl-7-(dimethylamino)-2,3,4,5-tetrahydro-4-hydroxy-1,1-dioxido-1-benzothiepin-5-yl]phenoxy]butyl]-4-aza-1-azoniabicyclo[2.2.2] octane methanesulfonate (SC-435). Based on food consumption, guinea pigs received 0, 0.8, 3.7, or 13.4 mg/kg/day of the ASBT inhibitor. The amount of cholesterol in the four diets was maintained at 0.17%, equivalent to 1200 mg/day in the human situation. Guinea pigs treated with 13.4 mg/kg/day SC-435 had 41% lower total cholesterol and 44% lower low-density lipoprotein (LDL)-cholesterol concentrations compared with control (P < 0.01), whereas no significant differences were observed with either of the lower doses of SC-435. Hepatic cholesterol esters were significantly reduced by 43, 56, and 70% in guinea pigs fed 0.8, 3.7, and 13.4 mg/kg/day of the ASBT inhibitor, respectively (P < 0.01). In addition, the highest dose of the inhibitor resulted in a 42% increase in the number of very low-density lipoprotein (VLDL) triacylglycerol molecules and a larger VLDL diameter compared with controls (P < 0.05). Acyl-CoA cholesterol/acyltransferase activity was 30% lower with the highest dose treatment, whereas cholesterol 7alpha-hydroxylase, the regulatory enzyme of bile acid synthesis, was 30% higher with the highest ASBT inhibitor dose (P < 0.05). Furthermore, bile acid excretion increased 2-fold with the highest dose of SC-435 compared with the control group (P < 0.05). These results suggest that the reduction in total and LDL-cholesterol concentrations by the ASBT inhibitor is a result of alterations in hepatic cholesterol metabolism due to modifications in the enterohepatic circulation of bile acids.

  16. Effects of xenobiotics and steroids on renal and hepatic estrogen metabolism in lake trout.

    PubMed

    Jurgella, Gail F; Marwah, Ashok; Malison, Jeffrey A; Peterson, Richard; Barry, Terence P

    2006-09-01

    Experiments were conducted to (1) elucidate the biochemical pathways of E2 metabolism in the lake trout (Salvelinus namaycush) kidney and liver, and (2) test the hypothesis that specific xenobiotics and endogenous steroids inhibit E2 metabolism by these tissues. Kidney and liver tissue fragments from immature lake trout were incubated in vitro in the presence of radiolabelled E2 plus various xenobiotics or steroids. E2 metabolites were identified by liquid chromatography/mass spectroscopy, and quantified by liquid scintillation spectroscopy. A major metabolite produced by both tissues was an unidentified hydroxylated estrogen metabolite (E2-OH) with a molecular mass of 288 that was not estriol (16-OH-E2), but possibly 7alpha-OH-E2 or 2-OH-E2 (catecholestrogen). Both tissues also produced estradiol-17-glucuronide (E2-17-G), estradiol-17-sulfate (E2-17-S), and estradiol-3-glucuronide (E2-3-G). Compared to the kidney, the liver produced half the amount of conjugated metabolites, but twofold more E2-OH. The following xenobiotics (at a concentration of 100 microM) inhibited the production of water-soluble (i.e., conjugated) E2 metabolites by both the kidney and liver: 4,4'-(OH)2-3,3',5,5'- tetrachlorobiphenyl (4,4'-OH-TCB), bisphenol A (BPA), tetrabromobisphenol A (TB-BPA), tetrachlorobisphenol A (TC-BPA), tribromophenol (TBP), trichlorophenol (TCP), and pentachlorophenol (PCP). The alkylphenols, 4-n-nonylphenol (NP), and 4-octylphenol (OP), and 2,2',4,4'-tetrabromodiphenyl ether (TBDE) had no significant effect on E2 metabolism by either tissue. Testosterone and 17alpha,20beta-dihydroxy-4-pregnen-3-one inhibited the production of conjugated E2 metabolites by both the kidney and liver. Cortisol and 11-ketotestosterone inhibited E2 metabolism by the liver only. The median inhibitory concentrations (IC50) for 4,4'-OH-TCB ranged from 7-32 microM in the kidney and 0.6-1.6 microM in the liver. For BPA, IC50's ranged from 40-108 microM in the kidney and 11-18 microM in the

  17. Man, apes, and Old World monkeys differ from other mammals in the expression of alpha-galactosyl epitopes on nucleated cells.

    PubMed

    Galili, U; Shohet, S B; Kobrin, E; Stults, C L; Macher, B A

    1988-11-25

    The study of the expression of alpha-galactosyl epitopes on various mammalian cells is of particular interest, since as much as 1% of circulating IgG antibodies in humans interact with this carbohydrate residue. This natural antibody, designated "anti-Gal," was previously found to bind to terminal Gal alpha 1----3Gal beta 1----4GlcNAc-R on biochemically defined glycolipids (Galili, U., Macher, B. A., Buehler, J., and Shohet, S. B. (1985) J. Exp. Med. 162, 573-582; Galili, U., Buehler, J., Shohet, S. B., and Macher, B. A. (1987) J. Exp. Med. 165, 693-704). The expression of anti-Gal binding epitopes on nucleated cells from various mammalian species was studied by immunostaining with this antibody. The binding of anti-Gal to various cells was correlated with the binding of the lectin Bandeiraea (Griffonia) simplicifolia IB4 (BS lectin). The BS lectin also interacts with alpha-galactosyl residues and particularly with high affinity with Gal alpha 1----3Gal beta 1----4GlcNAc residues. We observed a striking evolutionary pattern in the expression of these epitopes on mammalian nucleated cells. Fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, and lymphoid cells of nonprimate mammals, prosimians, and New World monkeys readily bound both anti-Gal and BS lectin. However, no such binding was detectable on cells of Old World monkeys, apes, and humans. Measurment of the binding of radiolabeled BS lectin to the various nucleated cells suggests that cells binding anti-Gal express 10(6) to 3.5 x 10(7) alpha-galactosyl epitopes, most of which, based on the anti-Gal specificity, seem to have the structure of Gal alpha 1----3Gal beta 1----4GlcNAc-R. The absence of these epitopes from human cells results from diminished activity of the enzyme alpha 1----3 galactosyltransferase, which catalyzes the following reaction. Gal beta 1----4GlcNAc-R + UDP-Gal(alpha 1----3-galactosyltransferase)----Gal alpha 1----3Gal beta 1----4GlcNAc-R + UDP This enzyme, which

  18. Down-regulation of intestinal scavenger receptor class B, type I (SR-BI) expression in rodents under conditions of deficient bile delivery to the intestine.

    PubMed Central

    Voshol, P J; Schwarz, M; Rigotti, A; Krieger, M; Groen, A K; Kuipers, F

    2001-01-01

    Scavenger receptor class B, type I (SR-BI) is expressed in the intestines of rodents and has been suggested to be involved in the absorption of dietary cholesterol. The aim of this study was to determine whether intestinal SR-BI expression is affected in animal models with altered bile delivery to the intestine and impaired cholesterol absorption. SR-BI protein and mRNA levels were determined in proximal and distal small intestine from control, bile-duct-ligated and bile-diverted rats and from control and bile-duct-ligated mice. Two genetically altered mouse models were studied: multidrug resistance-2 P-glycoprotein-deficient [Mdr2((-/-))] mice that produce phospholipid/cholesterol-free bile, and cholesterol 7alpha-hydroxylase-deficient [Cyp7a((-/-))] mice, which exhibit qualitative and quantitative changes in the bile-salt pool. Cholesterol-absorption efficiency was quantified using a dual-isotope ratio method. SR-BI was present at the apical membrane of enterocytes in control rats and mice and was more abundant in proximal than in distal segments of the intestine. In bile-duct-ligated animals, levels of SR-BI protein were virtually absent and mRNA levels were decreased by approximately 50%. Bile-diverted rats, Mdr2((-/-)) mice and Cyp7a((-/-)) mice showed decreased levels of intestinal SR-BI protein while mRNA levels were unaffected. Cholesterol absorption was reduced by >90% in bile-duct-ligated and bile-diverted animals and in Cyp7a((-/-)) mice, whereas Mdr2((-/-)) mice showed an approximately 50% reduction. This study shows that SR-BI is expressed at the apical membrane of enterocytes of rats and mice, mainly in the upper intestine where cholesterol absorption is greatest, and indicates that bile components play a role in post-transcriptional regulation of SR-BI expression. Factors associated with cholestasis appear to be involved in transcriptional control of intestinal SR-BI expression. The role of SR-BI in the cholesterol-absorption process remains to be

  19. SUPERNOVA REMNANT PROGENITOR MASSES IN M31

    SciTech Connect

    Jennings, Zachary G.; Williams, Benjamin F.; Dalcanton, Julianne J.; Gilbert, Karoline M.; Fouesneau, Morgan; Weisz, Daniel R.; Murphy, Jeremiah W.; Dolphin, Andrew E. E-mail: adolphin@raytheon.com

    2012-12-10

    Using Hubble Space Telescope photometry, we age-date 59 supernova remnants (SNRs) in the spiral galaxy M31 and use these ages to estimate zero-age main-sequence masses (M{sub ZAMS}) for their progenitors. To accomplish this, we create color-magnitude diagrams (CMDs) and employ CMD fitting to measure the recent star formation history of the regions surrounding cataloged SNR sites. We identify any young coeval population that likely produced the progenitor star, then assign an age and uncertainty to that population. Application of stellar evolution models allows us to infer the M{sub ZAMS} from this age. Because our technique is not contingent on identification or precise location of the progenitor star, it can be applied to the location of any known SNRs. We identify significant young star formation around 53 of the 59 SNRs and assign progenitor masses to these, representing a factor of {approx}2 increase over currently measured progenitor masses. We consider the remaining six SNRs as either probable Type Ia candidates or the result of core-collapse progenitors that have escaped their birth sites. In general, the distribution of recovered progenitor masses is bottom-heavy, showing a paucity of the most massive stars. If we assume a single power-law distribution, dN/dM{proportional_to}M{sup {alpha}}, then we find a distribution that is steeper than a Salpeter initial mass function (IMF) ({alpha} = -2.35). In particular, we find values of {alpha} outside the range -2.7 {>=} {alpha} {>=} -4.4 to be inconsistent with our measured distribution at 95% confidence. If instead we assume a distribution that follows a Salpeter IMF up to some maximum mass, then we find that values of M{sub Max} > 26 are inconsistent with the measured distribution at 95% confidence. In either scenario, the data suggest that some fraction of massive stars may not explode. The result is preliminary and requires more SNRs and further analysis. In addition, we use our distribution to estimate a

  20. FTIR difference and resonance Raman spectroscopy of rhodopsins with applications to optogenetics

    NASA Astrophysics Data System (ADS)

    Saint Clair, Erica C.

    The major aim of this thesis is to investigate the molecular basis for the function of several types of rhodopsins with special emphasis on their application to the new field of optogenetics. Rhodopsins are transmembrane biophotonic proteins with 7 alpha-helices and a retinal chromophore. Studies included Archaerhodopsin 3 (AR3), a light driven proton pump similar to the extensively studied bacteriorhodopsin (BR); channelrhodopsins 1 and 2, light-activated ion channels; sensory rhodopsin II (SRII), a light-sensing protein that modulates phototaxis used in archaebacteria; and squid rhodopsins (sRho), the major photopigment in squid vision and a model for human melanopsin, which controls circadian rhythms. The primary techniques used in these studies were FTIR difference spectroscopy and resonance Raman spectroscopy. These techniques, in combination with site directed mutagenesis and other biochemical methodologies produced new knowledge regarding the structural changes of the retinal chromophore, the location and function of internal water molecules as well as specific amino acids and peptide backbone. Specialized techniques were developed that allowed rhodopsins to be studied in intact membrane environments and in some cases in vivo measurements were made on rhodopsin heterologously expressed in E. coli thus allowing the effects of interacting proteins and membrane potential to be investigated. Evidence was found that the local environment of one or more internal water molecules in SRII is altered by interaction with its cognate transducer, HtrII, and is also affected by the local lipid environment. In the case of AR3, many of the broad IR continuum absorption changes below 3000 cm -1, assigned to networks of water molecules involved in proton transport through cytoplasmic and extracellular portions in BR, were found to be very similar to BR. Bands assigned to water molecules near the Schiff base postulated to be involved in proton transport were, however, shifted

  1. Na(v)1.7 and Na(v)1.3 are the only tetrodotoxin-sensitive sodium channels expressed by the adult guinea pig enteric nervous system.

    PubMed

    Sage, D; Salin, P; Alcaraz, G; Castets, F; Giraud, P; Crest, M; Mazet, B; Clerc, N

    2007-10-01

    The types of sodium channels that are expressed by neurons shape the rising phase of action potentials and influence patterns of action potential discharge. With regard to the enteric nervous system (ENS), there is uncertainty about which channels are expressed, and in particular it is unknown whether Na(v)1.7 is present. We designed specific probes for the guinea pig Na(v)1.7 alpha subunit as well as for the other tetrodotoxin (TTX)-sensitive alpha subunits (Na(v)1.1, Na(v)1.2, Na(v)1.3, and Na(v)1.6) in order to perform in situ hybridization (ISH) histochemistry on guinea pig myenteric ganglia. We established that only Na(v)1.7 mRNA and Na(v)1.3 mRNA are expressed in these ganglia. The ISH signal for Na(v)1.7 transcripts was found in seemingly all the myenteric neurons. The expression of the Na(v)1.3 alpha subunit was confirmed by immunohistochemistry in a large proportion (62%) of the myenteric neuron population. This population included enteric sensory neurons. Na(v)1.6 immunoreactivity, absent from myenteric neurons, was detected in glial cells only when a high anti-Na(v)1.6 antibody concentration was used. This suggests that the Na(v)1.6 alpha subunit and mRNA are present only at low levels, which is consistent with the fact that no Na(v)1.6 mRNA could be detected in the ENS by ISH. The fact that adult myenteric neurons are endowed with only two TTX-sensitive alpha subunits, namely, Na(v)1.3 and Na(v)1.7, emphasizes the singularity of the ENS. Both these subunits, known to have slow-inactivation kinetics, are well adapted for generating action potentials from slow excitatory postsynaptic potentials, a mode of synaptic transmission that applies to all ENS neuron types.

  2. Independent repression of bile acid synthesis and activation of c-Jun N-terminal kinase (JNK) by activated hepatocyte fibroblast growth factor receptor 4 (FGFR4) and bile acids.

    PubMed

    Yu, Chundong; Wang, Fen; Jin, Chengliu; Huang, Xinqiang; McKeehan, Wallace L

    2005-05-06

    The fibroblast growth factor (FGF) receptor complex is a regulator of adult organ homeostasis in addition to its central role in embryonic development and wound healing. FGF receptor 4 (FGFR4) is the sole FGFR receptor kinase that is significantly expressed in mature hepatocytes. Previously, we showed that mice lacking mouse FGFR4 (mR4(-/-)) exhibited elevated fecal bile acids, bile acid pool size, and expression of liver cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for canonical neutral bile acid synthesis. To prove that hepatocyte FGFR4 was a negative regulator of cholesterol metabolism and bile acid synthesis independent of background, we generated transgenic mice overexpressing a constitutively active human FGFR4 (CahR4) in hepatocytes and crossed them with the FGFR4-deficient mice to generate CahR4/mR4(-/-) mice. In mice expressing active FGFR4 in liver, fecal bile acid excretion was 64%, bile acid pool size was 47%, and Cyp7a1 expression was 10-30% of wild-type mice. The repressed level of Cyp7a1 expression was resistant to induction by a high cholesterol diet relative to wild-type mice. Expression of CahR4 in mR4(-/-) mouse livers depressed bile acid synthesis below wild-type levels from the elevated levels observed in mR4(-/-). Levels of phosphorylated c-Jun N-terminal kinase (JNK), which is part of a pathway implicated in bile acid-mediated repression of synthesis, was 30% of wild-type levels in mR4(-/-) livers, whereas CahR4 livers exhibited an average 2-fold increase. However, cholate still strongly induced phospho-JNK in mR4(-/-) livers. These results confirm that hepatocyte FGFR4 regulates bile acid synthesis by repression of Cyp7a1 expression. Hepatocyte FGFR4 may contribute to the repression of bile acid synthesis through JNK signaling but is not required for activation of JNK signaling by bile acids.

  3. Free fatty acids repress small heterodimer partner (SHP) activation and adiponectin counteracts bile acid-induced liver injury in superobese patients with nonalcoholic steatohepatitis.

    PubMed

    Bechmann, Lars P; Kocabayoglu, Peri; Sowa, Jan-Peter; Sydor, Svenja; Best, Jan; Schlattjan, Martin; Beilfuss, Anja; Schmitt, Johannes; Hannivoort, Rebekka A; Kilicarslan, Alpaslan; Rust, Christian; Berr, Frieder; Tschopp, Oliver; Gerken, Guido; Friedman, Scott L; Geier, Andreas; Canbay, Ali

    2013-04-01

    Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries and may proceed to steatohepatitis (NASH). Apoptosis and free fatty acid (FFA)-induced lipotoxicity are important features of NASH pathogenesis. We have shown a hepatoprotective effect of adiponectin in steatotic livers of hepatitis C virus (HCV) patients and recent data links bile acid (BA) metabolism to the pathogenesis of NAFLD. The aim of this study was to identify potential interactions between BA and FFA metabolism in NAFLD. Liver biopsies and serum samples from 113 morbidly obese patients receiving bariatric surgery, healthy individuals, and moderately obese NAFLD patients were studied. Serum FFA, BA, and M30 were increased in NASH versus simple steatosis, while adiponectin was significantly decreased. The NAFLD activity score (NAS) score correlated with BA levels and reversely with adiponectin. Adiponectin reversely correlated with CD95/Fas messenger RNA (mRNA) and hepatocellular apoptosis. The BA transporter high-affinity Na+ /taurocholate cotransporter (NTCP) and the BA synthesizing enzyme cholesterol 7 alpha-hydroxylase (CYP7A1) were significantly up-regulated in obese patients and hepatoma cells exposed to FFA. Up-regulation of NTCP and CYP7A1 indicate failure to activate small heterodimer partner (SHP) upon farnesoid X receptor (FXR) stimulation by increasing BA concentrations. In line with the NAS score, adiponectin levels were reversely correlated with BA levels. Adiponectin correlated with NTCP and affects Cyp7A1 expression both in vivo and in vitro. BA synthesis and serum BA levels correlated with disease severity in NAFLD, while adiponectin is reversely correlated. FFA exposure prevented SHP-mediated repression of NTCP and Cyp7A1 expression, which lead to increased BA synthesis and uptake. In NASH, BA accumulation induced hepatocyte cell death and late FXR activation failed to prevent hepatocyte injury due to decreased adiponectin levels. Early

  4. Effects of salvinorin A, a kappa-opioid hallucinogen, on a neuroendocrine biomarker assay in nonhuman primates with high kappa-receptor homology to humans.

    PubMed

    Butelman, Eduardo R; Mandau, Marek; Tidgewell, Kevin; Prisinzano, Thomas E; Yuferov, Vadim; Kreek, Mary Jeanne

    2007-01-01

    This study focused on the in vivo effects of the kappa-opioid hallucinogen salvinorin A, derived from the plant Salvia divinorum. The effects of salvinorin A (0.0032-0.056 mg/kg i.v.) were studied in a neuroendocrine biomarker assay of the anterior pituitary hormone prolactin in gonadally intact, adult male and female rhesus monkeys (n = 4 each). Salvinorin A produced dose- and time-dependent neuroendocrine effects, similar to the synthetic high-efficacy kappa-agonist U69,593 ((+)-(5alpha,7 alpha,8beta)-N-methyl-N-[7-(1-pyrrolidiniyl)-1-oxaspiro[4.5]dec-8yl]-benzeneacetamide), but of shorter duration than the latter. Salvinorin A was approximately equipotent to U69,593 in this endpoint (salvinorin A ED50, 0.015 mg/kg; U69,593 ED(50), 0.0098 mg/kg). The effects of i.v. salvinorin A were not prevented by a small dose of the opioid antagonist nalmefene (0.01 mg/kg s.c.) but were prevented by a larger dose of nalmefene (0.1 mg/kg); the latter nalmefene dose is sufficient to produce kappa-antagonist effects in this species. In contrast, the 5HT2 receptor antagonist ketanserin (0.1 mg/kg i.m.) did not prevent the effects of salvinorin A. As expected, the neuroendocrine effects of salvinorin A (0.0032 mg/kg i.v.) were more robust in female than in male subjects. Related studies focused on full-length cloning of the coding region of the rhesus monkey kappa-opioid receptor (OPRK1) gene and revealed a high homology of the nonhuman primate OPRK1 gene compared with the human OPRK1 gene, including particular C-terminal residues thought to be involved in receptor desensitization and internalization. The present studies indicate that the hallucinogen salvinorin A acts as a high-efficacy kappa-agonist in nonhuman primates in a translationally viable neuroendocrine biomarker assay.

  5. Regulation of pituitary gonadotropin-releasing hormone receptors by androgens in the male rabbit.

    PubMed

    Limonta, P; Ladizhenskaya, A; Gunsalus, G L; Bardin, C W; Thau, R B

    1986-01-01

    The regulation of pituitary GnRH receptors was studied in adult male rabbits after castration and androgen replacement with testosterone (T) or 7 alpha-methyl-19-nortestosterone acetate (U-15,614; T analog) supplied by Silastic capsules implanted sc. Castration increased pituitary GnRH receptors significantly, from 99.3 to 329.5 fmol/mg protein within 4 weeks, without a change in the equilibrium association constant. Serum LH concentrations increased from 0.45 to maximum levels of 2.6 ng/ml by day 8 after orchiectomy; these levels persisted throughout the 4 weeks of study. Serum FSH reached maximum levels of 33.6 ng/ml 5 days after castration. T replacement with 250, 500, and 1000 micrograms/kg X day, prevented a postcastration rise in both pituitary GnRH receptor concentrations and gonadotropin secretion, while 100 micrograms/kg X day prevented an increase in GnRH receptors, but did not completely inhibit hypersecretion of gonadotropins. Administration of T analog at doses of 6.25 and 12.5 micrograms/kg X day partially suppressed the castration-induced increase in pituitary GnRH receptor concentrations, while 25, 50, and 100 micrograms/kg X day suppressed GnRH-binding sites to the levels found in intact controls in 15 of 16 rabbits. By contrast, none of the T analog doses was able to prevent completely LH and FSH hypersecretion. The fact that both T and T analog induced dose-dependent stimulation of prostate and seminal vesicle weights indicates that there are tissue-specific differences in the sensitivity to androgens. We conclude that in the male rabbit 1) pituitary GnRH receptors significantly increase after castration; 2) this increase may partially mediate the postcastration hypersecretion of LH and FSH; 3) castration-induced effects can be prevented by androgen replacement. These results are similar to those obtained in rats, where castration increases LHRH receptors, but contrast with results in mice and hamsters, where castration either reduces or does not

  6. Xylobiose, an Alternative Sweetener, Ameliorates Diabetes-Related Metabolic Changes by Regulating Hepatic Lipogenesis and miR-122a/33a in db/db Mice.

    PubMed

    Lim, Eunjin; Lim, Ji Ye; Kim, Eunju; Kim, Yoo-Sun; Shin, Jae-Ho; Seok, Pu Reum; Jung, Sangwon; Yoo, Sang-Ho; Kim, Yuri

    2016-12-05

    Type 2 diabetes is a major public health concern worldwide. Xylobiose (XB) consists of two molecules of d-xylose and is a major disaccharide in xylooligosaccharides that are used as prebiotics. We hypothesized that XB could regulate diabetes-related metabolic and genetic changes via microRNA expression in db/db mice. For six weeks, C57BL/KsJ-db/db mice received 5% XB as part of the total sucrose content of their diet. XB supplementation improved glucose tolerance with reduced levels of OGTT AUC, fasting blood glucose, HbA1c, insulin, and HOMA-IR. Furthermore, XB supplementation decreased the levels of total triglycerides, total cholesterol, and LDL-C. The expression levels of miR-122a and miR-33a were higher and lower in the XB group, respectively. In the liver, expressions of the lipogenic genes, including, fatty acid synthase (FAS), peroxisome proliferator activated receptor γ (PPARγ), sterol regulatory element-binding protein-1C (SREBP-1C), sterol regulatory element-binding protein-2 (SREBP-2), acetyl-CoA carboxylase (ACC), HMG-CoA reductase (HMGCR), ATP-binding cassette transporter G5/G8 (ABCG5/8), cholesterol 7 alpha-hydroxylase (CYP7A1), and sterol 12-alpha-hydroxylase (CYP8B1), as well as oxidative stress markers, including superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), glutathione peroxidase (GPX), and catalase, were also regulated by XB supplementation. XB supplementation inhibited the mRNA expressions levels of the pro-inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1, as well as phosphorylation of c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK), p38 mitogen-activated protein kinases (MAPK), and extracellular signal-regulated kinases 1/2 (ERK1/2). These data demonstrate that XB exhibits anti-diabetic, hypolipogenic, and anti-inflammatory effects via regulation of the miR-122a/33a axis in db/db mice.

  7. Differential pattern of integrin receptor expression in differentiated and anaplastic thyroid cancer cell lines.

    PubMed

    Hoffmann, S; Maschuw, K; Hassan, I; Reckzeh, B; Wunderlich, A; Lingelbach, S; Zielke, A

    2005-09-01

    Adhesion of tumor cells to the extracellular matrix (ECM) is a crucial step for the development of metastatic disease and is mediated by specific integrin receptor molecules (IRM). The pattern of metastatic spread differs substantially among the various histotypes of thyroid cancer (TC). However, IRM have only occasionally been characterized in TC until now. IRM expression was investigated in 10 differentiated (FTC133, 236, 238, HTC, HTC TSHr, XTC, PTC4.0/4.2, TPC1, Kat5) and two anaplastic TC cell lines (ATC, C643, Hth74), primary cultures of normal thyroid tissue (Thy1,3), and thyroid cancer specimens (TCS). Expression of 16 IRM (beta1-4, beta7, alpha1-6, alphaV, alphaIIb, alphaL, alphaM, alphaX) and of four IRM heterodimers (alpha2beta1, alpha5beta1, alphaVbeta3, alphaVbeta5), was analyzed by fluorescent-activated cell sorter (FACS) and immunohistochemical staining. Thyroid tumor cell adhesion to ECM proteins and their IRM expression in response to thyrotropin (TSH) was assessed. Follicular TC cell lines presented high levels of integrins alpha2, alpha3, alpha5, beta1, beta3 and low levels of alpha1, whereas papillary lines expressed a heterogenous pattern of IRM, dominated by alpha5 and beta1. ATC mainly displayed integrins alpha2, alpha3, alpha5, alpha6, beta1 and low levels of alpha1, alpha4 and alphaV. Integrin heterodimers correlated with monomer expression. Evaluation of TCS largely confirmed these results with few exceptions, namely alpha4, alpha6, and beta3. The ability of TC cell lines to adhere to purified ECM proteins correlated with IRM expression. TSH induced TC cell adhesion in a dose-dependent fashion, despite an unchanged array of IRM expression or level of a particular IRM. Thyroid carcinoma cell lines of different histogenetic background display profoundly different patterns of IRM expression that appear to correlate with tumor aggressiveness. In vitro adhesion to ECM proteins and IRM expression concur. Finally, TSH-stimulated adhesion of

  8. Identification of QTL underlying vitamin E contents in soybean seed among multiple environments.

    PubMed

    Li, Haiyan; Liu, Huancheng; Han, Yingpeng; Wu, Xiaoxia; Teng, Weili; Liu, Guifeng; Li, Wenbin

    2010-05-01

    Vitamin E (VE) in soybean seed has value for foods, medicines, cosmetics, and animal husbandry. Selection for higher VE contents in seeds along with agronomic traits was an important goal for many soybean breeders. In order to map the loci controlling the VE content, F(5)-derived F(6) recombinant inbred lines (RILs) were advanced through single-seed-descent (SSD) to generate a population including 144 RILs. The population was derived from a cross between 'OAC Bayfield', a soybean cultivar with high VE content, and 'Hefeng 25', a soybean cultivar with low VE content. A total of 107 polymorphic simple sequence repeat markers were used to construct a genetic linkage map. Seed VE contents were analyzed by high performance liquid chromatography for multiple years and locations (Harbin in 2007 and 2008, Hulan in 2008 and Suihua in 2008). Four QTL associated with alpha-Toc (on four linkage groups, LGs), eight QTL associated with gamma-Toc (on eight LGs), four QTL associated with delta-Toc (on four LGs) and five QTL associated with total VE (on four LGs) were identified. A major QTL was detected by marker Satt376 on linkage group C2 and associated with alpha-Toc (0.0012 > P > 0.0001, 5.0% < R (2) < 17.0%, 25.1 < alpha-Toc < 30.1 microg g(-1)), total VE (P < 0.0001, 7.0% < R (2) < 10.0%, 118.2 < total VE < 478.3 microg g(-1)). A second QTL detected by marker Satt286 on LG C2 was associated with gamma-Toc (0.0003 > P > 0.0001, 6.0% < R (2) < 13.0%, 141.5 < gamma-Toc < 342.4 microg g(-1)) and total VE (P < 0.0001, 2.0% < R (2) < 9.0%, 353.9 < total VE < 404.0 microg g(-1)). Another major QTL was detected by marker Satt266 on LG D1b that was associated with alpha-Toc (0.0002 > P > 0.0001, 4.0% < R (2) < 6.0%, 27.7 < alpha-Toc < 43.7 microg g(-1)) and gamma-Toc (0.0032 > P > 0.0001, 3.0% < R (2) < 10.0%, 69.7 < gamma-Toc < 345.7 microg g(-1)). Since beneficial alleles were all from 'OAC Bayfield', it was concluded that these three QTL would have great potential value for marker

  9. Functional interaction of hepatic nuclear factor-4 and peroxisome proliferator-activated receptor-gamma coactivator 1alpha in CYP7A1 regulation is inhibited by a key lipogenic activator, sterol regulatory element-binding protein-1c.

    PubMed

    Ponugoti, Bhaskar; Fang, Sungsoon; Kemper, Jongsook Kim

    2007-11-01

    Insulin inhibits transcription of cholesterol 7alpha-hydroxylase (Cyp7a1), a key gene in bile acid synthesis, and the hepatic nuclear factor-4 (HNF-4) site in the promoter was identified as a negative insulin response sequence. Using a fasting/feeding protocol in mice and insulin treatment in HepG2 cells, we explored the inhibition mechanisms. Expression of sterol regulatory element-binding protein-1c (SREBP-1c), an insulin-induced lipogenic factor, inversely correlated with Cyp7a1 expression in mouse liver. Interaction of HNF-4 with its coactivator, peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), was observed in livers of fasted mice and was reduced after feeding. Conversely, HNF-4 interaction with SREBP-1c was increased after feeding. In vitro studies suggested that SREBP-1c competed with PGC-1alpha for direct interaction with the AF2 domain of HNF-4. Reporter assays showed that SREBP-1c, but not of a SREBP-1c mutant lacking the HNF-4 interacting domain, inhibited HNF-4/PGC-1alpha transactivation of Cyp7a1. SREBP-1c also inhibited PGC-1alpha-coactivation of estrogen receptor, constitutive androstane receptor, pregnane X receptor, and farnesoid X receptor, implying inhibition of HNF-4 by SREBP-1c could extend to other nuclear receptors. In chromatin immunoprecipitation studies, HNF-4 binding to the promoter was not altered, but PGC-1alpha was dissociated, SREBP-1c and histone deacetylase-2 (HDAC2) were recruited, and acetylation of histone H3 was decreased upon feeding. Adenovirus-mediated expression of a SREBP-1c dominant-negative mutant, which blocks the interaction of SREBP-1c and HNF-4, partially but significantly reversed the inhibition of Cyp7a1 after feeding. Our data show that SREBP-1c functions as a non-DNA-binding inhibitor and mediates, in part, suppression of Cyp7a1 by blocking functional interaction of HNF-4 and PGC-1alpha. This mechanism may be relevant to known repression of many other HNF-4 target genes upon

  10. CYP7A1 (-204 A>C; rs3808607 and -469 T>C; rs3824260) promoter polymorphisms and risk of gallbladder cancer in North Indian population.

    PubMed

    Srivastava, Anvesha; Choudhuri, Gourdas; Mittal, Balraj

    2010-06-01

    Cholesterol 7-alpha hydroxylase (CYP7A1), which is a rate-limiting enzyme for cholesterol catabolism and bile acid synthesis, may affect cholesterol homeostasis and result in gallstone formation that is a major risk factor for gallbladder cancer (GBC) pathogenesis. Genetic variations in CYP7A1 may influence its expression and thus may affect the risk of gallstone disease and GBC. We aimed to study the association of 2 promoter polymorphisms of CYP7A1 (-204 A>C [rs3808607] and -469 T>C [rs3824260]) in gallstone and GBC susceptibility in North Indian population. The study included 185 GBC patients, 195 symptomatic gallstone patients, and 200 healthy controls. Genotyping for both polymorphisms was done by polymerase chain reaction-restriction fragment length polymorphism method. Although the CC genotype of CYP7A1 -204 A>C was not significantly associated with gallstone disease (P = .083, odds ratio [OR] = 1.69, 95% confidence interval [CI] = 0.9-3.0), it was conferring higher risk for GBC (P = .018, OR = 2.05, 95% CI = 1.1-3.7). However, CYP7A1 -469 T>C was not associated with gallstone disease and GBC risk in our population. After subgroup stratifications on the basis of sex and gallstone status, CC genotype and variant allele of CYP7A1 -204 A>C imparted higher risk for GBC in women (P = .003, OR = 3.30, 95% CI = 1.5-7.2) and patients without gallstones (P = .045, OR = 1.91, 95% CI = 1.2-3.6). Haplotype analysis of the 2 polymorphisms showed that C,T (P = .045, OR = 1.84, 95% CI = 1.0-3.3) and C,C (P = .0001, OR = 3.10, 95% CI = 1.6-6.0) haplotypes had elevated risk of GBC predisposition. CYP7A1 -469 T>C is not associated with gallstone disease or GBC risk. Although CYP7A1 -204 A>C might play a modest role in gallstone susceptibility, it is an independent risk factor for GBC in North Indian population. Underlying mechanism for GBC susceptibility by CYP7A1 (-204 A>C and -469 T>C) haplotype appears to be independent of gallstone pathway and is believed to involve

  11. Mice expressing the human CYP7A1 gene in the mouse CYP7A1 knock-out background lack induction of CYP7A1 expression by cholesterol feeding and have increased hypercholesterolemia when fed a high fat diet.

    PubMed

    Chen, Jean Y; Levy-Wilson, Beatriz; Goodart, Sheryl; Cooper, Allen D

    2002-11-08

    Cholesterol 7alpha-hydroxylase (CYP7A1) catalyzes the rate-limiting step in the pathway responsible for the formation of the majority of bile acids. Transcription of the gene is regulated by the size of the bile acid pool and dietary and hormonal factors. The farnesoid X receptor and the liver X receptor (LXR) are responsible for regulation by bile acids and cholesterol, respectively. To study the effects of dietary cholesterol and fat upon expression of the human CYP7A1 gene, mice were generated by crossing transgenic mice carrying the human CYP7A1 gene with mice that were homozygous knock-outs (CYP7A1(-/-)). The mice (mCYP7A1(-/-)/hCYP7A1) expressed the human gene at much higher levels than did the transgenics bred in the wild-type background. A diet containing 1% cholic acid reduced the expression of the human gene in mCYP7A1(-/-)/hCYP7A1 mice to undetectable levels. Cholestyramine (5%) increased the level of expression of the human gene and the mouse gene. Thus, farnesoid X receptor-mediated regulation was preserved. A diet containing 2% cholesterol increased expression of the mouse gene in wild-type mice, but it did not affect expression of the human gene in mCYP7A1(-/-)/hCYP7A1 mice. None of the diets altered the serum cholesterol or triglyceride levels in these mice; 1% cholic acid caused a redistribution of cholesterol from the high density lipoprotein to the low density lipoprotein density in the humanized mice but not in wild-type mice. A diet containing 30% saturated fat and 2% cholesterol caused a decrease in CYP7A1 levels in mCYP7A1(-/-)/hCYP7A1 mice. The serum cholesterol levels rose in all mice fed this diet. The increase was greater in the mCYP7A1(-/-)/hCYP7A1 mice. Together, these data suggest that the lack of an LXR element in the region from -56 to -49 of the human CYP7A1 promoter may account for some of the differences in response to diets between humans and rodents.

  12. Xylobiose, an Alternative Sweetener, Ameliorates Diabetes-Related Metabolic Changes by Regulating Hepatic Lipogenesis and miR-122a/33a in db/db Mice

    PubMed Central

    Lim, Eunjin; Lim, Ji Ye; Kim, Eunju; Kim, Yoo-Sun; Shin, Jae-Ho; Seok, Pu Reum; Jung, Sangwon; Yoo, Sang-Ho; Kim, Yuri

    2016-01-01

    Type 2 diabetes is a major public health concern worldwide. Xylobiose (XB) consists of two molecules of d-xylose and is a major disaccharide in xylooligosaccharides that are used as prebiotics. We hypothesized that XB could regulate diabetes-related metabolic and genetic changes via microRNA expression in db/db mice. For six weeks, C57BL/KsJ-db/db mice received 5% XB as part of the total sucrose content of their diet. XB supplementation improved glucose tolerance with reduced levels of OGTT AUC, fasting blood glucose, HbA1c, insulin, and HOMA-IR. Furthermore, XB supplementation decreased the levels of total triglycerides, total cholesterol, and LDL-C. The expression levels of miR-122a and miR-33a were higher and lower in the XB group, respectively. In the liver, expressions of the lipogenic genes, including, fatty acid synthase (FAS), peroxisome proliferator activated receptor γ (PPARγ), sterol regulatory element-binding protein-1C (SREBP-1C), sterol regulatory element-binding protein-2 (SREBP-2), acetyl-CoA carboxylase (ACC), HMG-CoA reductase (HMGCR), ATP-binding cassette transporter G5/G8 (ABCG5/8), cholesterol 7 alpha-hydroxylase (CYP7A1), and sterol 12-alpha-hydroxylase (CYP8B1), as well as oxidative stress markers, including superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), glutathione peroxidase (GPX), and catalase, were also regulated by XB supplementation. XB supplementation inhibited the mRNA expressions levels of the pro-inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1, as well as phosphorylation of c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK), p38 mitogen-activated protein kinases (MAPK), and extracellular signal-regulated kinases 1/2 (ERK1/2). These data demonstrate that XB exhibits anti-diabetic, hypolipogenic, and anti-inflammatory effects via regulation of the miR-122a/33a axis in db/db mice. PMID:27929393

  13. Obeticholic acid, a selective farnesoid X receptor agonist, regulates bile acid homeostasis in sandwich-cultured human hepatocytes.

    PubMed

    Zhang, Yuanyuan; Jackson, Jonathan P; St Claire, Robert L; Freeman, Kimberly; Brouwer, Kenneth R; Edwards, Jeffrey E

    2017-08-01

    Farnesoid X receptor (FXR) is a master regulator of bile acid homeostasis through transcriptional regulation of genes involved in bile acid synthesis and cellular membrane transport. Impairment of bile acid efflux due to cholangiopathies results in chronic cholestasis leading to abnormal elevation of intrahepatic and systemic bile acid levels. Obeticholic acid (OCA) is a potent and selective FXR agonist that is 100-fold more potent than the endogenous ligand chenodeoxycholic acid (CDCA). The effects of OCA on genes involved in bile acid homeostasis were investigated using sandwich-cultured human hepatocytes. Gene expression was determined by measuring mRNA levels. OCA dose-dependently increased fibroblast growth factor-19 (FGF-19) and small heterodimer partner (SHP) which, in turn, suppress mRNA levels of cholesterol 7-alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for de novo synthesis of bile acids. Consistent with CYP7A1 suppression, total bile acid content was decreased by OCA (1 μmol/L) to 42.7 ± 20.5% relative to control. In addition to suppressing de novo bile acids synthesis, OCA significantly increased the mRNA levels of transporters involved in bile acid homeostasis. The bile salt excretory pump (BSEP), a canalicular efflux transporter, increased by 6.4 ± 0.8-fold, and the basolateral efflux heterodimer transporters, organic solute transporter α (OSTα ) and OSTβ increased by 6.4 ± 0.2-fold and 42.9 ± 7.9-fold, respectively. The upregulation of BSEP and OSTα and OSTβ, by OCA reduced the intracellular concentrations of d8 -TCA, a model bile acid, to 39.6 ± 8.9% relative to control. These data demonstrate that OCA does suppress bile acid synthesis and reduce hepatocellular bile acid levels, supporting the use of OCA to treat bile acid-induced toxicity observed in cholestatic diseases. © 2017 Intercept Pharmaceuticals. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and

  14. Permeation enhancement of octreotide by specific bile salts in rats and human subjects: in vitro, in vivo correlations.

    PubMed Central

    Fricker, G.; Fahr, A.; Beglinger, C.; Kissel, T.; Reiter, G.; Drewe, J.

    1996-01-01

    1. The potential of bile salts to improve the enteral absorption of octreotide, an orally active somatostatin analogue, was investigated by a combination of in vitro, in situ and in vivo experiments. 2. Incorporation of octreotide into lipid monolayers (as measured by area increase of the monolayer at constant surface pressure using a Langmuir-Blodgett trough set-up) depended on the type of bile salt used for monolayer pre-treatment. Addition of 20 microM octreotide to the subphase containing 20 microM of the dihydroxylated bile salt ursodeoxycholate (UDCA) causes a 9% increase in area, whereas addition of octreotide to the subphase containing the 7 alpha-enantiomer of UDCA, chenodeoxycholate (CDCA), resulted in an area increase of the lipid monolayer of 20%. Area increase by octreotide alone was not significantly different from the increase of octreotide and UDCA in combination. 3. CDCA and UDCA in combination with octreotide increased the permeability of liposomal membranes for rubidium ions, whereas octreotide alone did not significantly change the permeability. This indicates membrane distortion as a possible cause for the enhanced absorption of octreotide by bile salts. 4. In polarized Caco-2 cell monolayers octreotide exhibited a permeation coefficient of 0.008 +/- 0.004 cm h-1. Addition of 0.2-1% of UDCA to the apical incubation medium had no significant effect upon the permeation coefficient. In contrast, 0.2-1% CDCA in the incubation medium resulted in a significant increase (P < 0.05) of the monolayer permeability of octreotide (0.015-0.037 cm h-1). 5. Octreotide was absorbed as the intact peptide from the gastrointestinal tract in rats with an absorption efficiency of 0.26%. Coadministration of bile salt resulted in a dose-dependent increase in absorption efficiency of the peptide up to 20.2%. The observed effect was more pronounced for CDCA than for UDCA. 6. The effect of CDCA and UDCA on octreotide absorption in vivo was assessed in a pharmacokinetic

  15. Complex genetic control of HDL levels in mice in response to an atherogenic diet. Coordinate regulation of HDL levels and bile acid metabolism.

    PubMed Central

    Machleder, D; Ivandic, B; Welch, C; Castellani, L; Reue, K; Lusis, A J

    1997-01-01

    Inbred strains of mice differ in susceptibility to atherogenesis when challenged with a high fat, high cholesterol diet containing 0.5% cholic acid. Studies of recombinant inbred (RI) strains derived from the susceptible strain C57BL/6J (B6) and the resistant strains C3H/HeJ (C3H) and BALB/cJ have revealed an association between fatty streak lesion size and a decrease in high density lipoprotein (HDL) levels on the diet. To better understand the genetic factors contributing to HDL metabolism and atherogenesis in response to the diet, we studied mice derived from an intercross between B6 and C3H using a complete linkage map approach. A total of 185 female progeny were typed for 134 genetic markers spanning the mouse genome, resulting in an average interval of about 10 cM between markers. A locus on distal chromosome 1 containing the apolipoprotein AII gene was linked to HDL-cholesterol levels on both the chow and the atherogenic diets, but this locus did not contribute to the decrease in HDL-cholesterol in response to the diet. At least three distinct genetic loci, on chromosomes 3, 5, and 11, exhibited evidence of linkage to a decrease in HDL-cholesterol after a dietary challenge. Since a bile acid (cholic acid) is required for the diet induced changes in HDL levels and for atherogenesis in these strains, we examined cholesterol-7-alpha hydroxylase (C7AH) expression. Whereas B6 mice exhibited a large decrease in C7AH mRNA levels in response to the diet, C3H showed an increase. Among the intercross mice, multiple loci contributed to the regulation of C7AH mRNA levels in response to the diet, the most notable of which coincided with the loci on chromosomes 3, 5, and 11 controlling HDL levels in response to the diet. None of these loci were linked to the C7AH structural gene which we mapped to proximal chromosome 4. These studies reveal coordinate regulation of C7AH expression and HDL levels, and they indicate that the genetic factors controlling HDL levels are more

  16. Three-dimensional quantitative structure-activity relationships of steroid aromatase inhibitors

    NASA Astrophysics Data System (ADS)

    Oprea, Tudor I.; García, Angel E.

    1996-06-01

    Inhibition of aromatase, a cytochrome P450 that converts androgens to estrogens, is relevant in the therapeutic control of breast cancer. We investigate this inhibition using a three-dimensional quantitative structure-activity relationship (3D QSAR) method known as Comparative Molecular Field Analysis, CoMFA [Cramer III, R.D. et al., J. Am. Chem. Soc., 110 (1988) 5959]. We analyzed the data for 50 steroid inhibitors [Numazawa, M. et al., J. Med. Chem., 37 (1994) 2198, and references cited therein] assayed against androstenedione on human placental microsomes. An initial CoMFA resulted in a three-component model for log(1/Ki), with an explained variance r2 of 0.885, and a cross-validated q2 of 0.673. Chemometric studies were performed using GOLPE [Baroni, M. et al., Quant. Struct.-Act. Relatsh., 12 (1993) 9]. The CoMFA/GOLPE model is discussed in terms of robustness, predictivity, explanatory power and simplicity. After randomized exclusion of 25 or 10 compounds (repeated 25 times), the q2 for one component was 0.62 and 0.61, respectively, while r2 was 0.674. We demonstrate that the predictive r2 based on the mean activity (Ym) of the training set is misleading, while the test set Ym-based predictive r2 index gives a more accurate estimate of external predictivity. Using CoMFA, the observed differences in aromatase inhibition among C6-substituted steroids are rationalized at the atomic level. The CoMFA fields are consistent with known, potent inhibitors of aromatase, not included in the model. When positioned in the same alignment, these compounds have distinct features that overlap with the steric and electrostatic fields obtained in the CoMFA model. The presence of two hydrophobic binding pockets near the aromatase active site is discussed: a steric bulk tolerant one, common for C4, C6-alpha and C7-alpha substitutents, and a smaller one at the C6-beta region.

  17. Fasting levels of monoketonic bile acids in human peripheral and portal circulation.

    PubMed

    Björkhem, I; Angelin, B; Einarsson, K; Ewerth, S

    1982-09-01

    It has been suggested that large amounts of ketonic bile acids may be present in portal venous blood. We have therefore determined the approximate concentration of 3-oxo-, 7-oxo-, and 12-oxo-bile acids (monoketonic bile acids) in human peripheral and portal circulation. These compounds were converted into the corresponding 3alpha-, 7alpha-, and 12alpha-hydroxy bile acids by treatment with sodium borodeuteride, thus increasing the molecular weight of each bile acid formed by one mass unit. The ratio between deuterated and nondeuterated bile acid was determined by combined gas-liquid chromatography-mass spectrometry with use of selected ion monitoring. From the ratio obtained and from the concentration of unlabeled bile acid, determined by isotope dilution-mass spectrometry, the approximate concentration of the different ketonic bile acids could be calculated. This method underestimates 3-oxygenated bile acids by 4-8%, 7-oxygenated bile acids by 2-3%, and 12-oxygenated bile acids by about 25%. The approximate concentration of monoketonic 3,7-oxygenated bile acids was found to be 0.08 +/- 0.02 and 0.37 +/- 0.25 micro mol/l in the peripheral venous serum and the portal venous serum, respectively. The approximate concentration of monoketonic 3,12-oxygenated bile acids was found to be 0.07 +/- 0.02 and 0.32 +/- 0.12 micro mol/l in the peripheral venous serum and the portal venous serum, respectively. The approximate concentration of monoketonic 3,7,12-oxygenated bile acids was found to be 0.03 +/- 0.01 and 0.14 +/- 0.05 micro mol/l in the peripheral venous serum and in the portal venous serum, respectively. The total concentration of the ketonic bile acids constituted only 9 +/- 1% and 8 +/- 3% of the nonoxidized bile acids in the peripheral venous serum and in the portal venous serum, respectively. Thus it seems less likely that the portal inflow of ketonic bile acids is of significant physiological importance under normal conditions.-Björkhem, I., B. Angelin, K

  18. Preconditioning with oil mixes of high ratio Omega-9: Omega-6 and a low ratio Omega-6:Omega-3 in rats subjected to brain ischemia/reperfusion.

    PubMed

    Pinheiro, Petrúcia Maria Antero; Campelo, Ana Paula Bomfim Soares; Guimarães, Sérgio Botelho; Patrocínio, Régia Maria Vidal do; Valença Junior, José Telmo; Vasconcelos, Paulo Roberto Leitão de

    2011-01-01

    This study aimed to assess the effects of preconditioning with mixtures of oils containing high/low ratio of ω-6/ω-3 and ω-9/ω-6, respectively, in an experimental model of cerebral ischemia-reperfusion (I/R). Forty-two Wistar rats were randomly distributed into two groups: control (n=24) and test (n=18). Control group was subdivided in 4 subgroups (n=6): G1: Sham-Water; G2: I/R-Water; G3: Sham-Isolipidic and G4: I/R-Isolipid. The animals received water or a isolipid mixture containing ω-3 oils (8:1 ratio) and ω-9/ω-6 (0.4:1 ratio) by gavage for seven days. Test group included 3 subgroups (n=6) G5: I/R-Mix1, G: 6 I/R-Mix2 and G7: I/R-Mix3. Test group animals received oily mixtures of ω-3 (1.4:1 ratio) and ω-6 (3.4:1 ratio), differing only in source of ω-3: G5 (alpha-linolenic acid); G6 (alpha-linolenic, docosahexaenoic and eicosapentaenoic acids), and G7 (alpha-linolenic and docosahexaenoic acids). On day 7 I/R rats underwent cerebral ischemia with bilateral occlusion of common carotid arteries for 1 hour followed by reperfusion for 3 hours. G1 and G3 animals underwent sham operation. Concluded the experiment, animals were decapitated and their brains sliced for red neurons (RN) count in CA3 area of the hippocampus. Variables were compared using ANOVA-Tukey test. The use of different mix preparations promoted a decrease in red cell count in all three groups (G5/G6/G7), compared with G2/G4, confirming the protective effect of different oil blends, regardless of ω-3 source. Pre-conditioning with mixtures of oils containing high ratio ω-6/ω-3 and low ω-9/ω-6 relationship protects brain neurons against I/R injury in an experimental model.

  19. Characterization of the retina in the alpha7 nicotinic acetylcholine receptor knockout mouse

    NASA Astrophysics Data System (ADS)

    Smith, Marci L.

    Acetylcholine receptors (AChRs) are involved in visual processing and are expressed by inner retinal neurons in all species studied to date (Keyser et al., 2000; Dmitrieva et al., 2007; Liu et al., 2009), but their distribution in the mouse retina remains unknown. Reductions in alpha7 nicotinic AChRs (nAChRs) are thought to contribute to memory and visual deficits observed in Alzheimer's and schizophrenia (Coyle et al., 1983; Nordberg et al., 1999; Leonard et al., 2006). However, the alpha7 nAChR knockout (KO) mouse has a mild phenotype (Paylor et al., 1998; Fernandes et al., 2006; Young et al., 2007; Origlia et al., 2012). The purpose of this study was to determine the expression of AChRs in wildtype (WT) mouse retina and to assess whether up-regulation of other AChRs in the alpha7 nAChR KO retina may explain the minimal deficits described in the KO mouse. Reverse-transcriptase PCR (RT-PCR) showed that mRNA transcripts for alpha2-7, alpha 9, alpha10, beta2-4 nAChR subunits and m1-m5 muscarinic AChR (mAChR) subtypes were present in WT murine retina. Western blot analysis confirmed the presence of alpha3-5, alpha9, and m1-m5 AChR proteins and immunohistochemical analysis demonstrated nAChR and mAChR proteins expressed by subsets of bipolar, amacrine and ganglion cells. This is the first reported expression of alpha9 and alpha10 nAChR transcripts and alpha9 nAChR proteins in the retina of any species. Quantitative RT-PCR (qPCR) showed changes in AChR transcript expression in the alpha7 nAChR KO mouse retina relative to WT. Within whole retina alpha2, alpha9, alpha10, beta4, m1 and m4 AChR transcripts were up-regulated, while alpha5 nAChR transcripts were down-regulated. However, cell populations showed subtle differences; m4 mAChR transcripts were up-regulated in the ganglion cell layer and outer portion of the inner nuclear layer (oINL),while beta4 nAChR transcript up-regulation was limited to the oINL. Surprisingly, alpha2, alpha9, beta4, m2 and m4 transcripts were

  20. Functional analyses of AmpC beta-lactamase through differential stability.

    PubMed Central

    Beadle, B. M.; McGovern, S. L.; Patera, A.; Shoichet, B. K.

    1999-01-01

    Despite decades of intense study, the complementarity of beta-lactams for beta-lactamases and penicillin binding proteins is poorly understood. For most of these enzymes, beta-lactam binding involves rapid formation of a covalent intermediate. This makes measuring the equilibrium between bound and free beta-lactam difficult, effectively precluding measurement of the interaction energy between the ligand and the enzyme. Here, we explore the energetic complementarity of beta-lactams for the beta-lactamase AmpC through reversible denaturation of adducts of the enzyme with beta-lactams. AmpC from Escherichia coli was reversibly denatured by temperature in a two-state manner with a temperature of melting (Tm) of 54.6 degrees C and a van't Hoff enthalpy of unfolding (deltaH(VH)) of 182 kcal/mol. Solvent denaturation gave a Gibbs free energy of unfolding in the absence of denaturant (deltaG(u)H2O) of 14.0 kcal/mol. Ligand binding perturbed the stability of the enzyme. The penicillin cloxacillin stabilized AmpC by 3.2 kcal/mol (deltaTm = +5.8 degrees C); the monobactam aztreonam stabilized the enzyme by 2.7 kcal/mol (deltaTm = +4.9 degrees C). Both acylating inhibitors complement the active site. Surprisingly, the oxacephem moxalactam and the carbapenem imipenem both destabilized AmpC, by 1.8 kcal/mol (deltaTm = -3.2 degrees C) and 0.7 kcal/mol (deltaTm = -1.2 degrees C), respectively. These beta-lactams, which share nonhydrogen substituents in the 6(7)alpha position of the beta-lactam ring, make unfavorable noncovalent interactions with the enzyme. Complexes of AmpC with transition state analog inhibitors were also reversibly denatured; both benzo(b)thiophene-2-boronic acid (BZBTH2B) and p-nitrophenyl phenylphosphonate (PNPP) stabilized AmpC. Finally, a catalytically inactive mutant of AmpC, Y150F, was reversibly denatured. It was 0.7 kcal/mol (deltaTm = -1.3 degrees C) less stable than wild-type (WT) by thermal denaturation. Both the cloxacillin and the moxalactam

  1. Decreased cyclin A2 and increased cyclin G1 levels coincide with loss of proliferative capacity in rat Leydig cells during pubertal development.

    PubMed

    Ge, R S; Hardy, M P

    1997-09-01

    Postnatal development of Leydig cells can be divided into three distinct stages of differentiation: initially they exist as mesenchymal-like progenitors (PLC) by day 21; subsequently, as immature Leydig cells (ILC) by day 35, they acquire steroidogenic organelle structure and enzyme activities but metabolize most of the testosterone they produce; finally, as adult Leydig cells (ALC) by day 90 they actively produce testosterone. The aims of the present study were to determine whether changes in proliferative capacity are associated with progressive differentiation of Leydig cells, and if the proliferative capacity of Leydig cells is controlled by known hormonal regulators of testosterone biosynthesis: LH, insulin-like growth factor I (IGF-I), androgen, and estradiol (E2). Isolated PLC, ILC, and ALC were cultured in DMEM/F-12 for 24 h followed by an additional 24 h in the presence of LH (1 ng/ml), IGF-I (70 ng/ml), 7alpha-methyl-19-nortestosterone (MENT, 50 nM), a synthetic androgen that is not metabolized by 5alpha-reductase, or E2 (50 nM). Proliferative capacity was measured by assaying [3H]thymidine incorporation and labeling index (LI). Messenger RNA (mRNA) and protein levels for cyclin A2 and G1, which are putative intracellular regulators of Leydig cell proliferation and differentiation, were measured by RT-PCR and immunoblotting, respectively. Thymidine incorporation was highest in PLC (9.24 +/- 0.21 cpm/10(3) cell, mean +/- SE), intermediate in ILC (1.74 +/- 0.07) and lowest in ALC (0.24 +/- 0.03). Similarly, LI was highest in PLC (13.42 +/- 0.30%, mean +/- SE), intermediate in ILC (1.95 +/- 0.08%), and undetectable in ALC. Cyclin A2 mRNA levels, normalized to ribosomal protein S16 (RPS16), were highest in PLC (2.76 +/- 0.21, mean +/- SE), intermediate in ILC (1.79 +/- 0.14), and lowest in ALC (0.40 +/- 0.06). In contrast, cyclin G1 mRNA levels were highest in ALC (1.32 +/- 0.16), intermediate in ILC (0.47 +/- 0.07), and lowest in PLC (0.12 +/- 0.02). The

  2. Immunohistochemical analysis of androgen effects on androgen receptor expression in developing Leydig and Sertoli cells.

    PubMed

    Shan, L X; Bardin, C W; Hardy, M P

    1997-03-01

    Leydig and Sertoli cells are both targets of androgen action in the testis. Androgen exerts contrasting effects on the two cell types partially inhibiting steroidogenesis in adult Leydig cell and stimulating adult Sertoli cell functions required to support spermatogenesis. The developmental changes in the messenger RNA (mRNA) levels of androgen receptor (AR) also differ between Leydig and Sertoli cells, with Leydig cell AR mRNA being highest on day 35 postpartum, whereas Sertoli cell AR mRNA levels are highest on day 90. The purpose of the present study was to determine if the concentrations of AR in Leydig and Sertoli cells are differentially regulated during development using quantitative immunostaining. AR protein levels were measured in rat testes after hormonal treatments at three developmental stages: on days 21, 35, and 90 postpartum. At each age, five groups of animals were treated for 4 days with: 1) vehicle; 2) LHRH antagonist (NalGlu, 0.3 mg/kg BW.day) to suppress endogenous levels of androgen that accompany inhibition of LH and FSH secretion; 3) NalGlu + LH (0.2 mg/kg BW.day); 4) NalGlu + testosterone (T, at 7.5 mg/kg BW.day); and 5) NalGlu + MENT (a potent synthetic androgen, 7 alpha-methyl-19-nortestosterone, 0.7 mg/kg BW.day). AR protein was visualized by immunohistochemistry and measured by computer-assisted image analysis in Leydig and Sertoli cells using frozen sections of tests. After NalGlu treatment, AR levels in Leydig cells declined sharply to 42% and 31% of vehicle control (P < 0.01) in the 21 and 35 days postpartum age groups, respectively, but in 90-day-old rats there was no change. AR levels were partially maintained by exogenous LH, and completely maintained by exogenous androgen treatments in Leydig cells from 21- and 35-day-old rats, whereas in Leydig cells from 90-day-old rats, AR levels were unaffected in all treatment groups. In contrast, after NalGlu treatment, the AR concentration in Sertoli cells from 90-day-old rats were reduced

  3. High serum luteinizing hormone levels induce ovarian delta4 cytochrome P450c17alpha down-regulation in hirsute women: complete effect on 17-hydroxylase and partial effect on 17,20-lyase.

    PubMed

    Rieu, M; Mourrieras, F; Riveline, J P; Laplanche, S; Both, D; Kuhn, J M

    1998-09-01

    It is well known that normal and mildly elevated luteinizing hormone (LH) levels induce increased activity of ovarian 17-hydroxylase and 17,20-lyase, the cytochrome P450cl7alpha (P450) enzymes. This leads to increased ovarian 17alpha-hydroxyprogesterone (17-OHP) and androstenedione production. In contrast, it has been shown in both in vitro and in vivo studies in animals and in in vitro studies in women that high LH concentrations have opposite effects on these enzymes. These LH down-regulating effects appear to be more marked on 17,20-lyase than on 17-hydroxylase. Finally, these LH effects have not been reported in vivo in women. Therefore, we investigated the relationships between serum LH levels and serum 17-OHP and androstenedione concentrations in 263 consecutive hirsute women (HW) with normal serum 17-OHP responses to acute adrenocorticotropin (ACTH) stimulation. The patterns of basal serum steroid concentrations differed according to the basal serum LH levels. Indeed, for relationships between LH and 17-OHP concentrations, a positive correlation (P < 0.001) was found between the levels of these parameters when LH levels ranged from 0.2 to 9.0 IU/l. Conversely, for LH levels greater than 9.0 to 21.0 IU/l, LH values were negatively correlated (P<0.001) with 17-OHP concentrations. Similar results were observed for relationships between LH and androstenedione levels but the LH peak level related to decreasing androstenedione concentrations was 12.0 IU/l. Finally, the mean 17-OHP level in patients with LH levels which induced marked P450 down-regulation (i.e. more than 12 IU/l) was similar to that in patients with LH levels within the normal range (i.e. less than 6 IU/l). In contrast, the mean androstenedione level in the former patients was markedly higher (P<0.001) than that in the latter patients. In conclusion, as previously reported in in vitro studies, this in vivo study indicates that LH induces stimulating and down-regulating effects on both ovarian delta