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

  1. 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... canned dog food, each 61/2 ounce can containing 30 or 60 micrograms of mibolerone. (b) Conditions of use...) Indications for use. For the prevention of estrus (heat) in adult female dogs not intended primarily...

  2. 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... canned dog food, each 61/2 ounce can containing 30 or 60 micrograms of mibolerone. (b) Conditions of use...) Indications for use. For the prevention of estrus (heat) in adult female dogs not intended primarily...

  3. Efficacy and dosage titration study of mibolerone for treatment of pseudopregnancy in the bitch.

    PubMed

    Brown, J M

    1984-06-15

    A controlled, blind-labeled, dose-response field trial was conducted to evaluate the efficacy of mibolerone as a treatment for pseudopregnancy in the bitch. Bitches were treated orally for 5 consecutive days with one of the following dosages of mibolerone: 0.008, 0.016, or 0.025 mg/kg of body weight. Changes in psychologic signs (nesting behavior, mothering inanimate objects, and self-nursing) or physical signs (mammary gland enlargement and secretion of a liquid or milk, ie, galactorrhea) were noted. The period within which the improvement occurred also was noted. There were 63 cases distributed over the 3 dosages--19 at 0.008 mg/kg, 22 at 0.016 mg/kg, and 22 at 0.025 mg/kg. Seventeen bitches given a placebo served as controls. There were significant differences in improvement of clinical signs among the dosages for the combinations of psychologic (P less than 0.001), physical (P less than 0.01), psychologic or physical (P less than 0.001), and psychologic and physical (P less than 0.001). The projected optimal dosages were: 0.016 mg/kg, 0.013 mg/kg, 0.014 mg/kg, and 0.015 mg/kg for the psychologic, physical, psychologic or physical, and psychologic and physical signs, respectively. Of the 3 dosages used, 0.016 mg/kg (for 5 consecutive days) was estimated to be optimal for improvement of the physiologic signs of pseudopregnancy.

  4. 7 alpha-hydroxytrichodermol, a new trichothecene from Myrothecium roridum.

    PubMed Central

    Jarvis, B B; Lee, Y W; Yatawara, C S; Mazzocchi, D B; Flippen-Anderson, J L; Gilardi, R; George, C

    1985-01-01

    Five plant-pathogenic isolates of Myrothecium roridum from Florida produced only simple trichothecenes rather than the usual macrocyclic trichothecenes. The major metabolite was 7 alpha-hydroxytrichodermol. PMID:4091555

  5. The microbiological transformation of 7alpha-hydroxy-ent-kaur-16-ene derivatives by Gibberella fujikuroi.

    PubMed

    Fraga, Braulio M; Bressa, Carlo; González, Pedro; Guillermo, Ricardo; Hernández, Melchor G; Suárez, Sergio

    2007-06-01

    The biotransformation of 7alpha-hydroxy-ent-kaur-16-ene (epi-candol A) by the fungus Gibberella fujikuroi gave 7alpha,16alpha,17-trihydroxy-ent-kaur-16-ene and a seco-ring B derivative, fujenoic acid, whilst the incubation of candicandiol (7alpha,18-dihydroxy-ent-kaur-16-ene) and canditriol (7alpha,15alpha,18-trihydroxy-ent-kaur-16-ene) afforded 7alpha,18,19-trihydroxy-ent-kaur-16-ene and 7alpha,11beta,15alpha,18-tetrahydroxy-ent-kaur-16-ene, respectively. The presence of a 7alpha-hydroxyl group in epi-candol A avoids its biotransformation along the biosynthetic pathway of gibberellins, and directs it to the seco-ring B acids route. The 15alpha-hydroxyl group in canditriol inhibits oxidation at C-19 and direct hydroxylation at C-11(beta). The formation of fujenoic acid, from 7alpha-hydroxy-ent-kaur-16-ene, probably occurs via 7alpha-hydroxykaurenoic acid and 7-oxokaurenoic acid, with subsequent hydroxylation at the C-6(beta) position.

  6. Synthesis of 7 alpha- and 7 beta-spermidinylcholesterol, squalamine analogues.

    PubMed

    Choucair, B; Dherbomez, M; Roussakis, C; El-kihel, L

    2004-08-16

    Stereoselective synthesis of squalamine dessulfates analogues, 7 alpha and 7 beta-N-[3N-(4-aminobutyl) aminopropyl]aminocholesterol are reported, using 7 alpha and 7 beta-aminocholesterol as a key intermediate. It's the first example in which the position of spermidine is modified at the steroid ring. These molecules showed a comparable antibacteria and fungi activities to squalamine. Then, they have a cytotoxic activity on a human non-small cell bronchopulmonary carcinoma line (NSCLC-N6). PMID:15261272

  7. 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.

  8. 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.

  9. [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.

  10. Improved assay for cholesterol 7 alpha-hydroxylase activity using phospholipid liposome solubilized substrate

    SciTech Connect

    Junker, L.H.; Story, J.A.

    1985-10-01

    A persistent problem in measurement of cholesterol 7 alpha-hydroxylase (7 alpha-OHase) activity by isotope incorporation has been solubilization of cholesterol substrate. Solubilization with Tween 20, for example, resulted in a 75% reduction in 7 alpha-OHase activity after a 60 min incubation of substrate with microsomes. Incorporation of cholesterol substrate into small, unilamellar phospholipid vesicles (liposomes) prevented this effect, resulting in a 50% increase in activity over the same 60 min incubation at optimal concentrations. Using cholesterol in liposomes as substrate, standard assay conditions were determined to be: preparation of liposomes with 180 microM cholesterol substrate and 0.5 mg phospholipid/assay; incubation of these liposomes with 0.5 mg microsomal protein at 37 C for 60 min; addition of a NADPH generating system to start the reaction, and incubation at 37 C for 30 min before stopping the reaction and determining the amount of 7 alpha-hydroxycholesterol formed. This method provides a sensitive and reliable alternative to methods which require more sophisticated equipment and allows total control of substrate concentration in a form readily accessible to the enzyme.

  11. 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.

  12. Cholesterol 7{alpha}-hydroxylase is phosphorylated at multiple amino acids

    SciTech Connect

    Stroup, D. . E-mail: dstroup1@kent.edu; Ramsaran, J.R.

    2005-04-15

    The activity of cholesterol 7{alpha}-hydroxylase (gpCYP7A1), the rate limiting enzyme in bile acid synthesis, has been postulated to be regulated by phosphorylation/dephosphorylation. This study has found that several kinase activators rapidly reduce the amount of bile acid produced by the human hepatoma cell line, HepG2, and that gpCYP7A1 from HepG2 cell extracts eluted in the phosphoprotein fraction of FeIII columns. After incubating the HepG2 cells with radioactive orthophosphate, the band identified as gpCYP7Al on immunoblots was strongly labeled. Recombinant gpCYP7A was expressed as 6x HIS fusion polypeptides and subjected to kinase assays. The locations of phosphorylation were mapped further by screening synthetic peptides against AMP-activated protein kinase (AMPK), c-Jun N-terminal kinase, protein kinase A, and a panel of nine protein kinase C isoforms. AMPK, also known as 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase, phosphorylated cholesterol 7{alpha}-hydroxylase, suggesting a potential mechanism of coordination of cholesterol synthesis and degradation.

  13. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  15. Linkage between cholesterol 7alpha-hydroxylase and high plasma low-density lipoprotein cholesterol concentrations.

    PubMed Central

    Wang, J; Freeman, D J; Grundy, S M; Levine, D M; Guerra, R; Cohen, J C

    1998-01-01

    Interindividual differences in plasma low-density lipoprotein cholesterol (LDL-C) levels reflect both environmental variation and genetic polymorphism, but the specific genes involved and their relative contributions to the variance in LDL-C are not known. In this study we investigated the relationship between plasma LDL-C concentrations and three genes with pivotal roles in LDL metabolism: the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and cholesterol 7alpha-hydroxylase (CYP7). Analysis of 150 nuclear families indicated statistically significant linkage between plasma LDL-C concentrations and CYP7, but not LDLR or APOB. Further sibling pair analyses using individuals with high plasma LDL-C concentrations as probands indicated that the CYP7 locus was linked to high plasma LDL-C, but not to low plasma LDL-C concentrations. This finding was replicated in an independent sample. DNA sequencing revealed two linked polymorphisms in the 5' flanking region of CYP7. The allele defined by these polymorphisms was associated with increased plasma LDL-C concentrations, both in sibling pairs and in unrelated individuals. Taken together, these findings indicate that polymorphism in CYP7 contributes to heritable variation in plasma LDL-C concentrations. Common polymorphisms in LDLR and APOB account for little of the heritable variation in plasma LDL-C concentrations in the general population. PMID:9502769

  16. Partial purification and characterization of 7 alpha-hydroxysteroid dehydrogenase from rat liver microsomes.

    PubMed

    Amuro, Y; Yamade, W; Yamamoto, T; Maebo, A; Hada, T; Higashino, K

    1987-01-13

    An NADPH-dependent 7 alpha-hydroxysteroid dehydrogenase acting on 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid was partially purified 160-fold with a yield of 13% from rat liver microsomes using DEAE-cellulose, hydroxyapatite and Affi-Gel Blue column chromatography. The specific activity of the purified enzyme was 91.3 nmol chenodeoxycholic acid formed/min per mg of protein. The reaction was reversible, and the optimum pH of the enzyme for the oxidation was about 8.5, whereas that for the reduction was about 5.0 A molecular weight of the enzyme was estimated to be about 130,000 by Superose 6TM gel filtration chromatography. The apparent Km value for 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid was 35.7 microM and that for NADPH was 90.9 microM. The preferred substrate for the enzyme was 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid rather than 3 alpha,12 alpha-dihydroxy-7-keto-5 beta-cholanoic acid, a 7-keto-bile acid analogue. The enzyme also preferred the unconjugated form to the conjugated forms. The enzyme activity was inhibited by p-chloromercuribenzoate; however, the inhibition was prevented by addition of reduced form of glutathione to the reaction mixture, indicating that the enzyme requires a sulfhydryl group for activity. PMID:3466650

  17. Characterization and regulation of the NADP-linked 7 alpha-hydroxysteroid dehydrogenase gene from Clostridium sordellii.

    PubMed Central

    Coleman, J P; Hudson, L L; Adams, M J

    1994-01-01

    A bile acid-inducible NADP-linked 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) from Clostridium sordellii ATCC 9714 was purified 310-fold by ion-exchange, gel filtration, and dye-ligand affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed one predominant peptide band (30,000 Da). The N-terminal sequence was determined, and the corresponding oligonucleotides were synthesized and used to screen EcoRI and HindIII genomic digests of C. sordellii. Two separate fragments (4,500 bp, EcoRI; 3,200 bp, HindIII) were subsequently cloned by ligation to pUC19 and transformation into Escherichia coli DH5 alpha-MCR. The EcoRI fragment was shown to contain a truncated 7 alpha-HSDH gene, while the HindIII fragment contained the entire coding region. E. coli clones containing the HindIII insert expressed high levels of an NADP-linked 7 alpha-HSDH. Nucleotide sequence analyses suggest that the 7 alpha-HSDH is encoded by a monocistronic transcriptional unit, with DNA sequence elements resembling rho-independent terminators located in both the upstream and downstream flanking regions. The transcriptional start site was located by primer extension analysis. Northern (RNA) blot analysis indicated that induction is mediated at the transcriptional level in response to the presence of bile acid in the growth medium. In addition, growth-phase-dependent expression is observed in uninduced cultures. Analysis of the predicted protein sequence indicates that the enzyme can be classified in the short-chain dehydrogenase group. Images PMID:8050999

  18. 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.

  19. 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.

  20. Cholesterol 7 alpha-hydroxylase activity is increased by dietary modification with psyllium hydrocolloid, pectin, cholesterol and cholestyramine in rats.

    PubMed

    Matheson, H B; Colón, I S; Story, J A

    1995-03-01

    Sources of dietary fiber known to alter cholesterol metabolism and/or bile acid pool size were fed to rats, and activity of the rate-limiting step in bile acid synthesis, cholesterol 7 alpha-hydroxylase, was measured. In the first experiment, semipurified diets containing 5% cellulose, psyllium hydrocolloid, pectin or oat bran as dietary fiber sources or 2% cholestyramine were fed to groups of 10 male Wistar rats for 4 wk. In the second experiment, groups of six rats were fed diets containing 5% cellulose, rice bran, oat bran or psyllium with and without 0.25% cholesterol. In the first experiment, the activity of cholesterol 7 alpha-hydroxylase (pmol.min-1.mg protein-1) was highest in the cholestyramine-treated group (95.6 +/- 3.6), followed by groups fed psyllium (35.5 +/- 3.5) or pectin (36.0 +/- 4.5), which exhibited more than twice the enzyme activity of groups fed cellulose (16.9 +/- 1.9) or oat bran (12.3 +/- 2.0). In the second experiment, feeding cholesterol resulted in significantly higher enzyme activity when cellulose (65%), oat bran (118%) and rice bran (60%) were fed, but no difference in activity was observed when cholesterol was added to the psyllium-containing diet. Higher activity of cholesterol 7 alpha-hydroxylase when pectin or psyllium rather than cellulose was fed may explain the almost twofold higher bile acid pool sizes previously reported in response to feeding either of these fibers. These data support the hypothesis that the hypocholesterolemic effect of soluble fibers is modulated through increased synthesis and therefore pool size of bile acids.

  1. Isocholic acid formation from 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid with human liver enzyme.

    PubMed

    Amuro, Y; Yamade, W; Yamamoto, T; Kudo, K; Fujikura, M; Maebo, A; Hada, T; Higashino, K

    1986-12-01

    The formation of isocholic acid from 7 alpha, 12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid by human liver preparations was examined in vitro. Liver preparations were incubated with 7 alpha, 12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid at pH 7.4 in a phosphate buffer containing NADPH or NADH. The products formed were analyzed by gas chromatography and gas chromatography/mass spectrometry. Results showed that 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid was reduced mainly to isocholic acid and to cholic acid in a smaller amount in the presence of NADPH, while it was reduced only to cholic acid in the presence of NADH. The reducing enzyme participating in the formation of isocholic acid was localized largely in the cytosol and had more specificity to the unconjugated form as substrate than to the conjugated forms. 3-Keto bile acid analogues, 3-keto-5 beta-cholanoic and 7 alpha-hydroxy-3-keto-5 beta-cholanoic acids were not reduced to the corresponding iso-bile acids by the cytosol in the same conditions used in the isocholic acid formation and the activity of the enzyme catalyzing the reduction of 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid to isocholic acid was not inhibited by the addition of 3-keto-5 beta-cholanoic acid or 7 alpha-hydroxy-3-keto-5 beta-cholanoic acid to the reaction mixture. Furthermore, on column chromatography of Affi-Gel Blue, the peak of the enzyme catalyzing the reduction of 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid to isocholic acid was clearly distinguished from that of the enzyme catalyzing the reduction of 3-keto-5 beta-cholanoic acid to isolithocholic acid and that of alcohol dehydrogenase. These results indicate that this enzyme catalyzing the reduction of 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid to isocholic acid is different from the enzyme(s) catalyzing the reduction 3-keto-5 beta-cholanoic and 7 alpha-hydroxy-3-keto-5 beta-cholanoic acids to the corresponding iso-bile acids

  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

    2004-12-01

    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. Identification and characterization of cis-acting elements conferring insulin responsiveness on hamster cholesterol 7alpha-hydroxylase gene promoter.

    PubMed Central

    De Fabiani, E; Crestani, M; Marrapodi, M; Pinelli, A; Golfieri, V; Galli, G

    2000-01-01

    Bile acid biosynthesis occurs primarily through a pathway initiated by the 7alpha-hydroxylation of cholesterol, catalysed by cholesterol 7alpha-hydroxylase (encoded by CYP7A1). Insulin down-regulates CYP7A1 transcription. The aim of our study was to characterize the sequences of hamster CYP7A1 promoter, mediating the response to insulin. We therefore performed transient transfection assays with CYP7A1 promoter/luciferase chimaeras mutated at putative response elements and studied protein-DNA interactions by means of gel electrophoresis mobility-shift assay. Here we show that two sequences confer insulin responsiveness on hamster CYP7A1 promoter: a canonical insulin response sequence TGTTTTG overlapping a binding site for hepatocyte nuclear factor 3 (HNF-3) (at nt -235 to -224) and a binding site for HNF-4 at nt -203 to -191. In particular we show that the hamster CYP7A1 insulin response sequence is part of a complex unit involved in specific interactions with multiple transcription factors such as members of the HNF-3 family; this region does not bind very strongly to HNF-3 and as a consequence partly contributes to the transactivation of the gene. Another sequence located at nt -138 to -128 binds to HNF-3 and is involved in the tissue-specific regulation of hamster CYP7A1. The sequence at nt -203 to -191 is not only essential for insulin effect but also has a major role in the liver-specific expression of CYP7A1; it is the target of HNF-4. Therefore the binding sites for liver-enriched factors, present in the hamster CYP7A1 proximal promoter in close vicinity and conserved between species, constitute a regulatory unit important for basal hepatic expression and tissue restriction of the action of hormones such as insulin. PMID:10727413

  4. 7alpha- and 12alpha-Hydroxysteroid dehydrogenases from Acinetobacter calcoaceticus lwoffii: a new integrated chemo-enzymatic route to ursodeoxycholic acid.

    PubMed

    Giovannini, Pier Paolo; Grandini, Alessandro; Perrone, Daniela; Pedrini, Paola; Fantin, Giancarlo; Fogagnolo, Marco

    2008-12-22

    We report the very efficient biotransformation of cholic acid to 7-keto- and 7,12-diketocholic acids with Acinetobacter calcoaceticus lwoffii. The enzymes responsible of the biotransformation (i.e. 7alpha- and 12alpha-hydroxysteroid dehydrogenases) are partially purified and employed in a new chemo-enzymatic synthesis of ursodeoxycholic acid starting from cholic acid. The first step is the 12alpha-HSDH-mediated total oxidation of sodium cholate followed by the Wolf-Kishner reduction of the carbonyl group to chenodeoxycholic acid. This acid is then quantitatively oxidized with 7alpha-HSDH to 7-ketochenodeoxycholic acid, that was chemically reduced to ursodeoxycholic acid (70% overall yield).

  5. Identification of 7alpha-hydroxypregnenolone, a novel bioactive amphibian neurosteroid stimulating locomotor activity, and its physiological roles in the regulation of locomotion.

    PubMed

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

    2010-09-01

    We now know that steroids can be synthesized de novo by the brain and the peripheral nervous system. Such steroids are called neurosteroids and de novo neurosteroidogenesis from cholesterol is a conserved property of vertebrate brains. Our studies over the past decade have demonstrated that the brain expresses several kinds of steroidogenic enzymes and produces a variety of neurosteroids in sub-mammalian species. However, neurosteroid biosynthetic pathways in amphibians, as well as other vertebrates may still not be fully mapped. We first found that the newt brain actively produces 7alpha-hydroxypregnenolone, a previously undescribed amphibian neurosteroid. We then demonstrated that 7alpha-hydroxypregnenolone acts as a novel bioactive neurosteroid to stimulate locomotor activity of newt by means of the dopaminergic system. Subsequently, we analyzed the physiological roles of 7alpha-hydroxypregnenolone in the regulation of locomotor activity of newt. This paper summarizes the advances made in our understanding of 7alpha-hydroxypregnenolone, a newly discovered bioactive amphibian neurosteroid stimulating locomotor activity, and its physiological roles in the regulation of locomotion in newt.

  6. Role of the 7 alpha-methoxy and side-chain carboxyl of moxalactam in beta-lactamase stability and antibacterial activity.

    PubMed Central

    Murakami, K; Yoshida, T

    1981-01-01

    The effects of the alpha-carboxyl of the phenylmalonyl side chain and the 7 alpha-methoxy group in moxalactam (6059-S) (7 beta-[2-carboxy-2-(4-hydroxyphenyl) acetamido]-7 alpha-methoxy-3[[(1-methyl-1H-tetrazol-5-y])thio] methyl]-1-oxa-1-dethia-3-cephem-4-carboxylic acid) and in the 1-sulfur congener on the stability to beta-lactamase were investigated by spectrophotometric and microbiological assays. The 7 alpha-methoxy substituent stabilized the compounds against penicillinase hydrolysis, and the alpha-carboxyl group stabilized them against cephalosporinase. An exception is the beta-lactamase produced by Proteus vulgaris, an inducible cephalosporinase, which hydrolyzed compounds having the alpha-carboxyl group but not those having the 7 alpha-methoxy group. Both substituents exerted their stabilizing effects independently, and compounds with both substituents, e.g., moxalactam (6059-S) and its 1-sulfur congener, were resistant to both penicillinases and cephalosporinases. The stabilization of the compounds to beta-lactamase hydrolysis improved their antibacterial activity against beta-lactamase-producing strains. PMID:6454378

  7. Dietary fish oil up-regulates cholesterol 7alpha-hydroxylase mRNA in mouse liver leading to an increase in bile acid and cholesterol excretion.

    PubMed

    Bérard, Annie M; Dumon, Marie-France; Darmon, Michel

    2004-02-13

    To investigate the molecular events controlling reverse cholesterol transport, we compared gene expression of normal mouse liver to that of mice fed a long chain (LC) omega-3 fatty acid-enriched diet. Using cDNA microarrays, we assessed expression levels of 1176 genes, and we found that D-site binding protein (DBP) was three-fold increased in mice on a LC omega-3 fatty acid-rich diet compared to controls. DBP is known to increase transcriptional level of cholesterol 7alpha-hydroxylase (C7alpha), the rate-limiting enzyme for bile acid production and cholesterol excretion, and we found that C7alpha mRNA was also up-regulated by LC omega-3 fatty acids. Moreover, liver X receptor-alpha, another transcription factor up-regulating C7alpha, was three- to four-fold increased in liver of treated mice. On the other hand, we demonstrated that bile acid and cholesterol excretion were two-fold increased. These results show that LC omega-3 fatty acids control cholesterol metabolism in mice at a new endpoint.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  9. 21 CFR 558.348 - Mibolerone.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ..., AND RELATED PRODUCTS NEW ANIMAL DRUGS FOR USE IN ANIMAL FEEDS Specific New Animal Drugs for Use in...) Indications for use. For the prevention of estrus (heat) in adult female dogs not intended primarily for breeding purposes. (3) Limitations. Administer daily at least 30 days before expected initiation of...

  10. 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...

  11. 21 CFR 520.1430 - Mibolerone.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 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...

  12. 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...

  13. 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...

  14. 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.

  15. A putative role of micro RNA in regulation of cholesterol 7alpha-hydroxylase expression in human hepatocytes.

    PubMed

    Song, Kwang-Hoon; Li, Tiangang; Owsley, Erika; Chiang, John Y L

    2010-08-01

    Cholesterol 7alpha-hydroxylase (CYP7A1) plays a critical role in regulation of bile acid synthesis in the liver. CYP7A1 mRNAs have very short half-lives, and bile acids destabilize CYP7A1 mRNA via the 3'-untranslated region (3'-UTR). However, the underlying mechanism of translational regulation of CYP7A1 mRNA remains unknown. Screening of a human micro RNA (miRNA) microarray has identified five differentially expressed miRNAs in human primary hepatocytes treated with chenodeoxycholic acid, GW4064, or fibroblast growth factor (FGF)19. These compounds also significantly induced the expression of miR-122a, a liver-specific and the predominant miRNA in human hepatocytes. The putative recognition sequences for miR-122a and miR-422a were localized in the 3'-UTR of human CYP7A1 mRNA. The miR-122a and miR-422a mimics inhibited, whereas their inhibitors stimulated CYP7A1 mRNA expression. These miRNAs specifically inhibited the activity of the CYP7A1-3'-UTR reporter plasmids, and mutations of miRNA binding sites in 3'-UTR abrogated miRNA inhibition of reporter activity. These results suggest that miR-122a and miR-422a may destabilize CYP7A1 mRNA to inhibit CYP7A1 expression. However, these miRNAs did not play a role in mediating FGF19 inhibition of CYP7A1 transcription. Under certain conditions, miRNA may reduce CYP7A1 mRNA stability to inhibit bile acid synthesis, and the miR-122a antagomirs may stimulate bile acid synthesis to reduce serum cholesterol and triglycerides.

  16. 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.

  17. Ketoconazole blocks bile acid synthesis in hepatocyte monolayer cultures and in vivo in rat by inhibiting cholesterol 7 alpha-hydroxylase.

    PubMed Central

    Princen, H M; Huijsmans, C M; Kuipers, F; Vonk, R J; Kempen, H J

    1986-01-01

    In cultured hepatocytes conversion of [4-14C]cholesterol into bile acids was dose dependently reduced by the antimycotic drug ketoconazole, giving half-maximal inhibition at 10 microM ketoconazole in rat hepatocytes and at 1 microM in human hepatocytes. No change was observed in the ratio of produced cholic, beta-muricholic, and chenodeoxycholic acid with increasing amounts of the drug. Conversion of [4-14C]7 alpha-hydroxycholesterol, an intermediate of bile acid pathway, to bile acids was not affected by ketoconazole. These results together with kinetic studies with rat liver microsomes, demonstrating noncompetitive inhibition (Ki = 0.4 microM), indicate that cholesterol 7 alpha-hydroxylase is the main site of inhibition. In bile-diverted rats a single dose of ketoconazole (50 mg/kg) dramatically impaired bile flow and biliary bile acid output (92% inhibition). A similar blockade was observed using [4-14C]cholesterol as precursor for bile acid synthesis. Therefore, treatment of patients with this drug may inhibit bile acid synthesis, resulting in a reduction of the bile acid pool size after long-term ketoconazole therapy. PMID:3760182

  18. Fragrance material review on (3aalpha,4alpha,6alpha,7alpha,7aalpha)-3a,4,5,6,7,7a-hexahydro-3-methyl-5-methylene-4,7-methano-1H-inden-6-yl acetate.

    PubMed

    Bhatia, S P; Jones, L; Letizia, C S; Api, A M

    2008-12-01

    A toxicologic and dermatologic review of (3aalpha,4alpha,6alpha,7alpha,7aalpha)-3a,4,5,6,7,7a-hexahydro-3-methyl-5-methylene-4,7-methano-1H-inden-6-yl acetate when used as a fragrance ingredient is presented.

  19. 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.

  20. Chemical synthesis and hepatic biotransformation of 3 alpha,7 alpha-dihydroxy-7 beta-methyl-24-nor-5 beta-cholan-23-oic acid, a 7-methyl derivative of norchenodeoxycholic acid: studies in the hamster.

    PubMed

    Yoshii, M; Mosbach, E H; Schteingart, C D; Hagey, L R; Hofmann, A F; Cohen, B I; McSherry, C K

    1991-11-01

    A new bile acid analogue, 3 alpha,7 alpha-dihydroxy-7 beta-methyl-24-nor-5 beta-cholan-23-oic acid (7-Me-norCDCA) was synthesized from the methyl ester of norursodeoxycholic acid, and its hepatic biotransformation was defined in the hamster. To synthesize 7-Me-norCDCA, the 3 alpha-hydroxyl group of methyl norursodeoxycholate was protected as the hemisuccinate, and the 7 beta-hydroxyl group was oxidized with CrO3 to form the 7-ketone. A Grigard reaction with methyl magnesium iodide followed by alkaline hydrolysis gave 7-Me-norCDCA (greater than 70% yield). The structure of the new compound was confirmed by proton magnetic resonance and mass spectrometry. After intraduodenal administration of the 14C-labeled compound into the anesthetized biliary fistula hamster, it was rapidly and efficiently secreted into the bile; 80% of radioactivity was recovered in 2 h. After intravenous infusion, the compound was efficiently extracted by the liver and secreted into the bile (greater than 75% in 3 h). Most (93%) of the biliary radioactivity was present in biotransformation products. The major biotransformation product (48.7 +/- 6.0%) was a new compound, assigned the structure of 3 alpha,5 beta,7 alpha- trihydroxy-7 beta-methyl-24-nor-5 beta-cholan-23-oic acid (5 beta-hydroxy-7- Me-norCDCA). In addition, conjugates of 7-Me-norCDCA with taurine (13.7 +/- 5.0%), sulfate (10.3 +/- 3.0%), or glucuronide (5.1 +/- 1.7%) were formed. 7-Me-norCDCA was strongly choleretic in the hamster; during its intravenous infusion, bile flow increased 2 to 3 times above the basal level, and the calculated choleretic activity of the compound (and its metabolic products) was much greater than that of many natural bile acids, indicating that the compound induced hypercholeresis. It is concluded that the biotransformation and physiological properties of 7-Me-norCDCA closely resemble those of norCDCA. Based on previous studies, the major biological effect of the 7-methyl group in 7-Me-norCDCA is to

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

    PubMed Central

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

    2014-01-01

    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. PMID:24582860

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

    PubMed

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

    2014-12-23

    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.

  3. 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.

  4. Hepatobiliary disposition of 3alpha,6alpha,7alpha,12alpha-tetrahydroxy-cholanoyl taurine: a substrate for multiple canalicular transporters.

    PubMed

    Megaraj, Vandana; Iida, Takashi; Jungsuwadee, Paiboon; Hofmann, Alan F; Vore, Mary

    2010-10-01

    Tetrahydroxy bile acids become major biliary bile acids in Bsep(-/-) mice and Fxr(-/-) mice fed cholic acid; we characterized disposition of these novel bile acids that also occur in patients with cholestasis. We investigated mouse Mrp2 (mMrp2) and P-glycoprotein [(P-gp) mMdr1a]-mediated transport of a tetrahydroxy bile acid, 6α-OH-taurocholic acid (6α-OH-TC), and its biliary excretion in wild-type and Mrp2(-/-) mice in the presence or absence of N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), a P-gp and breast cancer resistance protein inhibitor. 6α-OH-TC was rapidly excreted into bile of wild-type mice (78% recovery); coinfusion of GF120918 had no significant effect. In Mrp2(-/-) mice, biliary excretion was decreased (52% recovery) and coinfusion of GF120918 further decreased these values (34% recovery). In wild-type, but not Mrp2(-/-), mice, 6α-OH-TC increased bile flow 2.5-fold. Membrane vesicle transport studies of 6α-OH-TC (0.05-0.75 mM) yielded saturation kinetics with a higher apparent affinity for mMrp2 (K(m) = 0.13 mM) than for mMdr1a (K(m) = 0.33 mM); mBsep transported 6α-OH-TC with positive cooperativity (Hill slope = 2.1). Human multidrug resistance-associated protein (MRP) 2 and P-gp also transported 6α-OH-TC but with positive cooperativity (Hill slope = 3.6 and 1.6, respectively). After intraileal administration, the time course of 6α-OH-TC biliary recovery was similar to that of coinfused taurocholate, implying that 6α-OH-TC can undergo enterohepatic cycling. Thus, Mrp2 plays a key role in 6α-OH-TC biliary excretion, whereas P-glycoprotein plays a secondary role; Bsep likely mediates excretion of 6α-OH-TC in the absence of Mrp2 and P-gp. In Bsep(-/-) mice, efficient synthesis of tetrahydroxy bile acids that are Mrp2 and P-gp substrates can explain the noncholestatic phenotype.

  5. [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.

  6. (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.

  7. Axon diameter and axonal transport: In vivo and in vitro effects of androgens

    PubMed Central

    Pesaresi, M; Soon-Shiong, R; French, L; Kaplan, DR; Miller, FD; Paus, T.

    2015-01-01

    Testosterone is a sex hormone involved in brain maturation via multiple molecular mechanisms. Previous human studies described age-related changes in the overall volume and morphological properties of white matter during male puberty. Based on this work, we have proposed that testosterone may induce an increase of radial growth and, possibly, modulate axonal transport. In order to determine whether this is the case we have used two different experimental approaches. With electron microscopy, we have evaluated sex differences in the structural properties of axons in the corpus callosum (splenium) of young rats, and tested consequences of castration carried out after weaning. Then we examined in vitro the effect of the non-aromatizable androgen Mibolerone on the structure and bidirectional transport of wheat-germ agglutinin vesicles in the axons of cultured sympathetic neurons. With electron microscopy, we found robust sex differences in axonal diameter (males>females) and g ratio (males>females). Removal of endogenous testosterone by castration was associated with lower axon diameter and lower g ratio in castrated (vs. intact) males. In vitro, Mibolerone influenced the axonal transport in a time- and dose-dependent manner, and increased the axon caliber as compared with vehicle-treated neurons. These findings are consistent with the role of testosterone in shaping the axon by regulating its radial growth, as predicted by the initial human studies. PMID:25956809

  8. 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

  9. Hydroxylated gedunin derivatives from Cedrela sinensis.

    PubMed

    Mitsui, Kumiko; Saito, Hiroaki; Yamamura, Ryota; Fukaya, Haruhiko; Hitotsuyanagi, Yukio; Takeya, Koichi

    2006-09-01

    Four new limonoids, 11alpha-hydroxygedunin (1), 11beta-hydroxygedunin (2), 7-deacetoxy-7alpha,11alpha-dihydroxygedunin (3), and 7-deacetoxy-7alpha,11beta-dihydroxygedunin (4), were isolated from the cortex of Cedrela sinensis, together with three known compounds, gedunin (5), 7-deacetoxy-7alpha-hydroxygedunin (6), and 11-oxogedunin (7). The structures of 1-4 were determined by a combination of 2D NMR experiments and chemical methods and by X-ray crystallography of 1 and 2. PMID:16989525

  10. 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.

  11. Development and validation of an HPLC method for the determination of spironolactone and its metabolites in paediatric plasma samples.

    PubMed

    Sandall, J M; Millership, J S; Collier, P S; McElnay, J C

    2006-07-24

    An HPLC method has been developed and validated for the determination of spironolactone, 7 alpha-thiomethylspirolactone and canrenone in paediatric plasma samples. The method utilises 200 microl of plasma and sample preparation involves protein precipitation followed by Solid Phase Extraction (SPE). Determination of standard curves of peak height ratio (PHR) against concentration was performed by weighted least squares linear regression using a weighting factor of 1/concentration2. The developed method was found to be linear over concentration ranges of 30-1000 ng/ml for spironolactone and 25-1000 ng/ml for 7 alpha-thiomethylspirolactone and canrenone. The lower limit of quantification for spironolactone, 7 alpha-thiomethylspirolactone and canrenone were calculated as 28, 20 and 25 ng/ml, respectively. The method was shown to be applicable to the determination of spironolactone, 7 alpha-thiomethylspirolactone and canrenone in paediatric plasma samples and also plasma from healthy human volunteers. PMID:16510319

  12. 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.

  13. In vivo and in vitro effect of novel 4,16-pregnadiene-6,20-dione derivatives, as 5alpha-reductase inhibitors.

    PubMed

    Bratoeff, Eugene; Cabeza, Marisa; Pérez-Ornelas, Victor; Recillas, Sergio; Heuze, Ivonne

    2008-09-01

    In this study, we report the synthesis and biological evaluation of several new 3-substituted pregna-4,16-diene-6,20-dione derivatives (11a-11d). These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological effect of these steroids was demonstrated in in vivo and in vitro experiments. In the in vivo experiments, we measured the activity of the 11a-11d on the weight of the prostate gland of gonadectomized hamsters treated with testosterone plus finasteride or with the new steroids. For the studies in vitro, we determined the IC50 values by measuring the steroid concentration that inhibits 50% of the activity of 5alpha-reductase present in human prostate. In order to study the mechanism of action of 11a-11d, we also determined the capacity of these steroids to bind to the androgen receptor (AR) present in the rat prostate cytosol using labeled mibolerone as a tracer. The results from this work indicated that compounds 11a-11d significantly decreased the weight of the prostate as compared to testosterone treated animals and this reduction of the weight of the prostate was comparable to that produced by the finasteride. On the other hand 11a-11d exhibited a high inhibitory activity for the human 5alpha-reductase enzyme with IC50 values of 1.4 x 10(-8), 1.8 x 10(-9), 1.0 x 10(-8) and 4 x 10(-5) respectively. However the IC50 value of 11a (1.8 x 10(-9)) was the only one lower than that of finasteride (8.5 x 10(-9)). Nevertheless this compound did not show a higher potency in vivo as compared to that of compounds 11b-11d. The competition analysis for the androgen receptor indicated that the IC50 value of non-labeled mibolerone used in this experiment was 1nM, whereas steroids 10, 11a-11d did not inhibit the labeled mibolerone binding to the androgen receptor. On the other hand, steroid 10 did not show any activities in vitro or in vivo, and for this reason these steroidal derivatives (11a-11d) cannot be considered as

  14. Astrocytes and neurosteroids: metabolism of pregnenolone and dehydroepiandrosterone. Regulation by cell density

    PubMed Central

    1993-01-01

    The rat central nervous system (CNS) has previously been shown to synthesize pregnenolone (PREG) and convert it to progesterone (PROG) and 7 alpha-hydroxy-PREG (7 alpha-OH PREG). Astrocytes, which participate to the regulation of the CNS function, might be involved in the metabolism of neurosteroids. Purified type 1 astrocytes were obtained from fetal rat forebrain with the use of selective culture conditions and were identified by immunostaining with specific antibodies (GFAP+, A2B5-). They were plated at low, intermediate, or high densities (2.5-5 x 10(5), 1-2 x 10(6), or 4-8 x 10(6) cells/dish, respectively) and maintained for 21 d. They were then incubated with 14C-PREG and 14C-DHEA for 24 h and the steroids extracted from cells and media were analyzed. Most radioactive derivatives were released into incubation media. Two metabolic pathways were mainly observed. PREG and DHEA were oxidized to PROG and androstenedione (ADIONE), respectively, [3 beta-hydroxysteroid-dehydrogenase, delta 5-->4 3- ketosteroid-isomerase (3 beta-HSD) activity], and converted to 7 alpha- OH PREG and 7 alpha-OH DHEA, respectively (7 alpha-hydroxylase activity). After low density plating, the formation of PROG and ADIONE was approximately 10% of incubated radioactivity, tenfold larger than that of 7 alpha-hydroxylated metabolites. In contrast, after high density plating, low levels of PROG and ADIONE were formed, whereas the conversion to either 7 alpha-OH PREG or 7 alpha-OH DHEA was > or = 50%. The results expressed per cell indicated that the 3 beta-HSD activity was almost completely inhibited at high cell density, in contrast to the 7 alpha-hydroxylation which was maintained or increased. The pattern of steroid metabolism was related to cell density at the time of measurement and not to an early commitment of cells: when primary cultures were plated at high density (8 x 10(6) cells/dish), then subcultured after several dilutions (3-, 9-, or 27-fold), the 3 beta- HSD activity was

  15. Hydroxycholesterols in serum from hypercholesterolaemic patients with and without bile acid sequestrant therapy.

    PubMed

    van Doormaal, J J; Smit, N; Koopman, B J; van der Molen, J C; Wolthers, B G; Doorenbos, H

    1989-05-31

    To assess the effect of bile acid sequestrant therapy on bile acid precursors in plasma, we determined hydroxycholesterols in serum from patients with primary hypercholesterolaemia. Compared with a group of 5 male and 12 female patients without any lipid-lowering drug therapy, which has normal to slightly elevated 7 alpha-hydroxycholesterol, normal 7 beta-hydroxycholesterol and high normal to elevated 26-hydroxycholesterol levels, a group of 5 male and 9 female patients, using colestipol had higher 7 alpha-hydroxycholesterol without overlap, and higher 7 beta-hydroxycholesterol levels, but similar levels of 26-hydroxycholesterol. In the latter group, the ratio between 7 alpha-hydroxycholesterol and total cholesterol in serum was also higher without overlap. Both groups did not differ for age, body weight, body mass index and serum lipid levels. In the group of patients without lipid-lowering drug therapy, 7 alpha-hydroxycholesterol correlated positively with total and low-densitylipoprotein cholesterol, 7 beta-hydroxycholesterol negatively with body weight and body mass index, and 26-hydroxycholesterol positively with body weight. In both groups, 7 alpha-hydroxycholesterol correlated positively with 7 beta-hydroxycholesterol. These results suggest that (1) bile acid sequestrants enhance bile acid synthesis via the 7 alpha-hydroxylation but not via the 26-hydroxylation pathway, (2) serum 7 alpha-hydroxycholesterol level and the ratio between this hydroxycholesterol and total cholesterol in serum might be suitable parameters to check intake of bile acid sequestrants irrespective of dose, and (3) 7 beta-hydroxycholesterol is unlikely to be the result of cholesterol auto-oxidation in vitro.

  16. 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.

  17. Demonstration of 26-hydroxylation of C27-steroids in human skin fibroblasts, and a deficiency of this activity in cerebrotendinous xanthomatosis.

    PubMed Central

    Skrede, S; Björkhem, I; Kvittingen, E A; Buchmann, M S; Lie, S O; East, C; Grundy, S

    1986-01-01

    26-Hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and other C27-steroids was demonstrated in cultured skin fibroblasts from healthy individuals. Activities in skin fibroblasts were approximately 5-10% of those previously found in human liver homogenates, and were inhibited by CO. The apparent Km was lowest for 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol (1.3 mumol/liter) and highest for 5-cholestene-3 beta, 7 alpha-diol (12 mumol/liter). The rate of 26-hydroxylation was highest with 7 alpha-hydroxy-4-cholesten-3-one. These characteristics are similar to those of hepatic mitochondrial C27-steroid 26-hydroxylase. In skin fibroblasts from three patients with cerebrotendinous xanthomatosis (CTX), 26-hydroxylation of C27-steroids proceeded at a rate of only 0.2-2.5% of healthy controls. No accumulation of endogenous 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol could be demonstrated in these cells, and the lowered formation of radioactive, 26-hydroxylated products could not be explained by dilution of the labeled exogenous substrate. The present results add strong evidence to the concept that the primary metabolic defect in CTX is a deficiency of C27-steroid 26-hydroxylase. PMID:3745434

  18. Collision-induced dissociation pathways of anabolic steroids by electrospray ionization tandem mass spectrometry.

    PubMed

    Guan, Fuyu; Soma, Lawrence R; Luo, Yi; Uboh, Cornelius E; Peterman, Scott

    2006-04-01

    Anabolic steroids are structurally similar compounds, and their product-ion spectra obtained by tandem mass spectrometry under electrospray ionization conditions are quite difficult to interpret because of poly-ring structures and lack of a charge-retaining center in their chemical structures. In the present study, the fragmentation of nine anabolic steroids of interest to the racing industry was investigated by using triple quadrupole mass spectrometer, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, and a linear ion trap instrument. With the aid of an expert system software (Mass Frontier version 3.0), accurate mass measurements, and multiple stage tandem mass spectrometric (MS(n)) experiments, fragmentation pathways were elucidated for boldenone, methandrostenolone, tetrahydrogestrinone (THG), trenbolone, normethandrolone and mibolerone. Small differences in the chemical structures of the steroids, such as an additional double-bond or a methyl group, result in significantly different fragmentation pathways. The fragmentation pathways proposed in this paper allow interpretation of major product ions of other anabolic steroids reported by other researchers in a recent publication. The proposed fragmentation pathways are helpful for characterization of new steroids. The approach used in this study for elucidation of the fragmentation pathways is helpful in interpretation of complicated product-ion spectra of other compounds, drugs and their metabolites. PMID:16488153

  19. 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. PMID:20146354

  20. 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

  1. 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. PMID:26862856

  2. (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.

  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. Influence of food on the bioavailability of spironolactone.

    PubMed

    Overdiek, H W; Merkus, F W

    1986-11-01

    Nine healthy volunteers received a single oral dose of 200 mg spironolactone, once during fasting conditions and once immediately after a standardized breakfast. Serum concentrations of spironolactone and its metabolites 7 alpha-thiomethylspirolactone, 6 beta-hydroxy-7 alpha-thiomethylspirolactone, and canrenone were determined by HPLC for 24 hours after dosing. By taking spironolactone with food, the mean (+/- SD) AUC (0 to 24 hours) of the parent drug increased from 288 +/- 138 (empty stomach) to 493 +/- 105 ng X ml-1 X hr (P less than 0.001). The AUC (0 to 24 hours) of the three metabolites together also increased significantly from 8511 +/- 2062 (empty stomach) to 11219 +/- 2471 ng X ml-1 X hr (P less than 0.01). The mean (+/- SD) percent increase in AUC (0 to 24 hours) of spironolactone when it was given with food, compared with the ingestion on an empty stomach (95.4% +/- 66.9%), was much more pronounced than the corresponding increase of 7 alpha-thiomethylspirolactone (45.4% +/- 33.7%), 6 beta-hydroxy-7 alpha-thiomethylspiro-lactone (21.8% +/- 21.5%), and canrenone (40.7% +/- 26.3%). These observations indicate that food promotes the absorption of spironolactone and possibly decreases its first-pass metabolism.

  6. Formal synthesis of squalamine from desmosterol.

    PubMed

    Okumura, Kazuo; Nakamura, Yutaka; Takeuchi, Seiji; Kato, Isao; Fujimoto, Yoshinori; Ikekawa, Nobuo

    2003-10-01

    The key intermediate to squalamine, (5alpha,7alpha,24R)-7,24-dihydroxy-cholestan-3-one, was synthesized from the 3-O-acetyl-24R,25-dihydroxy derivative of desmosterol via 10 steps in 16% overall yield and squalamine was also prepared via two further steps in 7.4% total yield from the desmosterol derivative. PMID:14519925

  7. Side chain hydroxylation of C27-steroids and vitamin D3 by a cytochrome P-450 enzyme system isolated from human liver mitochondria

    SciTech Connect

    Oftebro, H.; Saarem, K.; Bjoerkhem, I.; Pedersen, J.I.

    1981-11-01

    The present study was undertaken to obtain information on the involvement of cytochrome P-450 in the 26-hydroxylation on bile acid intermediates and in the 25-hydroxylation of vitamin D3 in human liver mitochondria. Cytochrome P-450 was solubilized from human liver mitochondria and purified two times to a specific content of 0.125 nmol per mg protein. Furthermore, a ferredoxin was isolated from the mitochondria and partly purified. This iron-sulfur protein had properties similar to bovine adrenal ferredoxin. A mitochondrial NADPH-ferredoxin reductase was also isolated and purified to homogeneity. This enzyme was a flavoprotein with properties very similar to the bovine adrenal NADPH-ferredoxin reductase. The cytochrome P-450 preparation catalyzed 26-hydroxylation of C27-steroids and 25-hydroxylation of vitamin D3 when reconstructed with NADPH, the ferredoxin and the ferredoxin reductase. With different substrates the following turnover numbers (nmol product X nmol P-450(-1) X min-1) were found: cholesterol, 8; 5-cholestene-3 beta, 7 alpha-diol, 10; 7 alpha-hydroxy-4-cholesten-3-one, 23; 7 alpha, 12 alpha-dihydroxy-4-cholesten-3-one, 27; 5 beta-cholestane-3 alpha, 7 alpha-diol, 28; 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, 41; and vitamin D3, 0.16. The hydroxylation reactions were inhibited by CO and metyrapone. The human liver mitochondrial ferredoxin and ferredoxin reductase could be replaced by adrenal ferredoxin and adrenal ferredoxin reductase without reduction of activity, but they could not be replaced by microsomal NADPH-cytochrome P-450 reductase. It is concluded that human liver mitochondria contain cytochrome P-450 involved in the oxidation of the side chain of C27-steroids and vitamin D3.

  8. Biological activity of novel progesterone derivatives having a bulky ester side chains at C-3.

    PubMed

    Cabeza, Marisa; Bratoeff, Eugene; Ramírez, Elena; Heuze, Ivonne; Recillas, Sergio; Berrios, Hilda; Cruz, Angel; Cabrera, Olmo; Perez, Victor

    2008-09-01

    Antiandrogens are widely used agents for the treatment of androgen dependent diseases as inhibitors of androgen receptors (AR) action. Although the precise mechanism of antiandrogen action is not yet elucidated, recent studies indicate the involvement of the structure of the ligand in relation with the nuclear co-repressors. In the present study, we investigated the relationship between logP (the partition coefficient) of four pregnane derivatives 9a-9d and their biological activity. For this purpose, we determined the relative binding affinity (RBA) of steroids 9a-9d to androgen receptor (AR) obtained from rat prostate cytosol, using labeled mibolerone (MIB) as ligand. The IC(50) value of each compound was calculated according to the plots of concentration versus percentage of binding. The in vivo effect of 9a-9d was determined on the weight of the prostate and seminal vesicles from castrated hamsters treated with dihydrotestosterone. The four compounds bind to the androgen receptor with different relative binding affinity (RBA). Compound 9d having a logP of 4.17 showed the highest RBA>100% as compared to compound 9a having a logP of 2.92 which exhibited a RBA of only 2.85%. These data show a very good correlation between the lipophilicity of these compounds represented by logP and the percentage of RBA. The in vivo experiments showed that all new compound 9a-9d reduced the weight of the prostate gland as well as the seminal vesicles. Steroids 9c and 9d having a logP of 3.75 and 4.17, respectively, showed the highest antiandrogenic effect. PMID:18472120

  9. 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.

  10. 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.

  11. Proteomic-Coupled-Network Analysis of T877A-Androgen Receptor Interactomes Can Predict Clinical Prostate Cancer Outcomes between White (Non-Hispanic) and African-American Groups

    PubMed Central

    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. PMID:25409505

  12. Mechanism of androgen action in cultured dermal papilla cells derived from human hair follicles with varying responses to androgens in vivo.

    PubMed

    Randall, V A; Thornton, M J; Hamada, K; Messenger, A G

    1992-06-01

    Androgens are major regulators of human hair growth, but their effects vary: many follicles are stimulated by androgens, e.g., beard; some remain unaffected, e.g., eyelashes; whereas scalp follicles undergo regression and balding in genetically disposed individuals. Because the dermal papilla controls many aspects of the hair follicle, androgens may act via the dermal papilla, affecting the other follicular components indirectly. In this hypothesis androgens would alter dermal papilla cell production of regulatory substances, e.g., growth factors and/or extracellular matrix components. To test this theory the mechanism of androgen action has been compared in primary lines of dermal papilla cells cultured from androgen-dependent follicles and relatively androgen-independent non-balding scalp. Androgen receptor levels were assayed by saturation analysis (9-10 points; 0.05-10 nmol/l) using the synthetic androgen [3H]-mibolerone and specificity was confirmed by competition studies. Androgen metabolism was investigated both intracellularly and in the media after a 2-h incubation with 5 nM [3H]-testosterone. Carrier and [14C] steroids were added to the extracts before separation by thin-layer chromatography; steroid identity was confirmed by recrystallization. Dermal papilla cells from androgen-dependent follicles contained higher levels of specific, high-affinity, low-capacity androgen receptors than non-balding scalp cells. Testosterone metabolism also varied with beard, public and scalp cells containing testosterone and androstenedione intracellularly, but only beard cells producing 5 alpha-dihydrotestosterone, in line with the scanty beard growth found in 5 alpha-reductase deficiency. Elsewhere we have shown that cultured dermal papilla cells produce extracellular matrix components and mitogenic factors. These results all concur with our original hypothesis and suggest that further studies of such cells may elucidate the paradoxical effects of androgens on human hair

  13. Fbw7 and Usp28 regulate myc protein stability in response to DNA damage.

    PubMed

    Popov, Nikita; Herold, Steffi; Llamazares, Maria; Schülein, Christina; Eilers, Martin

    2007-10-01

    The cellular levels of the Myc oncoprotein are critical determinants of cell proliferation, cell growth and apoptosis and are tightly regulated by external growth factors. Levels of Myc oncoprotein also decline in response to intracellular stress signals such as DNA damage. We show here that this decline is in part due to proteasomal degradation and that it is mediated by the Fbw7 ubiquitin ligase. We have shown previously that the ubiquitin-specific protease Usp28, binds to the nucleoplasmic isoform of Fbw7, Fbw7alpha, and counteracts its function in mammalian cells. Usp28 dissociates from Fbw7alpha in response to UV irradiation, providing a mechanism how Fbw7-mediated degradation of Myc is enhanced upon DNA damage. Our data extend previous observations that link Myc function to the cellular response to DNA damage.

  14. Kaempferol acetylrhamnosides from the rhizome of Dryopteris crassirhizoma and their inhibitory effects on three different activities of human immunodeficiency virus-1 reverse transcriptase.

    PubMed

    Min, B S; Tomiyama, M; Ma, C M; Nakamura, N; Hattori, M

    2001-05-01

    Three new kaempferol glycosides, called crassirhizomosides A (1), B (2) and C (3), were isolated from the rhizome of Dryopteris crassirhizoma (Aspidiaceae), together with the known kaempferol glycoside, sutchuenoside A (4). The structures of 1-3 were determined as kaempferol 3-alpha-L-(2,4-di-O-acetyl)rhamnopyranoside-7-alpha-L-rhamnopyranoside, kaempferol 3-alpha-L-(3,4-di-O-acetyl)rhamnopyranoside, and kaempferol 3-alpha-L-(2,3-di-O-acetyl)rhamnopyranosside-7-alpha-L-rhamnopyranoside, respectively, by chemical and spectroscopic means. Inhibitory effects of 1-4 and kaempferol on human immunodeficiency virus reverse transcriptase-associated DNA polymerase (RNA-dependent DNA polymerase and DNA-dependent DNA polymerase) and RNase H activities were investigated.

  15. Guaianolide sesquiterpenes from Pulicaria crispa (Forssk.) Oliv.

    PubMed

    Stavri, Michael; Mathew, K T; Gordon, Andrew; Shnyder, Steven D; Falconer, Robert A; Gibbons, Simon

    2008-06-01

    A phytochemical study of the asteraceous herb Pulicaria crispa (Forssk.) Oliv. resulted in the characterisation of three guaianolide sesquiterpenes, 2alpha,4alpha-dihydroxy-7alphaH,8alphaH,10alphaH-guaia-1(5),11(13)-dien-8beta,12-olide (1), 1alpha,2alpha-epoxy-4beta-hydroxy-5alphaH,7alphaH,8alphaH,10alphaH-guaia-11(13)-en-8beta,12-olide (2) and 5,10-epi-2,3-dihydroaromatin (3). The structures were assigned on the basis of extensive 1 and 2D NMR experiments. Compound 3 exhibited weak antimycobacterial activity against Mycobacterium phlei with a minimum inhibitory concentration of 0.52 mM and cytotoxicity (IC50 of 5.8+/-0.2 microM) in a human bladder carcinoma cell line, EJ-138.

  16. New polyoxygenated steroids from the South China Sea gorgonian Echinogorgia aurantiaca.

    PubMed

    Qiu, Yunqi; Qi, Shuhua; Zhang, Si; Yang, Jin; Xiao, Zhihui

    2006-07-01

    Three new polyoxygenated steroids, named 3beta,7alpha,9alpha-trihydroxy-cholestan-6-one (1), 3beta,5alpha,6beta-trihydroxycholestan-1-one (2) and cholestane-3beta,5alpha,6beta,11beta-tetrol (3), along with four known steroids (4-7) were isolated from the South China Sea gorgonian Echinogorgia aurantiaca. The structures of 1-3 were established by extensive spectroscopic analysis, including 1D and 2D NMR data.

  17. Bile acids of snakes of the subfamily Viperinae and the biosynthesis of C-23-hydroxylated bile acids in liver homogenate fractions from the adder, Vipera berus (Linn.).

    PubMed Central

    Ikawa, S; Tammar, A R

    1976-01-01

    1. Analysis of bile salts of four snakes of the subfamily Viperinae showed that their bile acids consisted mainly of C-23-hydroxylated bile acids. 2. Incubations of 14C-labelled sodium cholate (3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oate) and deoxycholate (3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oate) with whole and fractionated adder liver homogenates were carried out in the presence of molecular oxygen and NADPH or an NADPH-generating system. The formation of C-23-hydroxylated bile acids, namely bitocholic acid (3 alpha, 12 alpha, 23xi-trihydroxy-5 beta-cholan-24-oic acid) and 3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-cholanic acid (3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-5 beta-cholan-24-oic acid), was observed mainly in the microsomal fraction and partly in the mitochondrial fraction. 3. Biosynthetic pathways of C-23-hydroxylated bile acids are discussed. PMID:6007

  18. 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.

  19. Highly hydroxylated guaianolides of Achillea asiatica and Middle European Achillea species.

    PubMed

    Glasl, S; Presser, A; Gunbilig, D; Werner, I; Narantuya, S; Haslinger, E; Jurenitsch, J; Kubelka, W

    2001-12-01

    From flower heads of Achillea asiatica (L.) Serg., three new guaianolides were isolated by repeated column chromatography and HPLC. The constitution and the stereochemistry of these new, labile compounds were determined by MS, one ((1)H, (13)C, selective (1)H-TOCSY and (1)H-NOESY) and two-dimensional NMR experiments ((1)H, (1)H-COSY, (1)H, (13)C-HSQC, (1)H, (13)C-HMBC). The substances were identified as 8 alpha-angeloxy-2 alpha, 4 alpha,10 beta-trihydroxy-6 beta H,7 alpha H, 11 beta H-1(5)-guaien-12,6 alpha-olide (1), 8 alpha-angeloxy-1 beta,2 beta:4 beta,5 beta-diepoxy-10 beta-hydroxy-6 beta H, 7 alpha H, 11 beta H-12,6 alpha-guaianolide (2) and 8 alpha-angeloxy-4 alpha,10 beta-dihydroxy-2-oxo-6 beta H,7 alpha H, 11 beta H-1(5)-guaien-12,6 alpha-olide (3). They were also detected in Middle European species (Achillea collina, Achillea ceretanica (2x and 4x), Achillea roseoalba, Achillea asplenifolia) by HPLC, TLC and off line MS and have not been described before. The possibility that these compounds might be products of an oxidation process is discussed.

  20. Chromatographic determination of riboflavin and its derivatives in food.

    PubMed

    Gliszczyńska-Swigło, A; Koziołowa, A

    2000-06-01

    Three elution methods on two different reversed-phase C18 columns were developed to determine flavin derivatives in raw egg white, raw egg yolk, egg powder, pasteurised milk, fermented milk products and liver (chicken, calf and pig). Additionally, 11 thin-layer chromatography solvent systems were used to confirm presence of flavins detected in assessed products. It was found that an Alphabond C18 column was not as effective as a Symmetry C18 column. Method A (mobile phase gradient of methanol-0.05 M ammonium acetate, pH 6.0 applied on an Alphabond C18 column) can be used for determination of flavin adenine dinucleotide, flavin mononucleotide, riboflavin 4',5'-cyclic phosphate, riboflavin, 10-formylmethylflavin and 10-hydroxyethylflavin in products that do not contain 7alpha-hydroxyriboflavin. Method B (mobile phase gradient of methanol-demineralized water, on an Alphabond C18 column) can be useful to separate flavin coenzymes from other flavin compounds or to confirm the presence of 7alpha-hydroxyriboflavin and 10-hydroxyethylflavin in analysed samples. Method C (mobile phase gradient of methanol-0.05 M ammonium acetate, pH 6.0, on a Symmetry C18 column) allows separation of all flavins detected in tested products: flavin adenine dinucleotide, flavin mononucleotide, riboflavin 4',5'-cyclic phosphate, riboflavin, 10-formylmethylflavin, 10-hydroxyethylflavin, 7alpha-hydroxyriboflavin, riboflavin-beta-D-galactoside and riboflavin-alpha-D-glucoside. PMID:10905712

  1. 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.

  2. Activation of two mutant androgen receptors from human prostatic carcinoma by adrenal androgens and metabolic derivatives of testosterone.

    PubMed

    Culig, Z; Stober, J; Gast, A; Peterziel, H; Hobisch, A; Radmayr, C; Hittmair, A; Bartsch, G; Cato, A C; Klocker, H

    1996-01-01

    The androgen receptor (AR) plays a central regulatory role in prostatic carcinoma and is a target of androgen ablation therapy. Recent detection of mutant receptors in tumor specimens suggest a contribution of AR alterations to progression towards androgen independence. In a specimen derived from metastatic prostate cancer we have reported a point mutation in the AR gene that leads to a single amino acid exchange in the ligand binding domain of the receptor. Another amino acid exchange resulting from a point mutation was also identified 15 amino acids away from our mutation. This mutation was detected in the AR gene isolated from an organ-confined prostatic tumor. Here we report the functional characterization of the two mutant receptors in the presence of adrenal androgens and testosterone metabolites. These studies were performed by cotransfecting androgen-responsive reporter genes and either the wild-type or mutant AR expression vectors into receptor negative DU-145 and CV-1 cells. The indicator genes used consisted of the promoter of the androgen-inducible prostate-specific antigen gene or the C' Delta9 enhancer fragment from the promoter of the mouse sex-limited protein driving the expression of the bacterial chloramphenicol acetyl transferase gene. Cotransfection-transactivation assays revealed that the adrenal androgen androstenedione and two products of testosterone metabolism, androsterone and androstandiol, induced reporter gene activity more efficiently in the presence of the mutant receptors than in the presence of the wild-type receptor. No difference between wild-type and mutant receptors was observed in the presence of the metabolite androstandione. The interaction of receptor-hormone complexes with target DNA was studied in vitro by electrophoretic mobility shift assays (EMSA). Dihydrotestosterone and the synthetic androgen mibolerone induced a faster migrating complex with all receptors, whereas the androgen metabolite androstandione induced this

  3. 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

  4. The role of alpha-methylacyl-CoA racemase in bile acid synthesis.

    PubMed

    Cuebas, Dean A; Phillips, Christopher; Schmitz, Werner; Conzelmann, Ernst; Novikov, Dmitry K

    2002-05-01

    According to current views, the second peroxisomal beta-oxidation pathway is responsible for the degradation of the side chain of bile acid intermediates. Peroxisomal multifunctional enzyme type 2 [peroxisomal multifunctional 2-enoyl-CoA hydratase/(R)-3-hydroxyacyl-CoA dehydrogenase; MFE-2] catalyses the second (hydration) and third (dehydrogenation) reactions of the pathway. Deficiency of MFE-2 leads to accumulation of very-long-chain fatty acids, 2-methyl-branched fatty acids and C(27) bile acid intermediates in plasma, but bile acid synthesis is not blocked completely. In this study we describe an alternative pathway, which allows MFE-2 deficiency to be overcome. The alternative pathway consists of alpha-methylacyl-CoA racemase and peroxisomal multifunctional enzyme type 1 [peroxisomal multifunctional 2-enoyl-CoA hydratase/(S)-3-hydroxyacyl-CoA dehydrogenase; MFE-1]. (24E)-3alpha,7alpha,12alpha-Trihydroxy-5beta-cholest-24-enoyl-CoA, the presumed physiological isomer, is hydrated by MFE-1 with the formation of (24S,25S)-3alpha,7alpha,12alpha,24-tetrahydroxy-5beta-cholestanoyl-CoA [(24S,25S)-24-OH-THCA-CoA], which after conversion by a alpha-methylacyl-CoA racemase into the (24S,25R) isomer can again be dehydrogenated by MFE-1 to 24-keto-3alpha,7alpha,12alpha-trihydroxycholestanoyl-CoA, a physiological intermediate in cholic acid synthesis. The discovery of the alternative pathway of cholesterol side-chain oxidation will improve diagnosis of peroxisomal deficiencies by identification of serum 24-OH-THCA-CoA diastereomer profiles.

  5. 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.

  6. 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. PMID:17207824

  7. 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.

  8. Monoterpenoid glucoindole alkaloids and iridoids from Pterocephalus pinardii.

    PubMed

    Gülcemal, Derya; Masullo, Milena; Alankuş-Calişkan, Ozgen; Karayildirim, Tamer; Senol, Serdar G; Piacente, Sonia; Bedir, Erdal

    2010-03-01

    A new secondary metabolite, pterocephaline, along with the known cantleyoside, 7alpha-morroniside, 3beta,5alpha-tetrahydrodesoxycordifoline lactam, 5S-5-carboxyvincoside, sweroside, and loganin have been isolated from the aerial parts of P. pinardii (Dipsacaceae). Moreover, cantleyoside-methyl-hemiacetal and cantleyoside-dimethyl-acetal were obtained as seco-iridoid artifacts. The structures were elucidated by extensive spectroscopic methods including 1D-((1)H, (13)C and TOCSY) and 2D-NMR (DQF-COSY, HSQC and HMBC). Monoterpenoid glucoindole alkaloids were encountered for the first time in Dipsacaceae family.

  9. 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 %).

  10. 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.

  11. New guaian-type sesquiterpene from Wikstroemia indica.

    PubMed

    Kato, Mamoru; He, Yu-Min; Dibwe, Dya Fta; Li, Feng; Awale, Suresh; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2014-01-01

    From a MeOH extract of powdered roots of Wikstroemia indica, we isolated a new guaian-type sesquiterpene (1) and two known guaian-type sesquiterpenes [oleodaphnal (2), 1alpha,7alpha,10alphaH-guaia-4,11-dien-3-one (3)], together with twelve known compounds: (+)-arctigenin, (+)-matairesinol, (+)-trachelogenin, (+)-nortrachelogenin, (+)-hinokinin, (+)-kusunokinin, 7-methoxycoumarin, 7-hydroxycoumarin (umbelliferone), daphnogitin, daphnoretin, salicifoliol, and (-)-pinoresinol. The structure of compound 1 was determined to be 4,10,11-guaiatrien-3-one-14-oic acid, by the analyses of spectral data. PMID:24660446

  12. 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.

  13. Emergence of a cefepime- and cefpirome-resistant Citrobacter freundii clinical isolate harbouring a novel chromosomally encoded AmpC beta-lactamase, CMY-37.

    PubMed

    Ahmed, Ashraf M; Shimamoto, Tadashi

    2008-09-01

    Citrobacter freundii strain 4306 was isolated from a urine specimen of a patient in March 2006 in Palestine. This strain showed a unique multidrug resistance phenotype, as it was resistant both to 7-alpha-methoxy- and oxyimino-cephalosporins, including cefepime, cefpirome and monobactams, in addition to quinolones, streptomycin and trimethoprim/sulfamethoxazole. Clavulanic acid did not act synergistically with cephalosporins by the double-disk synergy test. Molecular characterisation showed that the resistance to 7-alpha-methoxy- and oxyimino-cephalosporins was due to a novel AmpC beta-lactamase, designated CMY-37, with an isoelectric point of approximately 9.0. CMY-37 is a variant of C. freundii chromosomal AmpC enzymes with at least seven amino acid substitutions. One of these substitutions, L316I, is located within the R2 loop that is considered the hotspot region responsible for the extended substrate spectrum in class C beta-lactamases. The blaCMY-37 gene was cloned and expressed in Escherichia coli TG1. CMY-37 is chromosomally encoded and is not associated with ISEcp1-like element. Phylogenetic analysis suggested that CMY-37 is the origin of many plasmid-mediated AmpC beta-lactamases. This study highlights the emergence of cefepime and cefpirome resistance in C. freundii owing to a new type of AmpC beta-lactamase.

  14. Delivery of testosterone replacement therapy.

    PubMed

    Hameed, Asjad; Brothwood, Theresa; Bouloux, Pierre

    2003-10-01

    Optimal testosterone replacement therapy remains a considerable challenge for the estimated five out of 1000 men in the general community with androgen deficiency. Oral delivery is not possible due to rapid first pass metabolism and short half-life. Testosterone derivatives have been developed to enhance intrinsic androgenic potency, prolong duration of action, or improve oral bioavailability of synthetic androgens. Structural modification of testosterone include 17 beta-esterification, 17 alpha-alkylation, 1-methylation, addition of a 19-normethyl group, and 7 alpha-methylation. Currently, oral (testosterone undecanoate), transcutaneous (Andropatch, Virormone, Testoderm (ALZA Corp), Testogel), sublingual (testosterone cyclodextrin), intramuscular (Sustanon, Primoteston Depot), and fused crystalline testosterone pellet preparations are available for clinical use. Transbuccal testosterone systems have also been developed for clinical use and require twice daily application. Suspensions of biodegradable microspheres consisting of a polyglycolide-lactide matrix laden with testosterone can deliver stable, physiological levels of testosterone for 2 to 3 months. Micronized testosterone has low oral bioavailability requiring high daily doses. 7 alpha-Methyl 19-nortestosterone, a potent, synthetic androgen free of hepatotoxicity, has tissue-specific selectivity, being susceptible to aromatization but not 5 alpha-reduction, thereby potentially avoiding intraprostatic androgen amplification. PMID:14649214

  15. The pea gene NA encodes ent-kaurenoic acid oxidase.

    PubMed

    Davidson, Sandra E; Elliott, Robert C; Helliwell, Chris A; Poole, Andrew T; Reid, James B

    2003-01-01

    The gibberellin (GA)-deficient dwarf na mutant in pea (Pisum sativum) has severely reduced internode elongation, reduced root growth, and decreased leaflet size. However, the seeds develop normally. Two genes, PsKAO1 and PsKAO2, encoding cytochrome P450 monooxygenases of the subfamily CYP88A were isolated. Both PsKAO1 and PsKAO2 had ent-kaurenoic acid oxidase (KAO) activity, catalyzing the three steps of the GA biosynthetic pathway from ent-kaurenoic acid to GA(12) when expressed in yeast (Saccharomyces cerevisiae). In addition to the intermediates ent-7alpha-hydroxykaurenoic acid and GA(12)-aldehyde, some additional products of the pea KAO activity were detected, including ent-6alpha,7alpha-dihydroxykaurenoic acid and 7beta-hydroxykaurenolide. The NA gene encodes PsKAO1, because in two independent mutant alleles, na-1 and na-2, PsKAO1 had altered sequences and the five-base deletion in PsKAO1 associated with the na-1 allele cosegregated with the dwarf na phenotype. PsKAO1 was expressed in the stem, apical bud, leaf, pod, and root, organs in which GA levels have previously been shown to be reduced in na plants. PsKAO2 was expressed only in seeds and this may explain the normal seed development and normal GA biosynthesis in seeds of na plants.

  16. Delta 4-3-oxosteroid 5 beta-reductase deficiency causing neonatal liver failure and hemochromatosis.

    PubMed

    Shneider, B L; Setchell, K D; Whitington, P F; Neilson, K A; Suchy, F J

    1994-02-01

    Neonatal liver failure was evaluated in two infants. Neither infant had evidence of congenital infection, galactosemia, alpha 1-antitrypsin deficiency, tyrosinemia, Zellweger syndrome, or hemophagocytic lymphohistiocytosis. Abnormal levels of iron were detected in the minor salivary glands of the first infant and in the explanted liver of the second. Analyses of urinary bile salts by fast-atom bombardment ionization mass spectrometry and gas chromatography-mass spectrometry revealed a paucity of primary bile acids and a predominance of 7 alpha-hydroxy-3-oxo-4-cholenoic and 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acids. These findings are consistent with delta 4-3-oxosteroid 5 beta-reductase deficiency, a primary genetic defect in bile acid synthesis. Postmortem evaluation of the first infant revealed significant iron deposition in the liver, pancreas, thyroid, adrenal glands, myocardium, stomach, and submucosal glands of the respiratory tract. In both infants examination of the liver revealed extensive loss of hepatic parenchyma. These cases expand the clinical spectrum of bile acid metabolism defects to include neonatal liver failure with associated hemochromatosis. PMID:8301429

  17. Synthesis of 24-nor-5 beta-cholan-23-oic acid derivatives: a convenient and efficient one-carbon degradation of the side chain of natural bile acids.

    PubMed

    Schteingart, C D; Hofmann, A F

    1988-10-01

    An efficient procedure for obtaining nor-bile acids from natural (C24) bile acids is described. Treatment of formylated bile acids with sodium nitrite in a mixture of trifluoroacetic anhydride with trifluoroacetic acid gives, through a "second order" Beckmann rearrangement, 24-nor-23-nitriles. These compounds, on alkaline hydrolysis, afford the corresponding nor-bile acids in high yields. The sequence was successfully applied to the synthesis of 3 alpha-hydroxy-24-nor-5 beta-cholan-23-oic (norlithocholic) acid, 3 alpha,6 alpha- (norhyodeoxycholic), 3 alpha,7 alpha- (norchenodeoxycholic), 3 alpha,7 beta- (norursodeoxycholic), and 3 alpha,12 alpha-dihydroxy-24-nor-5 beta-cholan-23-oic (nordeoxycholic) acids, as well as 3 alpha,7 alpha,12 alpha-trihydroxy-24-nor-5 beta-cholan-23-oic (norcholic) acid. 13C-NMR spectra of their methyl esters are reported. The procedure provides a more rapid alternative to the Barbier-Wieland degradation for shortening by one methylene group the side chain of natural (C24) bile acids.

  18. 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.

  19. 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

  20. Determination of chenodiol bioequivalence using an immobilized multi-enzyme bioluminescence technique.

    PubMed

    Rossi, S S; Clayton, L M; Hofmann, A F

    1986-03-01

    Measurement of the bioequivalence of formulations of chenodiol, a bile acid which is used for gallstone dissolution, is difficult because its high first-pass clearance results in low plasma levels after ingestion of usual dosages. To solve this problem, a new method was developed to determine the bioequivalence of several chenodiol formulations. The method included the following steps: isolation of all bile acids from serum by absorption to a hydrophobic resin, elution of bile acids from the resin by methanol, separation of the unconjugated bile acid fraction by an ion-exchange procedure, and bioluminescence measurement of the unconjugated 7 alpha-hydroxy bile acids using Sepharose beads containing co-immobilized 7 alpha-hydroxysteroid dehydrogenase, diaphorase, and luciferase. The isolation method gave complete recovery, and the bioluminescence procedure was simple, rapid, and sensitive. The peak level of systemic chenodiol occurred 1 to 2 h following oral ingestion and ranged from 4 to 8 microM. This method appears superior to previously reported methods for determining the bioequivalence of chenodiol preparations. In principle, the method is suitable for measurement of the bioequivalence of other bile acids provided the appropriate hydroxysteroid dehydrogenase is available. PMID:3701613

  1. Sterol synthesis. A novel reductive rearrangement of an alpha,beta-unsaturated steroidal epoxide; a new chemical synthesis of 5alpha-cholest-8(14)-en-3beta, 15alpha-diol.

    PubMed

    Parish, E J; Schroepfer, G J

    1977-04-01

    Reduction of 3beta-benzoyloxy-14alpha,15alpha-epoxy-5alpha-cholest-7-ene with either lithium triethylboro-hydride or lithium aluminum hydride (4 molar excess) gave 5-alpha-cholest-8(14)-en-3beta,15alpha-diol in high yield. Reduction of the epoxy ester with lithium triethylborodeuteride or lithium aluminum deuteride (4 molar excess) gave [7alpha-2-H]-5alpha-cholest-8(14)-en-3beta,15alpha-diol. Reduction of 2beta-benzoyloxy-14alpha,15alpha-epoxy-5alpha-cholest-7-ene with a large excess (24 molar excess) of lithium aluminum hydride gave, in addition to the expected 5alpha-cholest-8(14)-en-3beta,15alpha-diol, a significant yield (33%) of 5alpha-cholest-8(14)-en-3beta-o1. Reduction of the epoxy ester with a large excess (24 molar excess) of lithium aluminum deuteride gave [7alpha-2H]-5alpha-cholest-8(14)-en-3beta,15alpha-diol and 5alpha-cholest-8(14)-en-3beta-o1 which contained two atoms of stably bound deuterium. PMID:858170

  2. Effect of diet and Lactobacillus acidophilus supplements on human fecal bacterial enzymes.

    PubMed

    Goldin, B R; Swenson, L; Dwyer, J; Sexton, M; Gorbach, S L

    1980-02-01

    The effect of diet and Lactobacillus acidophilus supplements on fecal microflora enzyme activity was studied in humans. The bacterial enzymes that were investigated are known to catalyze reactions that may result in formation of proximal carcinogens. Compared to vegetarians, omnivores eating a "Western-type" diet had higher levels of beta-glucuronidase, nitroreductase, azoreductase, and steroid 7-alpha-dehydroxylase in their fecal microflora. Removal of red meat or addition of fiber in the form of bran or wheat germ to the diet of omnivores for 30 days had no effect on beta-glucuronidase, nitroreductase, or azoreductase activity. However, removal of red meat or addition of fiber reduced fecal steroid 7-alpha-dehydroxylase activity. The addition of viable Lactobacillus acidophilus supplements to the diet of omnivores significantly decreased fecal bacterial beta-glucuronidase and nitroreductase activities. Thirty days after Lactobacillus supplements were curtailed, fecal enzyme levels returned to normal base-line activities. These findings suggested that the metabolic activity of the fecal microflora was influenced by diet and could be altered by Lactobacillus supplements and to a lesser extent by dietary fiber.

  3. Polyhydroxy steroids and saponins from China Sea starfish Asterina pectinifera and their biological activities.

    PubMed

    Peng, Yan; Zheng, Jianxian; Huang, Riming; Wang, Yifei; Xu, Tunhai; Zhou, Xuefeng; Liu, Qiuying; Zeng, Fanli; Ju, Huaiqiang; Yang, Xianwen; Liu, Yonghong

    2010-06-01

    A new polyhydroxy sterol ester, (25S)-5alpha-cholestane-3beta,6alpha,7alpha,8,15alpha,16beta-hexahydroxyl-26-O-14'Z-eicosenoate (1), together with seven known steroid derivatives (2-8), were isolated from the EtOH extract of the whole body of China Sea starfish Asterina pectinifera. The structure of 1 was determined by using extensive spectra analysis (IR, 1D and 2D NMR, and MS), chemical degradation, and comparison with the known compound (25S)-5alpha-cholestane-3beta,6alpha,7alpha,8,15alpha,16beta,26-heptol (2). All the isolates were evaluated for their antiviral activity against herpes simplex virus type 1 (HSV-1) and their cytotoxicity against human liver carcinoma HepG2 cell line in vitro. Compounds 3-6, and 8 exhibited antiviral activity against HSV-1 virus with the minimal inhibitory concentration (MIC) values of 0.2, 0.05, 0.2, 0.22, and 0.07 microM, respectively. While compounds 4 and 5 exhibited cytotoxicity against HepG2 cells with IC(50) values of 0.2 and 1.6 microM, respectively.

  4. [Multiplex PCR for detecting genotypes of deletional alpha-thalassemia].

    PubMed

    Wu, Jie-Ying; Liao, Can; Li, Jian; Huang, Yi-Ning

    2004-08-01

    To investigate the clinical application of multiplex PCR in detecting genotypes of deletional alpha-thalassemia in South China and observe the distribution frequency of alpha-globin gene deletion, 145 patients with silent carrier, alpha thalassemia trait or HbH were identified by M-PCR and 1.2% agarose gel electrophoresis. There are 1.3, 1.6, 1.8 and 2.0 kb bands which indicate --(SEA), -alpha(4.2), alphaalpha and -alpha(3.7), respectively. The results showed that among 145 patients, 100 patients with --(SEA)/alphaalpha (68.9%), 15 with -alpha(3.7)/alphaalpha (10.3%), 8 with -alpha(4.2)/alphaalpha (5.52%), 2 with -alpha(3.7)/-alpha(4.2) (1.38%), 1 with -alpha(3.7)/-alpha(3.7) (0.69%), 1 with -alpha(4.2)/-alpha(4.2) (0.69%), 14 with --(SEA)/-alpha(3.7) (9.65%), 2 with --(SEA)/-alpha(4.2) (1.38%) were found. Two patients prenatal diagnosed were confirmed with Bart's hydrops fetuses. In conclusion, M-PCR analysis is a simple, rapid and accurate method for detection of alpha-thalassemia gene deletion. This technique is helpful in screening, carrier identification and prenatal diagnosis of deletional alpha-thalassemia.

  5. Solubility of calcium salts of unconjugated and conjugated natural bile acids.

    PubMed

    Gu, J J; Hofmann, A F; Ton-Nu, H T; Schteingart, C D; Mysels, K J

    1992-05-01

    The approximate solubility products of the calcium salts of ten unconjugated bile acids and several taurine conjugated bile acids were determined. The formation of micelles, gels, and/or precipitates in relation to Ca2+,Na+, and bile salt concentration was summarized by "phase maps." Because the ratio of Ca2+ to bile salt in the precipitates was ca. 1:2, and the activity of Ca2+ but not that of bile salt (BA-) could be measured, the ion product of aCa2+ [BA-]2 was calculated. The ion product (= Ksp) ranged over nine orders of magnitude and the solubility thus ranged over three orders of magnitude; its value depended on the number and orientation of the hydroxyl groups in the bile acid. Ion products (in units of 10(-9) mol/l)3 were as follows: cholic (3 alpha OH,7 alpha OH,12 alpha OH) 640; ursocholic (3 alpha OH,7 beta OH,12 alpha OH) 2300; hyocholic (3 alpha OH,6 alpha OH,7 alpha OH) 11; ursodeoxycholic (3 alpha OH,7 beta OH) 91; chenodeoxycholic (3 alpha OH,7 alpha OH) 10; deoxycholic (3 alpha OH,12 alpha OH) 1.5; 12-epideoxycholic (lagodeoxycholic, 3 alpha OH,12 beta OH) 2.2; hyodeoxycholic (3 alpha OH,6 alpha OH) 0.7; and lithocholic (3 alpha OH) 0.00005. The critical micellization temperature of the sodium salt of murideoxycholic acid (3 alpha OH,6 beta OH) was greater than 100 degrees C, and its Ca2+ salt was likely to be very insoluble. Taurine conjugates were much more soluble than their corresponding unconjugated derivatives: chenodeoxycholyltaurine, 384; deoxycholyltaurine, 117; and cholyltaurine, greater than 10,000. Calcium salts of unconjugated bile acids precipitated rapidly in contrast to those of glycine conjugates which were metastable for months. Thus, hepatic conjugation of bile acids with taurine or glycine not only enhances solubility at acidic pH, but also at Ca2+ ion concentrations present in bile and intestinal content.

  6. Investigation of gene expressions related to cholesterol metabolism in rats fed diets enriched in n-6 or n-3 fatty acid with a cholesterol after long-term feeding using quantitative-competitive RT-PCR analysis.

    PubMed

    Fukushima, M; Shimada, K; Ohashi, E; Saitoh, H; Sonoyama, K; Sekikawa, M; Nakano, M

    2001-06-01

    We have developed a method to quantitate hepatic apolipoprotein (apo) B, LDL receptor, 3-hydroxy-3-methylglutary coenzyme A reductase (HMG-CoA reductase) and cholesterol 7alpha-hydroxylase mRNA expression in rats fed a cholesterol-enriched diet after long-term feeding using competitive RT-RCR. Rats (8 wk of age) fed a conventional diet were shifted to diets containing 10% perilla oil (PEO, oleic acid+linoleic acid+alpha-linolenic acid), borage oil (BRO, oleic acid+linoleic acid+gamma-linolenic acid), evening primrose oil (EPO, linoleic acid+gamma-linolenic acid), mixed oil (MIO, oleic acid+linoleic acid+gamma-linolenic acid+alpha-linolenic acid), or palm oil (PLO, palmitic acid+oleic acid+linoleic acid) with 0.5% cholesterol for 15 wk. There were no significant differences in the food intake and body weight gain among the groups. The liver weight in the PEO and PLO groups was significantly higher than other groups. The serum total cholesterol and very low density lipoprotein (VLDL)+intermediate density lipoprotein (IDL)+low density lipoprotein (LDL)-cholesterol concentrations were consistently higher in PLO group than in the other groups. The serum high density lipoprotein cholesterol concentration was significantly lower in the PEO group than in the other groups. The liver cholesterol concentration group was significantly higher in the PEO than in the other groups. There were no significant differences in the hepatic LDL receptor mRNA level among the groups. Hepatic apo B, HMG-CoA reductase and cholesterol 7alpha-hydroxylase mRNA levels were not affected by the experimental conditions. However, hepatic cholesterol 7alpha-hydroxylase mRNA level in the PEO and MIO groups tended to be higher than in the other groups. The fecal cholesterol extraction was significantly higher in the MIO and PLO groups than in the PEO and EPO groups and the total bile acid extraction was significantly higher in the PEO and MIO groups than in the PLO group. The results of this study

  7. Investigation of gene expressions related to cholesterol metabolism in rats fed diets enriched in n-6 or n-3 fatty acid with a cholesterol after long-term feeding using quantitative-competitive RT-PCR analysis.

    PubMed

    Fukushima, M; Shimada, K; Ohashi, E; Saitoh, H; Sonoyama, K; Sekikawa, M; Nakano, M

    2001-06-01

    We have developed a method to quantitate hepatic apolipoprotein (apo) B, LDL receptor, 3-hydroxy-3-methylglutary coenzyme A reductase (HMG-CoA reductase) and cholesterol 7alpha-hydroxylase mRNA expression in rats fed a cholesterol-enriched diet after long-term feeding using competitive RT-RCR. Rats (8 wk of age) fed a conventional diet were shifted to diets containing 10% perilla oil (PEO, oleic acid+linoleic acid+alpha-linolenic acid), borage oil (BRO, oleic acid+linoleic acid+gamma-linolenic acid), evening primrose oil (EPO, linoleic acid+gamma-linolenic acid), mixed oil (MIO, oleic acid+linoleic acid+gamma-linolenic acid+alpha-linolenic acid), or palm oil (PLO, palmitic acid+oleic acid+linoleic acid) with 0.5% cholesterol for 15 wk. There were no significant differences in the food intake and body weight gain among the groups. The liver weight in the PEO and PLO groups was significantly higher than other groups. The serum total cholesterol and very low density lipoprotein (VLDL)+intermediate density lipoprotein (IDL)+low density lipoprotein (LDL)-cholesterol concentrations were consistently higher in PLO group than in the other groups. The serum high density lipoprotein cholesterol concentration was significantly lower in the PEO group than in the other groups. The liver cholesterol concentration group was significantly higher in the PEO than in the other groups. There were no significant differences in the hepatic LDL receptor mRNA level among the groups. Hepatic apo B, HMG-CoA reductase and cholesterol 7alpha-hydroxylase mRNA levels were not affected by the experimental conditions. However, hepatic cholesterol 7alpha-hydroxylase mRNA level in the PEO and MIO groups tended to be higher than in the other groups. The fecal cholesterol extraction was significantly higher in the MIO and PLO groups than in the PEO and EPO groups and the total bile acid extraction was significantly higher in the PEO and MIO groups than in the PLO group. The results of this study

  8. Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site

    SciTech Connect

    Trehan, I.; Beadle, B.M.; Shoichet, B.K.

    2010-03-08

    {beta}-Lactamases hydrolyze {beta}-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health. {beta}-Lactams that contain a bulky 6(7){alpha} substituent, such as imipenem and moxalactam, actually inhibit serine {beta}-lactamases and are widely used for this reason. Although mutant serine {beta}-lactamases have arisen that hydrolyze {beta}-lactamase resistant {beta}-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine {beta}-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity. Structural and thermodynamic studies suggest that the 6(7){alpha} substituents of these inhibitors form destabilizing contacts within the covalent adduct with the conserved Asn152 in class C {beta}-lactamases (Asn132 in class A {beta}-lactamases). This unfavorable interaction may be crucial to inhibition. To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C {beta}-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consistent with the hypothesis, the Asn152 {yields} Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes. However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme. To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 {angstrom}. Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces {beta}-lactams with 6(7){alpha} substituents out of

  9. [Anthracycline antibiotics from genetically modified streptomycetes. The isolation, spectroscopic structural elucidation and biologic effects of beta-rhodomycin I].

    PubMed

    Ihn, W; Schlegel, B; Fleck, W F; Tresselt, D; Gutsche, W; Sedmera, P; Vokoun, J

    1984-03-01

    By mutagenic treatment and selection procedures the mutant ZIMET 43678 was obtained from a population of the interspecific recombinant Streptomyces violaceus subsp. iremyceticus ZIMET 43615, which showed a changed spectrum of secondary metabolites. The main component isolated from the fermentation broth was a pure anthracycline evidenced by TLC. By means of acid hydrolysis, identification of the degradation products and also by spectroscopic UV/VIS-, IR-, MS-, 1H/13C-NMR- and CD-investigations with intact anthracycline the structure 7-(alpha-L- rhodosaminyl )-beta- rhodomycinon with the absolute configuration 7S, 9R , 10R was found. The anthracycline called beta- rhodomycin -1 (1) exhibits antimicrobial and cytostatic activity in vitro and is also effective on tumour cells in tumour bearing animals. PMID:6427794

  10. (+)-Gibberellin C: hydrogen-bonding pattern of the monohydrate of a non-racemic pentacyclic diterpenoid.

    PubMed

    Thompson, H W; Brunskill, A P; Lalancette, R A

    2000-12-01

    In the monohydrate of the title compound, (+)-2beta, 4aalpha-dihydroxy-1,7-dimethyl-8-oxo-4bbeta,7alpha- gibbane-1alpha, 10beta-dicarboxylic acid-1,4a-lactone, C(19)H(24)O(6).H(2)O, intermolecular hydrogen bonding progresses helically along b from carboxyl to ketone [O...O = 2.694 (5) A]. The carboxyl and lactone carbonyl groups in translationally related molecules within a helix both accept hydrogen bonds from the same water of hydration. The oxygen of this water in turn accepts a hydrogen bond from the hydroxyl group of a third screw-related molecule in an adjacent counterdirectionally oriented helix, yielding a complex three-dimensional hydrogen-bonding array. Intermolecular O...H-C close contacts were found to the carboxyl and lactone carbonyls, the hydroxyl, and the water. PMID:11119009

  11. 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.

  12. 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.

  13. Biosynthesis of riboflavin in Bacillus subtilis: origin of the four-carbon moiety.

    PubMed Central

    Le Van, Q; Keller, P J; Bown, D H; Floss, H G; Bacher, A

    1985-01-01

    We studied the incorporation of [1-13C]ribose and [1,3-13C2]glycerol into the riboflavin precursor 6,7-dimethyl-8-ribityllumazine, using a riboflavin-deficient mutant of Bacillus subtilis. The formation of the pyrazine ring requires the addition of a four-carbon moiety to a pyrimidine precursor. The results show that C-6 alpha, C-6, C-7, and C-7 alpha of 6,7-dimethyl-8-ribityllumazine were biosynthetically equivalent to C-1, C-2, C-3, and C-5 of a pentose phosphate. C-4 of the pentose precursor was lost through an intramolecular skeletal rearrangement. Thus, the last steps in the biosynthesis of 6,7-dimethyl-8-ribityllumazine apparently involve the same mechanism in bacteria as in fungi. PMID:3922947

  14. 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.

  15. Compositions of royal jelly II. Organic acid glycosides and sterols of the royal jelly of honeybees (Apis mellifera).

    PubMed

    Kodai, Tetsuya; Umebayashi, Kazue; Nakatani, Takafumi; Ishiyama, Kaori; Noda, Naoki

    2007-10-01

    Two organic acid glycosides (1, 2) and 16 sterols were isolated from the royal jelly of honeybees (Apis mellifera). The former two were monoglucosides of 10-hydroxy-2E-decenoic and 10-hydroxydecanoic acids. They are the first examples of glycosides isolated from royal jelly. The latter 16 were sterols mainly composed of 28 or 29 carbons. Among them, four compounds were new isofucosterol derivatives, and their structures were characterized as (24Z)-stigmasta-5,24(28)-dien-3beta-ol-7-one (3), (24Z)-stigmasta-5,24(28)-diene-3beta,7beta-diol (4), (24Z)-stigmasta-5,24(28)-diene-3beta,7alpha-diol (5), and (24Z)-stigmast-24(28)-ene-3beta,5alpha,6beta-triol (6) on the basis of various NMR spectroscopic data.

  16. Definition of a novel growth factor-dependent signal cascade for the suppression of bile acid biosynthesis.

    PubMed

    Holt, Jason A; Luo, Guizhen; Billin, Andrew N; Bisi, John; McNeill, Y Yvette; Kozarsky, Karen F; Donahee, Mary; Wang, Da Yuan; Mansfield, Traci A; Kliewer, Steven A; Goodwin, Bryan; Jones, Stacey A

    2003-07-01

    The nuclear bile acid receptor FXR has been proposed to play a central role in the feedback repression of the gene encoding cholesterol 7 alpha-hydroxylase (CYP7A1), the first and rate-limiting step in the biosynthesis of bile acids. We demonstrate that FXR directly regulates expression of fibroblast growth factor-19 (FGF-19), a secreted growth factor that signals through the FGFR4 cell-surface receptor tyrosine kinase. In turn, FGF-19 strongly suppresses expression of CYP7A1 in primary cultures of human hepatocytes and mouse liver through a c-Jun N-terminal kinase (JNK)-dependent pathway. This signaling cascade defines a novel mechanism for feedback repression of bile acid biosynthesis and underscores the vital role of FXR in the regulation of multiple pathways of cholesterol catabolism in the liver.

  17. Complete 1H and 13C NMR assignments of three new polyhydroxylated sterols from the South China Sea gorgonian Subergorgia suberosa.

    PubMed

    Qi, Shu-Hua; Zhang, Si; Wang, Yi-Fei; Li, Ming-Yi

    2007-12-01

    Three new polyhydroxylated sterols, 3beta,6alpha,11,20beta,24-pentahydroxy- 9,11-seco-5alpha-24-ethylcholest-7,28-diene-9-one (1), 3-(1',2'-ethandiol)-24- methylcholest-8(9),22E-diene-3beta,5alpha,6alpha,7alpha,11alpha-pentaol (2), 24-methylcholest-7,22 E-diene-3beta,5alpha,6beta,25-tetraol (3) together with five known sterols, were isolated from the EtOH/CH2Cl2 extract of the South China Sea gorgonian Subergorgia suberosa. The complete assignments of the 1H and 13C NMR chemical shifts for these new compounds were achieved by means of 1D and 2D NMR techniques, including HSQC, HMBC, 1H--1H COSY, and NOESY spectra.

  18. Aminosterols from the dogfish shark Squalus acanthias.

    PubMed

    Rao, M N; Shinnar, A E; Noecker, L A; Chao, T L; Feibush, B; Snyder, B; Sharkansky, I; Sarkahian, A; Zhang, X; Jones, S R; Kinney, W A; Zasloff, M

    2000-05-01

    Seven new aminosterols related to squalamine (8) were isolated from the liver of the dogfish shark Squalus acanthias. Their structures (1-7) were determined using spectroscopic methods, including 2D NMR and HRFABMS. These aminosterols possess a relatively invariant cholestane skeleton with a trans AB ring junction, a spermidine or spermine attached equatorially at C3, and a steroidal side-chain that may be sulfated. The structure of the lone spermine conjugate, 7 (MSI-1436), was confirmed by its synthesis from (5alpha,7alpha, 24R)-7-hydroxy-3-ketocholestan-24-yl sulfate. Some members of this family of aminosterols exhibit a broad spectrum of antimicrobial activity comparable to squalamine. PMID:10843574

  19. Structure of the novel steroidal antibiotic squalamine determined by two-dimensional NMR spectroscopy.

    PubMed

    Wehrli, S L; Moore, K S; Roder, H; Durell, S; Zasloff, M

    1993-08-01

    Squalamine is a novel aminosterol recently isolated from the dogfish shark, Squalus acanthias. This water-soluble steroid exhibits potent antibacterial activity against both gram-negative and gram-positive bacteria. In addition, squalamine is fungicidal and induces osmotic lysis of protozoa. We report here the structural determination of squalamine, 3 beta-N-1-[N(3-[4-aminobutyl])-1,3 diaminopropane]-7 alpha,24 zeta-dihydroxy-5 alpha-cholestane 24-sulfate, which was deduced from the analysis of fast atom bombardment spectra and a series of two-dimensional nuclear magnetic resonance (NMR) spectra. Squalamine is a cationic steroid characterized by a condensation of an anionic bile salt intermediate with the polyamine, spermidine. This molecule is a potential host-defense agent in the shark, and provides insight into a new class of vertebrate antimicrobial molecules. PMID:8212087

  20. The synthesis of spermine analogs of the shark aminosterol squalamine.

    PubMed

    Shu, Youheng; Jones, Stephen R; Kinney, William A; Selinsky, Barry S

    2002-03-01

    Aminosterols isolated from the dogfish shark Squalus acanthias are promising therapeutic agents in the treatment of infection and cancer. One of these, MSI-1436, has been shown to possess antimicrobial activity slightly better than squalamine. In this study, a series of analogs of MSI-1436 have been synthesized from stigmasterol. The 7 alpha-hydroxy substituent of MSI-1436 was either omitted or the stereochemistry modified to the 7 beta position. Also, analogs of MSI-1436 with 24-sulfate, 24-amino, and 24-hydroxy substituents were synthesized in order to assess the importance of the side chain functional group on antimicrobial activity. All of the analogs possess significant antimicrobial activity, suggesting that substitution at C7 and C24 of the aminosterols plays a minor role in their antimicrobial potency. PMID:11856553

  1. Studies of cholesterol and bile acid metabolism, and early atherogenesis in hamsters fed GT16-239, a novel bile acid sequestrant (BAS).

    PubMed

    Wilson, T A; Nicolosi, R J; Rogers, E J; Sacchiero, R; Goldberg, D J

    1998-10-01

    The purpose of this study was to compare the efficacy of GT16-239, an alkylated, cross-linked poly(allylamine) bile acid sequestrant with cholestyramine on cholesterol and bile acid metabolism, and early aortic atherosclerosis in hypercholesterolemic male F1B Golden Syrian hamsters. In this controlled study, 42 hamsters were divided into six groups and were fed a chow-based hypercholesterolemic diet supplemented with a 10% oil blend (55% coconut/45% corn), 0.1% cholesterol (w/w) (control) and either 0.9 or 1.2% cholestyramine or 0.2, 0.4 or 0.6% GT16-239 for 13 weeks. Laboratory analyses included evaluating plasma lipoprotein cholesterol and triglyceride concentrations, hepatic HMG-CoA reductase and 7 alpha-hydroxylase activities, fecal excretion of bile acids and neutral sterols, hepatic cholesterol concentrations, and early atherosclerosis (aortic fatty streak area). Relative to the control diet, the 0.6% GT16-239 versus the 1.2% cholestyramine significantly inhibited the elevation of plasma lipoprotein total cholesterol (TC) (-69% vs -40%), high density lipoprotein-cholesterol (HDL-C) (-49% vs -30%), and non-HDL-C (-81 vs -48%) concentrations; increased the activities of both HMG-CoA reductase (1492% vs 62%) and 7 alpha-hydroxylase (175% vs 86%); lowered the concentration of hepatic cholesteryl ester (-94% vs -59%); increased fecal cholesterol concentration (+28% vs -10%); and decreased aortic fatty streak area (-100% vs -86%). Unexpected findings of this comparison were increased fecal concentrations of cholic acid (533%) and chenodeoxycholic acid (400%) and the reduction in lithocholic acid (-50%) in the 0.6% GT16-239 compared to the 1.2% cholestyramine group. In summary, GT16-239 had a greater impact on cholesterol metabolism and early atherosclerosis in hypercholesterolemic hamsters than cholestyramine.

  2. Nicotine activates and up-regulates nicotinic acetylcholine receptors in bronchial epithelial cells.

    PubMed

    Fu, Xiao Wen; Lindstrom, Jon; Spindel, Eliot R

    2009-07-01

    Prenatal nicotine exposure impairs normal lung development and leads to diminished pulmonary function after birth. Previous work from our laboratory has demonstrated that nicotine alters lung development by affecting a nonneuronal cholinergic autocrine loop that is expressed in lung. Bronchial epithelial cells (BECs) express choline acetyltransferase, the choline high-affinity transporter and nicotinic acetylcholine (ACh) receptor (nAChR) subunits. We now demonstrate through a combination of morphological and electrophysiological techniques that nicotine affects this autocrine loop by up-regulating and activating cholinergic signaling. RT-PCR showed the expression of alpha 3, alpha 4, alpha 7, alpha 9, alpha 10, beta2, and beta 4 nAChR mRNAs in rhesus monkey lung and cultured BECs. The expression of alpha 7, alpha 4, and beta2 nAChR was confirmed by immunofluorescence in the cultured BECs and lung. The electrophysiological characteristics of nAChR in BECs were determined using whole-cell patch-clamp on cultured BECs. Both ACh and nicotine evoked an inward current, with a rapid desensitizing current. Nicotine induced inward currents in a concentration-dependent manner, with an EC(50) of 26.7 microM. Nicotine-induced currents were reversibly blocked by the nicotinic antagonists, mecamylamine, dihydro-beta-erythroidine, and methyllcaconitine. Incubation of BECs with 1 microM nicotine for 48 hours enhanced nicotine-induced currents by roughly 26%. The protein tyrosine phosphorylation inhibitor, genistein, increased nicotine-induced currents by 58% and enhanced methyllcaconitine-sensitive currents (alpha 7 nAChR activities) 2.3-fold, whereas the protein tyrosine phosphatase inhibitor, pervanadate, decreased the effects of nicotine. These results demonstrate that chronic nicotine exposure up-regulates nAChR activity in developing lung, and that nAChR activity can be further modified by tyrosine phosphorylation.

  3. 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.

  4. Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphates.

    PubMed

    Nielsen, P; Neuberger, G; Fujii, I; Bown, D H; Keller, P J; Floss, H G; Bacher, A

    1986-03-15

    The xylene ring of riboflavin originates by dismutation of the precursor, 6,7-dimethyl-8-ribityllumazine. The formation of the latter compound requires a 4-carbon unit as the precursor of carbon atoms 6 alpha, 6, 7, and 7 alpha of the pyrazine ring. The formation of riboflavin from GTP and ribose phosphate by cell extract from Candida guilliermondii has been observed by Logvinenko et al. (Logvinenko, E. M., Shavlovsky, G. M., Zakal'sky, A. E., and Zakhodylo, I. V. (1982) Biokhimiya 47, 931-936). We have studied this enzyme reaction in closer detail using carbohydrate phosphates as substrates and synthetic 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione or its 5'-phosphate as cosubstrates. Several pentose phosphates and pentulose phosphates can serve as substrate for the formation of riboflavin with similar efficiency. The reaction requires Mg2+. Various samples of ribulose phosphate labeled with 14C or 13C have been prepared and used as enzyme substrates. Radioactivity was efficiently incorporated into riboflavin from [1-14C]ribulose phosphate, [3,5-14C]ribulose phosphate, and [5-14C]ribulose phosphate, but not from [4-14C]ribulose phosphate. Label from [1-13C]ribose 5-phosphate was incorporated into C6 and C8 alpha of riboflavin. [2,3,5-13C]Ribose 5-phosphate yielded riboflavin containing two contiguously labeled segments of three carbon atoms, namely 5a, 9a, 9 and 8, 7, 7 alpha. 5-Amino-6-[1'-14C] ribitylamino-2,4 (1H,3H)-pyrimidinedione transferred radioactivity exclusively to the ribityl side chain of riboflavin in the enzymatic reaction. It follows that the 4-carbon unit used for the biosynthesis of 6,7-dimethyl-8-ribityllumazine consists of the pentose carbon atoms 1, 2, 3, and 5 in agreement with earlier in vivo studies. PMID:3949782

  5. 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

  6. [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.

  7. Diverse function of aromatase and the N-terminal sequence deleted form.

    PubMed

    Osawa, Y; Higashiyama, T; Toma, Y; Yarborough, C

    1997-04-01

    The diverse function of human placental aromatase including estradiol 6alpha-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6alpha-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6alpha,7alpha-(3)H2,4-(14)C]estradiol, 7-ethoxycoumarin, and [N-methyl-(3)H3]cocaine. 6Alpha-hydroxy[7alpha-(3)H,4-(14)C]estradiol was isolated as the metabolite of estradiol and the 3H-water release method based on the 6alpha-3H label was established. The initial rate kinetics of the 6alpha-hydroxylation gave Km of 4.3 microM, Vmax of 4.02 nmol min(-1) mg(-1), and turnover rate of 0.27 min(-1). Testosterone competed dose-dependently with the 6alpha-hydroxylation and showed the Ki of 0.15 microM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed Km of 200 microM, Vmax of 12.5 nmol min(-1) mg(-1) and turnover rate of 1.06 min(-1). The N-demethylation of cocaine was analysed by the 3H-release method, giving Km of 670 microM, Vmax of 4.76 nmol min(-1) mg(-1), and turnover rate of 0.49 min(-1). All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 +/- 83 pmol min(-1) mg(-1) (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min(-1).

  8. 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid stimulates GABA release from interneurons projecting to CA1 pyramidal neurons in the rat hippocampus via pre-synaptic alpha7 acetylcholine receptors.

    PubMed

    Kanno, Takeshi; Yaguchi, Takahiro; Yamamoto, Satoshi; Yamamoto, Hideyuki; Fujikawa, Hirokazu; Nagata, Tetsu; Tanaka, Akito; Nishizaki, Tomoyuki

    2005-11-01

    Nicotinic acetylcholine (ACh) receptors, such as alpha7, alpha3beta4 and alpha4beta2 receptors in the hippocampus, are suggested to modulate neurotransmitter release. 8-[2-(2-Pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) (100 nM), a linoleic acid derivative, potentiated responses of alpha7, alpha3beta4 and alpha4beta2 ACh receptors expressed in Xenopus oocytes that are blocked by 3-(1-[dimethylaminopropyl] indol-3-yl)-4-[indol-3-yl] maleimide (GF109203X), a selective inhibitor of protein kinase C (PKC), except for alpha3beta4 ACh receptors. DCP-LA enhanced the nicotine-triggered release of GABA from rat hippocampal slices in the presence of tetrodotoxin in a bell-shaped dose-dependent manner at concentrations ranging from 10 nM to 10 microM, although DCP-LA by itself had no effect on GABA release. The DCP-LA action was inhibited by GF109203X or alpha-bungarotoxin, an inhibitor of alpha7 ACh receptors, but not by mecamylamine or dihydro-beta-erithroidine, an inhibitor of alpha3beta4 and alpha4beta2 ACh receptors. A similar effect on GABA release was obtained with 12-O-tetradecanoylphorbol 13-acetate, a PKC activator. DCP-LA (100 nM) also enhanced GABA release triggered by choline, an agonist of alpha7 ACh receptors, but not 3-[2(s)-azetidinylmethoxy] pyridine, an agonist of alpha4beta2 ACh receptors. In addition, DCP-LA (100 nM) increased the rate of nicotine-triggered GABA(A) receptor-mediated miniature inhibitory post-synaptic currents, monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was also inhibited by GF109203X or alpha-bungarotoxin but not by mecamylamine. Thus, the results of the present study indicate that DCP-LA stimulates GABA release by enhancing activity of pre-synaptic alpha7 ACh receptors present on the GABAergic terminals of interneurons that transmit to CA1 pyramidal neurons via a PKC pathway. PMID:16248884

  9. Iridoids from Crescentia alata.

    PubMed

    Valladares, María Guadalupe; Rios, María Yolanda

    2007-01-01

    Four new 11-nor-iridoids, 6beta,7beta,8alpha,10-tetrahydroxy-cis-2-oxabicyclo[4.3.0]nonan-3-one (1), 6beta,7beta,8alpha,10-tetra-p-hydroxybenzoyl-cis-2-oxabicyclo[4.3.0]nonan-3-one (2), 1beta,6beta,7alpha,8alpha,10-pentahydroxy-cis-2-oxabicyclo[4.3.0]nonane (3), and 6beta-hydroxy-2-oxabicyclo[4.3.0]Delta8-9-nonen-1-one (4), were isolated from the pulp of the fruits of Crescentia alata. Although a limited number of Crescentia species have been studied chemically, iridoids lacking C-11 have been isolated from the fruits of these species, and the isolation of compounds 1-4 from C. alata is in accordance with the constituents of the species previously analyzed. The structures of these compounds were established on the basis of IR, UV, 1H and 13C NMR, DEPT, COSY, HSQC, HMBC, MS, and X-ray data.

  10. 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

  11. Alpha7 nicotinic acetylcholine receptor activation ameliorates scopolamine-induced behavioural changes in a modified continuous Y-maze task in mice.

    PubMed

    Redrobe, John P; Nielsen, Elsebet Ø; Christensen, Jeppe K; Peters, Dan; Timmermann, Daniel B; Olsen, Gunnar M

    2009-01-01

    The alpha7 (alpha7) nicotinic acetylcholine receptor may represent a drug target for the treatment of disorders associated with working memory/attentional dysfunction. We investigated the effects of three distinct alpha7 nicotinic acetylcholine receptor agonists: 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941; 0.01-0.1 mg/kg), 4-bromophenyl 1,4-diazabicyclo(3.2.2) nonane-4-carboxylate (SSR180711; 0.3-3 mg/kg) and N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide (PNU-282987; 1-10 mg/kg), on scopolamine-induced deficits in a modified Y-maze procedure. Mice were forced to choose one of two visually distinct arms, and were confined there for a 5 min exploration period before being allowed to explore both arms for a 2 min test session, immediately thereafter. The time spent in each arm, entries and total distance travelled were recorded using an automated system. Characterisation experiments showed that scopolamine-treated (1 mg/kg) mice spent less time exploring the unfamiliar arm, when compared with vehicle-treated animals. Combination experiments showed that all three alpha7 agonists ameliorated scopolamine-induced changes in unfamiliar arm exploration. In conclusion, the present data support the idea that alpha7 nicotinic acetylcholine receptors may represent an interesting target for the treatment of conditions associated with attentional/working memory dysfunction.

  12. Conjugates of gonadotropin releasing hormone (GnRH) with carminic acid: Synthesis, generation of reactive oxygen species (ROS) and biological evaluation.

    PubMed

    Lev-Goldman, Vered; Mester, Brenda; Ben-Aroya, Nurit; Hanoch, Tamar; Rupp, Barbara; Stanoeva, Tsvetanka; Gescheidt, Georg; Seger, Rony; Koch, Yitzhak; Weiner, Lev; Fridkin, Mati

    2008-07-15

    We synthesized two carminic acid (7-alpha-d-glucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxo-2-anthracene carboxlic acid, CA)-GnRH conjugates to be used as a model for potential photoactive targeted compounds. CA was conjugated to the epsilon-amino group of [d-Lys(6)]GnRH through its carboxylic moiety or via a beta-alanine spacer (beta-ala). Redox potentials of CA and its conjugates were determined. We used electron spin resonance (ESR) and spin trapping techniques to study the light-stimulated redox properties of CA and its CA-GnRH conjugates. Upon irradiation, the compounds stimulated the formation of reactive oxygen species (ROS), that is, singlet oxygen ((1)O(2)) and oxygen radicals (O(2)(-*) and OH(*)). Both conjugates exhibited higher ROS production than the non-conjugated CA. The bioactivity properties of the CA conjugates and the parent peptide, [d-Lys(6)]GnRH, were tested on primary rat pituitary cells. We found that the conjugates preserved the bioactivity of GnRH as illustrated by their capability to induce ERK phosphorylation and LH release. PMID:18571926

  13. 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.

  14. 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.

  15. [Prevention of cholelithiasis with ascorbic acid. Experimental study in hamsters].

    PubMed

    Peraza, M; Méndez, N; Lagarriga, J; Cohen, J; Alcantar, M; Chiprut, R

    1979-01-01

    Cholesterol lithogenesis is the end result of hepatic microsomal enzymatic alterations which determine an increase in cholesterol synthesis (HMG CoA reductase) and a decrease in its transformation into bile salts (7 alpha hydroxylase). Therefore biliary cholesterol excretion is increased while bile salt excretion is diminished. Ascorbic Acid (A.A.) seems capable of reversing those enzymatic derrangements in scorbutic animals. Since hamsters are able to synthesize A.A., we evaluated its effect used in high doses during diet induced lithogenesis. Two groups of 6 weeks old, male hamsters, were fed with a lithogenic diet for 30 days. Group A received the usual amount of A.A. contained in the diet (0.25 mg/day/manster) while group B had supplementary A.A. added to drinking water (5 mg/day/hamster). Thirteen out of twenty of group A (65%) and 5 out of 20 of group B (25%) developed cholesterol calculi (p 0.05). Less stones were found in the gallbladders of hamsters fed with supplementary A.A. It is concluded that A.A. in this model, has an inhibitory effect on lithogenesis. The possible mechanism seems to be related to A.A. influence on the microsomal enzymes involved in lithogenesis. These findings, plus the lack of undesirable secondary effects of supplementary A.A. suggest a potential therapeutic role in human cholelithiasis. PMID:531439

  16. Dietary cancer risk conditional cancerogens in produce of livestock fed on species of spurge (Euphorbiaceae). I. Skin irritant and tumor-promoting ingenane-type diterpene esters in E. peplus, one of several herbaceous Euphorbia species contaminating fodder of livestock.

    PubMed

    Zayed, S M; Farghaly, M; Taha, H; Gotta, H; Hecker, E

    1998-01-01

    The hypothesis was proposed that there is a risk of dietary cancer from conditional cancerogens in produce of livestock polluted with irritants of the diterpene ester type, picked up by feeding on species of Euphorbiaceae (spurge). To challenge this, several herbaceous plants of the genus Euphorbia, widespread as weeds and contaminants of livestock fodder, were identified botanically and extracts of their aerial parts were tested for irritancy on the mouse ear. As compared to a standard probe of croton oil, the extracts of E. peplus, E. nubica and E. helioscopia displayed irritancy. The most active extract (that from E. peplus) was investigated by a fractionation procedure monitored by the mouse ear assay, and five molecularly uniform irritant Euphorbia factors Pe1-Pe5 were identified as diterpene ester-type toxins. Together these factors comprise at least 11 ppm in the aerial parts. They were characterized individually to carry the diterpene parent alcohols ingenol, 20-deoxyingenol, and 20-deoxyingenol-6 alpha, 7alpha-epoxide. The irritancy of the aerial plant parts was shown to be caused mainly by the Euphorbia factors Pe1 and Pe2 together. Upon chronic administration of these irritants and hyperplasiogens as principal cancerogenic risk factors in the mouse skin initiation/promotion bioassay, Pe1 and Pe2 were established as tumor promoters. These findings together support the initial hypothesis and suggest the need for further investigations to determine whether there is a consequent risk of dietary cancer.

  17. 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. PMID:26680486

  18. Biologic stability of tauro-23-[75Se] selena-25-homocholic acid.

    PubMed

    Monks, R; Boyd, G S

    1988-08-01

    The stability of tauro-23-[75Se]selena-25-homocholic acid (SeHCAT) towards deconjugation by the enzyme cholylglycine hydrolase was compared with that of taurocholate: whereas taurocholate underwent 58% deconjugation within 2 hr, SeHCAT suffered only 8% deconjugation plus 5% conversion to an unknown product within 24 hr. Incubation of SeHCAT under anaerobic conditions for 48 hr at 37 degrees C with human fecal organisms resulted in considerable deconjugation, 7 alpha-dehydroxylation, and dehydrogenation. Twenty-four hours after the simultaneous administration of SeHCAT and tauro-[24-14C]cholate to a rabbit the recovery of 75Se in bile was 90% of that of 14C. Forty-eight hours following administration of SeHCAT to a second rabbit residual bile radioactivity revealed 80% deconjugation and dehydroxylation and 60% reconjugation with glycine. Although SeHCAT is more resistant than taurocholate towards modification by fecal bacterial enzymes, within the rabbit it follows the principal metabolic pathways of the natural bile acids.

  19. Biologic stability of tauro-23-(/sup 75/Se) selena-25-homocholic acid

    SciTech Connect

    Monks, R.; Boyd, G.S.

    1988-08-01

    The stability of tauro-23-(/sup 75/Se)selena-25-homocholic acid (SeHCAT) towards deconjugation by the enzyme cholylglycine hydrolase was compared with that of taurocholate: whereas taurocholate underwent 58% deconjugation within 2 hr, SeHCAT suffered only 8% deconjugation plus 5% conversion to an unknown product within 24 hr. Incubation of SeHCAT under anaerobic conditions for 48 hr at 37 degrees C with human fecal organisms resulted in considerable deconjugation, 7 alpha-dehydroxylation, and dehydrogenation. Twenty-four hours after the simultaneous administration of SeHCAT and tauro-(24-/sup 14/C)cholate to a rabbit the recovery of /sup 75/Se in bile was 90% of that of /sup 14/C. Forty-eight hours following administration of SeHCAT to a second rabbit residual bile radioactivity revealed 80% deconjugation and dehydroxylation and 60% reconjugation with glycine. Although SeHCAT is more resistant than taurocholate towards modification by fecal bacterial enzymes, within the rabbit it follows the principal metabolic pathways of the natural bile acids.

  20. Membrane-permeabilizing activities of Bacillus thuringiensis coleopteran-active toxin CryIIIB2 and CryIIIB2 domain I peptide.

    PubMed Central

    Von Tersch, M A; Slatin, S L; Kulesza, C A; English, L H

    1994-01-01

    Bacillus thuringiensis toxin CryIIIB2 exhibits activity against two agriculturally important pests, the Colorado potato beetle, Leptinotarsa decemlineata, and the Southern corn rootworm, Diabrotica undecimpunctata. CryIIIB2 shows significant structural similarity to Colorado potato beetle-active toxin CryIIIA, whose crystal structure has been determined elsewhere [J. Li, J. Carrol, and D. J. Ellar, Nature (London) 353:815-821, 1991]. A clone limited to the putative 7-alpha-helical bundle domain I peptide of CryIIIB2 was constructed by PCR. The truncated protein was expressed at high levels in Escherichia coli. Domain I peptide was isolated and compared with native CryIIIB2 toxin in promoting ion efflux from synthetic phospholipid vesicles and formation of ion channels in black lipid membranes. The results showed that CryIIIB2 domain I peptide is sufficient for ion channel formation and promotes ion efflux. Both native CryIIIB2 toxin and domain I peptide were inefficient channel-forming proteins that produced noisy ion channels of various conductance states. In ion efflux assays, native toxin promoted greater ion efflux from synthetic vesicles than did the truncated peptide. Images PMID:7527203

  1. 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.

  2. 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. PMID:19585467

  3. {alpha}-cluster structure and density waves in oblate nuclei

    SciTech Connect

    Kanada-En'yo, Yoshiko; Hidaka, Yoshimasa

    2011-07-15

    Pentagon and triangle shapes in {sup 28}Si and {sup 12}C are discussed in relation to nuclear density waves. In the antisymmetrized molecular dynamics calculations, the K{sup {pi}=}5{sup -} band in {sup 28}Si and the K{sup {pi}=}3{sup -} band in {sup 12}C are described by the pentagon and triangle shapes, respectively. These negative-parity bands can be interpreted as the parity partners of the K{sup {pi}=}0{sup +} ground bands and they are constructed from the parity-asymmetric-intrinsic states. The pentagon and the triangle shapes originate in 7{alpha}- and 3{alpha}-cluster structures, respectively. In a mean-field picture, they are described also by the static one-dimensional density waves at the edge of the oblate states. In analyses with ideal {alpha}-cluster models using Brink-Bloch cluster wave functions and that with a simplified model, we show that the static edge density waves for the pentagon and triangle shapes can be understood by spontaneous breaking of axial symmetry, i.e., the instability of the oblate states with respect to the edge density waves. The density wave is enhanced in the Z=N nuclei due to the proton-neutron coherent density waves, while it is suppressed in Z{ne}N nuclei.

  4. 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.

  5. Effects of spironolactone on human blood mononuclear cells: mineralocorticoid receptor independent effects on gene expression and late apoptosis induction.

    PubMed

    Sønder, Søren Ulrik Salling; Mikkelsen, Marianne; Rieneck, Klaus; Hedegaard, Chris Juul; Bendtzen, Klaus

    2006-05-01

    1 Spironolactone (SPIR) binds to cytoplasmic mineralocorticoid receptors (MR) and functions as an aldosterone antagonist. Recently, the drug was shown to have an early suppressive effect on several immunoactive and proinflammatory cytokines. 2 To elucidate the mechanism behind this, the four MR-binding steroids SPIR, canrenone, 7alpha-thiomethyl-spironolactone and aldosterone (ALDO) were investigated for effects on lipopolysaccharide- and phytohemagglutinin-A-activated human blood mononuclear cells. Gene expression was examined after 4 h using microarrays, and SPIR affected 1018 transcripts of the (=) 22,000 probed. In contrast, the SPIR-related steroids affected 17 or fewer transcripts. Combining SPIR and ALDO resulted in 940 affected transcripts, indicating that SPIR has an early gene-regulatory effect independent of MR. 3 The affected genes encode a large number of signalling proteins and receptors, including immunoinflammatory response genes and apoptosis and antiapoptosis genes. Apoptosis was evident in CD3-, CD14- and CD19-positive cells, but only after 18 h of exposure to SPIR. 4 The transcriptional network involving the differentially regulated genes was examined and the results indicate that SPIR affects genes controlled by the transcription factors NF-kappaB, CEBPbeta and MYC. 5 These observations provide new insight into the non-MR-mediated effects of SPIR. PMID:16520746

  6. Synthesis, aggregation behavior and cholesterol solubilization studies of 16-epi-pythocholic acid (3 alpha,12 alpha,16 beta-trihydroxy-5 beta-cholan-24-oic acid).

    PubMed

    Nonappa; Maitra, Uday

    2010-07-01

    Synthesis, aggregation behavior and in vitro cholesterol solubilization studies of 16-epi-pythocholic acid (3 alpha,12 alpha,16 beta-trihydroxy-5 beta-cholan-24-oic acid, EPCA) are reported. The synthesis of this unnatural epimer of pythocholic acid (3 alpha,12 alpha,16 alpha-trihydroxy-5 beta-cholan-24-oic acid, PCA) involves a series of simple and selective chemical transformations with an overall yield of 21% starting from readily available cholic acid (CA). The critical micellar concentration (CMC) of 16-epi-pythocholate in aqueous media was determined using pyrene as a fluorescent probe. In vitro cholesterol solubilization ability was evaluated using anhydrous cholesterol and results were compared with those of other natural di- and trihydroxy bile acids. These studies showed that 16-epi-pythocholic acid (16 beta-hydroxy-deoxycholic acid) behaves similar to cholic acid (CA) and avicholic acid (3 alpha,7 alpha,16 alpha-trihydroxy-5 beta-cholan-24-oic acid, ACA) in its aggregation behavior and cholesterol dissolution properties.

  7. 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. PMID:20071329

  8. 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.

  9. 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

  10. 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.

  11. Identification and quantitative analysis of beta-sitosterol oxides in vegetable oils by capillary gas chromatography-mass spectrometry.

    PubMed

    Zhang, Xin; Julien-David, Diane; Miesch, Michel; Geoffroy, Philippe; Raul, Francis; Roussi, Stamatiki; Aoudé-Werner, Dalal; Marchioni, Eric

    2005-12-01

    As vegetable oils and phytosterol-enriched spreads are marketed for frying food or cooking purposes, temperature is one of the most important factors leading to the formation of phytosterol oxides in food matrix. A methodology based on saponification, organic solvent extraction, solid-phase extraction (SPE), followed by mass spectrometric identification and quantitation of beta-sitosterol oxides using capillary gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode was developed and characterized. Relative response factors of six beta-sitosterol oxides, including 7alpha-hydroxy, 7beta-hydroxy, 5,6alpha-epoxy, 5,6beta-epoxy, 7-keto, and 5alpha,6beta-dihydroxysitosterol, were calculated against authentic standards of 19-hydroxycholesterol or cholestanol. Linear calibration data, limit of detection, and sample recoveries during analytical process. Recoveries of these oxidation compounds in spiked samples ranged from 88 to 115%, while relative standard derivation (R.S.D.) values were below 10% in most cases. The analytical method was applied to quantify beta-sitosterol oxides formed in thermal-oxidized vegetable oils which were heated at different temperatures and for varying time periods. Sitosterol oxidation is strikingly higher in sunflower oil relative to olive oil under all conditions of temperature and heating time.

  12. Ligand-dependent coactivation of the human bile acid receptor FXR by the peroxisome proliferator-activated receptor gamma coactivator-1alpha.

    PubMed

    Savkur, Rajesh S; Thomas, Jeffrey S; Bramlett, Kelli S; Gao, Yunling; Michael, Laura F; Burris, Thomas P

    2005-01-01

    Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) has been shown to play an important role in energy metabolism by coordinating transcriptional programs involved in mitochondrial biogenesis, adaptive thermogenesis, gluconeogenesis, and fatty acid oxidation. PGC-1alpha also plays a crucial role in cholesterol metabolism by serving as a coactivator of the liver X receptor-alpha and inducing the expression of cholesterol 7-alpha-hydroxylase. Here, we demonstrate that PGC-1alpha also functions as an effective coactivator of farnesoid X receptor (FXR), the bile acid receptor. Transient cotransfection assays demonstrate that PGC-1alpha enhances ligand-mediated FXR transcription when either full-length FXR or Gal4 DNA binding domain-FXR-ligand binding domain chimeras were analyzed. Mammalian two-hybrid analyses, glutathione S-transferase affinity chromatography and biochemical coactivator recruitment assays demonstrate ligand-dependent interaction between the two proteins both in vivo and in vitro. PGC-1alpha-mediated coactivation of FXR was highly ligand-dependent and absolutely required an intact activation function-2 (AF-2) domain of FXR and the LXXLL motif in PGC-1alpha. The integrity of the charge clamp was required, further illustrating the role of the ligand binding domain of FXR in PGC-1alpha recognition. Together, these results indicate that PGC-1alpha functions as a potent coactivator for FXR and further implicates its role in the regulation of genes that are involved in bile acid and lipid metabolism.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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. PMID:14764638

  18. Enzymic measurement of primary bile acids and the primary bile acid ratio in serum with the IL-Multistat III Fluorescence Light-Scattering Centrifugal Analyzer.

    PubMed

    Papanastasiou-Diamandi, A; Diamandis, E P; Soldin, S J

    1984-08-01

    Enzymic fluorimetric methods are described for the determination of primary bile acids and of chenodeoxycholic acid (CDC) and cholic acid (C) in serum. Bile acids are extracted from 0.3 mL of serum in a simple 5-min step with use of Sep-Pak C cartridges. Total primary bile acids are measured by an equilibrium technique after reaction with beta-NAD in the presence of 7 alpha-hydroxysteroid dehydrogenase. Chenodeoxycholic acid (and its conjugates) is measured by a reaction-rate technique employing the same reaction as above but under different experimental conditions. A small contribution of cholic acid (and its conjugates) to the reaction rate is eliminated by simple calculations. Cholic acid is calculated by difference of the two determinations. In both assays NADH fluorescence is measured with the Multistat centrifugal analyzer. Absolute recovery of bile acids from serum was about 87%. Day-to-day standard deviations for CDC and C were 1.6 and 2.0 mumol/L at serum concentrations of 22.1 and 24.1 mumol/L respectively. Comparison data with a cholylglycine RIA procedure gave the following correlation coefficients (x = RIA, y = proposed method): r = 0.980 (RIA vs total primary bile acids), r = 0.918 (RIA vs CDC) and r = 0.989 (RIA vs C). The methods described appear more practical for use on a routine basis than methods in the literature for the calculation of the primary bile acid ratio. PMID:6090040

  19. 5 beta-hydroxylation by the liver. Identification of 3,5,7-trihydroxy nor-bile acids as new major biotransformation products of 3,7-dihydroxy nor-bile acids in rodents.

    PubMed

    Schteingart, C D; Hagey, L R; Setchell, K D; Hofmann, A F

    1993-05-25

    24-Norursodeoxycholic acid (nor-UDCA), when administered into the anesthetized biliary fistula hamster or injected into the perfusate of an isolated liver, was hydroxylated at C-5 to give 5 beta-hydroxynorursodeoxycholic acid 2 (3 alpha,5,7 beta-trihydroxy-24-nor-5 beta-cholan-23-oic acid), which was secreted into bile mainly as such. Similarly, 24-norchenodeoxycholic acid (nor-CDCA) was 5 beta-hydroxylated to give 5 beta-hydroxynor-chenodeoxycholic acid 4 (3 alpha,5,7 alpha-trihydroxy-24-nor-5 beta-cholan-23-oic acid), which was also secreted into bile without appreciable further biotransformation. The site of hydroxylation was assigned by 13C and 1H NMR and mass spectrometry. 5-Hydroxylation was a major biotransformation pathway at physiological bile acid loads. 5-Hydroxylation of UDCA also occurred in the perfused rat liver but to a lesser extent. 5-Hydroxylation of nor-UDCA was not observed in rabbit, dog, or man, indicating that its formation is species-specific. 5-Hydroxylation of nor-CDCA and nor-UDCA is the first reported example of hydroxylation of a tertiary carbon atom of bile acids. Nor-dihydroxy bile acids appear to be useful for the detection of minor hydroxylation pathways, because their prolonged hepatobiliary retention exposes them repeatedly to hydroxylases present in the hepatobiliary system.

  20. Effects of novel bile salts on cholesterol metabolism in rats and guinea-pigs.

    PubMed

    Fears, R; Brown, R; Ferres, H; Grenier, F; Tyrrell, A W

    1990-11-01

    Novel bile salts (quaternary ammonium conjugates) inhibited cholic acid binding and transport in everted ileal sacs in vitro. The cationic piperazine conjugate of lithocholic acid (di-iodide salt, compound 8, BRL 39924A) appeared most active, inhibiting binding by 29% and transport by 59% in guinea-pig ileum (200 microM). BRL 39924A also inhibited taurocholate uptake into guinea-pig ileal sacs and cholate uptake into rat ileal sacs and was selected for further study in vivo. In hyperlipidaemic rats, BRL 39924A significantly raised cholesterol 7 alpha-hydroxylase activity and decreased hepatic accumulation of exogenous cholic acid. HDL cholesterol concentration in the serum increased and the level of VLDL plus LDL cholesterol decreased. In hyperlipidaemic guinea-pigs. BRL 39924A lowered serum total cholesterol and triglyceride levels. Although metabolic changes were less than those achieved with the bile acid sequestrant, cholestyramine, the doses of BRL 39924A used were much lower (100-500 mg/kg body wt). Selective inhibition of receptor mediated bile acid uptake may be associated with local side-effects but these novel bile salts are useful pharmacological tools to examine the effects of receptor blockade on lipoprotein metabolism. PMID:2242032

  1. alpha-thalassemia mutations in Khuzestan Province, Southwest Iran.

    PubMed

    Zandian, Khodamorad; Nateghi, Jamal; Keikhaie, Bijan; Pedram, Mohammad; Hafezi-Nejad, Nima; Hadavi, Valeh; Oberkanins, Christian; Azarkeivan, Azita; Law, Hai-Yang; Najmabadi, Hossein

    2008-01-01

    Although alpha-thalassemia (alpha-thal) is the most common hereditary hemoglobin (Hb) disorder in Iran, no comprehensive data are so far available on the prevalence of the disease in the province of Khuzestan in Southwest Iran. This study investigates the spectrum of alpha-thal mutations in this region. One hundred and twenty-one subjects from Khuzestan Province, Iran, were initially tested for the three most common Iranian alpha-thal mutations (- alpha3.7, -alpha4.2, and --MED) by gap-polymerase chain reaction (gap-PCR). Reverse hybridization test strips and DNA sequencing were used to identify additional alpha-globin mutations. A total of 131 mutated alpha-globin alleles were identified in these patients. Of the 13 mutations that were detected in Khuzestan Province, Iran, the - alpha3.7 single gene deletion was the most frequently identified variant, representing 62.6% of the total; we also observed significant numbers of individuals with compound heterozygous mutations. On the basis of our results, we strongly recommend screening for the most common mutations to improve the molecular diagnosis of anemia in this region.

  2. 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. PMID:15582210

  3. 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.

  4. 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

  5. In Vivo Lipid Regulation Mechanism of Polygoni Multiflori Radix in High-Fat Diet Fed Rats

    PubMed Central

    Lin, Pei; He, Yan Ran; Lu, Jian Mei; Li, Na; Wang, Wan Gen; Gu, Wen; Yu, Jie; Zhao, Rong Hua

    2014-01-01

    Mechanisms of the water extracts of Polygoni Multiflori Radix (PMR) and its processed products (PMRP) on liver lipid metabolism were observed in this paper. Aqueous extract of PMR and PMRP was given to nonalcoholic fatty liver model rats, respectively. PMR was better in reducing the contents of very low density lipoprotein (VLDL) than PMRP and the positive control groups. In the aspect of regulating TG, medium dose PMR reduced the activity of diacylglycerol acyltransferase (DGAT) to 1536 ± 47.69 pg/mL (P < 0.001) and promoted the expression of hepatic lipase (HL) to 23.59 ± 0.2758 U/mL (P < 0.05). HL promotion ability of medium dose PMR was similar with the simvastatin positive control. Both medium and high dose of PMR showed significant alterations in TC, which were related to the downregulation effects on hydroxyl methyl-glutaryl coenzyme A reductase (HMGCR) and upregulation effects on cholesterol 7-alpha-hydroxylase or cytochrome P450 7A (CYP7A). Quantitative relationships research indicated that the prominent effect on inhibiting the content of HMGCR (r = 0.756, P < 0.05) was strongly positive correlated with to the TC regulation effects. Effects of PMR on enhancing decomposition rate or reducing de novo synthesis rate of TG and TC were better than PMRP. PMID:24876874

  6. 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.

  7. Insights into the role of the (alpha+beta) insertion in the TIM-barrel catalytic domain, regarding the stability and the enzymatic activity of chitinase A from Serratia marcescens.

    PubMed

    Zees, Athanassios C; Pyrpassopoulos, Serapion; Vorgias, Constantinos E

    2009-01-01

    Chitinase A (ChiA) from Serratia marcescens is a mesophilic enzyme with high catalytic activity and high stability. The crystal structure of ChiA has revealed a TIM-barrel fold of the catalytic domain, an (alpha+beta) insertion between the B7 beta-strand and A7 alpha-helix of the TIM-barrel, an FnIII domain at the N-terminus of the molecule and a hinge region that connects the latter to the catalytic domain. In this study, the role of the (alpha+beta) domain on the stability, catalytic activity and specificity of the enzyme was investigated by deleting this domain and studying the enzymatic and structural properties of the resulting truncated enzyme. The obtained data clearly show that by removing the (alpha+beta) domain, the thermal stability of the enzyme is substantially reduced, with an apparent T(m) of 42.0+/-1.0 degrees C, compared to the apparent T(m) of 58.1+/-1.0 degrees C of ChiA at pH 9.0. The specific activity of ChiADelta(alpha+beta) was substantially decreased, the pH optimum was shifted from 6.5 to 5.0 and the substrate and product specificities were altered.

  8. 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).

  9. Inhibition of stigmasterol oxidation by antioxidants in purified sunflower oil.

    PubMed

    Rudzińska, Magdalena; Korczak, Józef; Gramza, Anna; Wasowicz, Erwin; Dutta, Paresh C

    2004-01-01

    A study was conducted to analyze the effect of the antioxidants butylated hydroxytoluene, alpha-tocopherol, ethanolic extracts of rosemary, and green tea on stigmasterol resistance against degradation and formation of its oxidation products in purified triacylglycerols (TAG) from sunflower oil. The content of stigmasterol and its oxidation products 7alpha- and 7beta-hydroxy, alpha- and beta-epoxy, triol, and 7-ketostigmasterol were determined during incubation at 60 degrees C for 3, 6, and 9 days. In addition, peroxide value and fatty acid composition were also determined in the samples. Correlation between the levels of the accumulated stigmasterol oxides and peroxide value of the TAG with antioxidants during incubation was significant only for rosemary extract (R = 0.6799, p < 0.05). The lack of correlation precludes the use of peroxide values to determine the level of sterol oxidation products in the used model system. Correlation between stigmasterol content and the level of stigmasterol oxides was significant for all samples (R = 0.8874, p < 0.05). The total increase of the stigmasterol oxidation products was the lowest in samples with alpha-tocopherol, but the content of stigmasterol-triol increased the most in this sample. In all the analyzed samples, alpha-epoxy-stigmasterol was formed in the highest amounts among the analyzed stigmasterol oxidation products. PMID:15164847

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

    PubMed Central

    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

    1998-01-01

    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. PMID:9512491

  11. 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.

  12. 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.

  13. 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.

  14. 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

    2004-10-01

    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. PMID:15464075

  15. 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.

  16. Squalamine: an aminosterol antibiotic from the shark.

    PubMed Central

    Moore, K S; Wehrli, S; Roder, H; Rogers, M; Forrest, J N; McCrimmon, D; Zasloff, M

    1993-01-01

    In recent years, a variety of low molecular weight antibiotics have been isolated from diverse animal species. These agents, which include peptides, lipids, and alkaloids, exhibit antibiotic activity against environmental microbes and are thought to play a role in innate immunity. We report here the discovery of a broad-spectrum steroidal antibiotic isolated from tissues of the dogfish shark Squalus acanthias. This water-soluble antibiotic, which we have named squalamine, exhibits potent bactericidal activity against both Gram-negative and Gram-positive bacteria. In addition, squalamine is fungicidal and induces osmotic lysis of protozoa. The chemical structure of the antibiotic 3 beta-N-1-(N-[3-(4-aminobutyl)]- 1,3-diaminopropane)-7 alpha,24 zeta-dihydroxy-5 alpha-cholestane 24-sulfate has been determined by fast atom bombardment mass spectroscopy and NMR. Squalamine is a cationic steroid characterized by a condensation of an anionic bile salt intermediate with spermidine. The discovery of squalamine in the shark implicates a steroid as a potential host-defense agent in vertebrates and provides insights into the chemical design of a family of broad-spectrum antibiotics. Images PMID:8433993

  17. 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

  18. 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.

  19. Study of antimalarial activity of chemical constituents from Diospyros quaesita.

    PubMed

    Ma, Cui-Ying; Musoke, Sebisubi Fred; Tan, Ghee Teng; Sydara, Kongmany; Bouamanivong, Somsanith; Southavong, Bounhoong; Soejarto, D Doel; Fong, Harry H S; Zhang, Hong-Jie

    2008-11-01

    Bioassay-directed fractionation led to the isolation of seven compounds from a sample of the dried leaves, twigs, and branches of Diospyros quaesita Thw. (Ebenaceae). One of the isolates, betulinic acid 3-caffeate (1), showed in vitro antimalarial activity against Plasmodium falciparum clones D(6) (chloroquine-sensitive) and W(2) (chloroquine-resistant) with IC(50) values of 1.40 and 0.98 microM, respectively. Evaluation of compound 1 in the human oral epidermoid (KB) cancer cell line revealed cytotoxicity at ED(50) of 4.0 microM. In an attempt to reduce the cytotoxicity of 1, the acetylated derivative 1a and betulinic acid (1b) were prepared. Of the seven isolates, diospyrosin (2) was determined to be a new neolignan. In addition to 1, other known compounds isolated in this study were pinoresinol, lariciresinol, N-benzoyl-L-phenylalaninol, scopoletin, and poriferast-5-en-3beta,7alpha-diol. The structure of 2 was elucidated based on spectroscopic data analysis including 1D- and 2D-NMR, and HR-ESI-MS. PMID:19035573

  20. Identification and quantitative analysis of beta-sitosterol oxides in vegetable oils by capillary gas chromatography-mass spectrometry.

    PubMed

    Zhang, Xin; Julien-David, Diane; Miesch, Michel; Geoffroy, Philippe; Raul, Francis; Roussi, Stamatiki; Aoudé-Werner, Dalal; Marchioni, Eric

    2005-12-01

    As vegetable oils and phytosterol-enriched spreads are marketed for frying food or cooking purposes, temperature is one of the most important factors leading to the formation of phytosterol oxides in food matrix. A methodology based on saponification, organic solvent extraction, solid-phase extraction (SPE), followed by mass spectrometric identification and quantitation of beta-sitosterol oxides using capillary gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode was developed and characterized. Relative response factors of six beta-sitosterol oxides, including 7alpha-hydroxy, 7beta-hydroxy, 5,6alpha-epoxy, 5,6beta-epoxy, 7-keto, and 5alpha,6beta-dihydroxysitosterol, were calculated against authentic standards of 19-hydroxycholesterol or cholestanol. Linear calibration data, limit of detection, and sample recoveries during analytical process. Recoveries of these oxidation compounds in spiked samples ranged from 88 to 115%, while relative standard derivation (R.S.D.) values were below 10% in most cases. The analytical method was applied to quantify beta-sitosterol oxides formed in thermal-oxidized vegetable oils which were heated at different temperatures and for varying time periods. Sitosterol oxidation is strikingly higher in sunflower oil relative to olive oil under all conditions of temperature and heating time. PMID:16038955

  1. Enhanced bile formation induced by experimental dicrocoeliosis in the hamster.

    PubMed

    Sánchez-Campos, S; Tuñón, M J; González, P; Marín, J J; González-Gallego, J

    1998-01-01

    The purpose of this investigation was to determine the effects of experimental dicrocoeliosis on bile formation in the hamster. Studies were carried out at 120 days after infection with an oral dose of 40 metacercariae of Dicrocoelium dendriticum. A significant elevation in bile flow (+20%) and in the biliary output of glutathione (+34%), bile acid (+59%), cholesterol (+108%), phospholipids (+99%) and alkaline phosphatase (+36%) was observed in the infected animals. The bile-to-plasma [14C] mannitol ratio increased to values greater than 1 and there was a reduced contribution (-26%) of biliary tree to bile formation. Those data suggest that enhancement in choleresis had a canalicular origin. The presence of oxidative stress, evidenced by the increased oxidized/reduced glutathione ratio and TBARS concentrations, may contribute to the elevated glutathione efflux into bile. Enhancement in bile acid output was not due to qualitative or quantitative changes in bile acid metabolism, as indicated by the absence of significant modification in liver cholesterol 7alpha-hydroxylase activity and bile acid profile in bile. Increase in the ability of the canalicular membrane to export bile acids was not involved, since maximal secretion rate for exogenously administered taurocholate was decreased. When bile flow, bile acid and biliary lipid secretion was determined in colchicine-pretreated animals differences between control and infected animals were abolished, suggesting that stimulation of the transcytotic vesicle pathway plays an important role in the alteration of the biliary function caused by dicrocoeliosis.

  2. Decommissioning of a mixed oxide fuel fabrication plant at Winfrith Technolgy Centre

    SciTech Connect

    Pengelly, M.G.A.

    1994-01-01

    The Alpha Materials Laboratory (Building A52) at Winfrith contained a mixed oxide fuel fabrication plant which had a capability of producing 10 te/yr of pelleted/compacted fuel and was in operation from 1962 until 1980, when the requirement for this type of fuel in the UK diminished, and the plant became surplus to requirements. A program to develop decommissioning techniques for plutonium plants was started in 1983, addressing the following aspects of alpha plant decommissioning: (1) Re-usable containment systems, (2) Strippable coating technology, (3) Mobile air filtration plant, (4) Size reduction primarily using cold cutting, (5) techniques, (6) Waste packing, and (7) Alpha plant decommissioning methodology. The technology developed has been used to safely and efficiently decommission radioactive plant and equipment including Pu contaminated glove boxes. (63 glove boxes to date) The technology has been widely adopted in the United Kingdom and elsewhere. This paper outlines the general strategies adopted and techniques used for glove box decommissioning in building A52.

  3. 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. PMID:26479901

  4. Isolation, structure, and bioactivities of abiesadines A-Y, 25 new diterpenes from Abies georgei Orr.

    PubMed

    Yang, Xian-Wen; Feng, Lin; Li, Su-Mei; Liu, Xiao-Hua; Li, Yong-Li; Wu, Liang; Shen, Yun-Heng; Tian, Jun-Mian; Zhang, Xi; Liu, Xin-Ru; Wang, Ning; Liu, Yonghong; Zhang, Wei-Dong

    2010-01-15

    Twenty-five new (abiesadines A-Y, 1-25) and 29 known (26-54) diterpenes were isolated from the aerial parts of Abies georgei. Abiesadine A (1) is a novel 8,14-seco-abietane, while abiesadine B (2) is a novel 9,10-seco-abietane. The structures of the new compounds were established on the basis of spectroscopic data analysis. Manool (52) showed the strongest effect against LPS-induced NO production in RAW264.7 macrophages with the IC(50) value of 11.0microg/mL. In another anti-inflammatory assay against TNFalpha-triggered NF-kappaB activity, (12R,13R)-8,12-epoxy-14-labden-13-ol (54) exhibited the strongest effect (IC(50)=8.7microg/mL). For antitumor assays, pomiferin A (26) and 8,11,13-abietatriene-7alpha,18-diol (29) both showed the most significant activity against LOVO cells (IC(50)=9.2microg/mL). While 7-oxocallitrisic acid (46) exhibited significant cytotoxicity against QGY-7703 tumor cells (IC(50)=10.2microg/mL).

  5. Variants of the melanocortin-1 receptor: do they matter clinically?

    PubMed

    Haddadeen, Ciara; Lai, Chester; Cho, Shin-Young; Healy, Eugene

    2015-01-01

    The melanocortin 1 receptor (MC1R) gene encodes for a seven-pass transmembrane receptor primarily expressed on melanocytes and melanoma cells. Single nucleotide polymorphisms (SNPs, also termed variants) in MC1R frequently cause red hair, fair skin and are associated with melanoma and keratinocyte-derived skin cancer development. Activation of wild-type (WT) MC1R in skin assists cutaneous photoprotection whereas reduced MC1R signalling, seen with MC1R variants, impairs ultraviolet radiation (UVR)-protective responses. As ancestral humans migrated out of Africa, the evolutionary advantage of MC1R variants may have related to improved cutaneous vitamin D synthesis and higher birthweight reported with certain MC1R variants. Reduced photoprotection secondary to MC1R dysfunction involves pigmentary and non-pigmentary mechanisms (reduced DNA repair, effects on cell proliferation and possibly immunological parameters), leading to clonal expansion of mutated cells within skin and subsequent carcinogenesis. Recent investigations suggest an association between MC1R genotype and vitiligo, with preliminary evidence that a MC1R agonist, [Nle4-D-Phe7]-alpha-MSH, in combination with UVB, assists repigmentation. Future development of compounds to correct defective MC1R responses secondary to MC1R variants could result in photoprotective benefits for fair-skinned individuals and reduce their skin cancer risk. PMID:25219681

  6. Squalamine: an aminosterol antibiotic from the shark.

    PubMed

    Moore, K S; Wehrli, S; Roder, H; Rogers, M; Forrest, J N; McCrimmon, D; Zasloff, M

    1993-02-15

    In recent years, a variety of low molecular weight antibiotics have been isolated from diverse animal species. These agents, which include peptides, lipids, and alkaloids, exhibit antibiotic activity against environmental microbes and are thought to play a role in innate immunity. We report here the discovery of a broad-spectrum steroidal antibiotic isolated from tissues of the dogfish shark Squalus acanthias. This water-soluble antibiotic, which we have named squalamine, exhibits potent bactericidal activity against both Gram-negative and Gram-positive bacteria. In addition, squalamine is fungicidal and induces osmotic lysis of protozoa. The chemical structure of the antibiotic 3 beta-N-1-(N-[3-(4-aminobutyl)]- 1,3-diaminopropane)-7 alpha,24 zeta-dihydroxy-5 alpha-cholestane 24-sulfate has been determined by fast atom bombardment mass spectroscopy and NMR. Squalamine is a cationic steroid characterized by a condensation of an anionic bile salt intermediate with spermidine. The discovery of squalamine in the shark implicates a steroid as a potential host-defense agent in vertebrates and provides insights into the chemical design of a family of broad-spectrum antibiotics. PMID:8433993

  7. Melanoma-targeting properties of (99m)technetium-labeled cyclic alpha-melanocyte-stimulating hormone peptide analogues.

    PubMed

    Chen, J; Cheng, Z; Hoffman, T J; Jurisson, S S; Quinn, T P

    2000-10-15

    Preliminary reports have demonstrated that (99m)technetium (Tc)-labeled cyclic [Cys(3,4,10), D-Phe7]alpha-MSH(3-13) (CCMSH) exhibits high tumor uptake and retention values in a murine melanoma mouse model. In this report, the tumor targeting mechanism of 99mTc-CCMSH was studied and compared with four other radiolabeled alpha-melanocyte stimulating hormone (alpha-MSH) peptide analogues: 125I-(Tyr2)-[Nle4, D-Phe7]alpha-MSH [125I-(Tyr2)-NDP]; 99mTc-CGCG-NDP; 99mTc-Gly11-CCMSH; and 99mTc-Nle11-CCMSH. In vitro receptor binding, internalization, and cellular retention of radiolabeled alpha-MSH analogues in B16/F1 murine cell line demonstrated that >70% of the receptor-bound radiolabeled analogues were internalized together with the receptor. Ninety % of the internalized 125I-(Tyr2)-NDP, whereas only 36% of internalized 99mTc-CCMSH, was released from the cells into the medium during a 4-h incubation at 37 degrees C. Two mouse models, C57 mice and severe combined immunodeficient (Scid) mice, inoculated s.c. with B16/F1 murine and TXM-13 human melanoma cells were used for the in vivo studies. Tumor uptake values of 11.32 and 2.39 [% injected dose (ID)/g] for 99mTc-CCMSH at 4 h after injection, resulted in an uptake ratio of tumor:blood of 39.0 and 11.5 in murine melanoma-C57 and human melanoma-Scid mouse models, respectively. Two strategies for decreasing the nonspecific kidney uptake of 99mTc-CCMSH, substitution of Lys11 in CCMSH with Gly11 or Nle11, and lysine coinjection, were evaluated. The biodistribution data for the modified peptides showed that Lys11 replacement dramatically decreased the kidney uptake, whereas the tumor uptakes of 99mTc-Nle11- and 99mTc-Gly11-CCMSH were significantly lower than that of 99mTc-CCMSH. Lysine coinjection significantly decreased the kidney uptake (e.g., from 14.6% ID/g to 4.5% ID/g at 4 h after injection in murine melanoma-C57 mice) without significantly changing the value of tumor uptake of 99mTc-CCMSH. In conclusion, the compact

  8. Postnatal rat lung retinoic acid receptor (RAR) mRNA expression and effects of dexamethasone on RAR beta mRNA.

    PubMed

    Grummer, M A; Zachman, R D

    1995-10-01

    Retinoids exert multiple effects upon lung differentiation and growth. Although the mechanisms involved are presently poorly understood, increasing evidence points to a central role of nuclear retinoic acid receptors (RAR). The purpose of this study was to determine RAR mRNA expression profile during postnatal alveolarization, compared with the expression in prenatal and adult rat lung, and to describe the effects of dexamethasone (DEX) and oxygen on postnatal lung RAR gene expression. Total RNA was isolated from lungs of Sprague-Dawley rats on prenatal day 19, on postnatal days 1, 3, 7, 10, and 14 of life, and from adults. One subgroup of littermate pups was treated with DEX daily for 3 or 7 days. In a second experiment, rats were exposed to room air or to 95% oxygen for 72 hours, and received either DEX or saline. Northern hybridization showed that the levels of all RAR subtypes in fetal lung were 45% or less of levels at postnatal day 1. The 3.7 kb RAR alpha transcript levels were lower than day 1 on days 10 and 14 (relative to day 1, day 10 = 0.54 +/- 0.05; day 14 = 0.54 +/- 0.08), but there was no change in a 2.7 kb RAR alpha transcript over this time period. By contrast, RAR beta mRNA levels were significantly higher at days 3, 10, and 14 compared with day 1 (day 3 = 1.79 +/- 0.19; day 10 = 1.41 +/- 0.14; day 14 = 1.53 +/- 0.05). Similarly, RAR gamma mRNA expression levels were higher on day 10 (1.45 +/- 0.09), but by day 14 there was no difference from day 1. Adult lung 3.7 kb RAR alpha, 2.7 kb RAR alpha, and RAR gamma were lower than day 1, but RAR beta was significantly greater (3.7 alpha = 0.52 +/- 0.05; 2.7 alpha = 0.49 +/- 0.26; gamma = 0.74 +/- 0.06; beta = 1.63 +/- 0.22). Treatment with DEX prevented the rise in RAR beta mRNA occurring on day 3 and significantly lowered (0.65 +/- 0.06) the amount of RAR beta mRNA in day 7 lung. Exposure of rat pups to oxygen caused an increase in RAR beta mRNA (1.21 +/- 0.03). DEX treatment again decreased RAR beta m

  9. Seminal plasma proteome of electroejaculated Bos indicus bulls.

    PubMed

    Rego, J P A; Crisp, J M; Moura, A A; Nouwens, A S; Li, Y; Venus, B; Corbet, N J; Corbet, D H; Burns, B M; Boe-Hansen, G B; McGowan, M R

    2014-07-01

    The present study describes the seminal plasma proteome of Bos indicus bulls. Fifty-six, 24-month old Australian Brahman sires were evaluated and subjected to electroejaculation. Seminal plasma proteins were separated by 2-D SDS-PAGE and identified by mass spectrometry. The percentage of progressively motile and morphologically normal sperm of the bulls were 70.4 ± 2.3 and 64 ± 3.2%, respectively. A total of 108 spots were identified in the 2-D maps, corresponding to 46 proteins. Binder of sperm proteins accounted for 55.8% of all spots detected in the maps and spermadhesins comprised the second most abundant constituents. Other proteins of the Bos indicus seminal plasma include clusterin, albumin, transferrin, metalloproteinase inhibitor 2, osteopontin, epididymal secretory protein E1, apolipoprotein A-1, heat shock 70 kDa protein, glutathione peroxidase 3, cathelicidins, alpha-enolase, tripeptidyl-peptidase 1, zinc-alpha-2-glycoprotein, plasma serine protease inhibitor, beta 2-microglobulin, proteasome subunit beta type-4, actin, cathepsins, nucleobinding-1, protein S100-A9, hemoglobin subunit alpha, cadherin-1, angiogenin-1, fibrinogen alpha and beta chain, ephirin-A1, protein DJ-1, serpin A3-7, alpha-2-macroglobulin, annexin A1, complement factor B, polymeric immunoglobulin receptor, seminal ribonuclease, ribonuclease-4, prostaglandin-H2 d-isomerase, platelet-activating factor acetylhydrolase, and phosphoglycerate kinase 1. In conclusion, this work uniquely portrays the Bos indicus seminal fluid proteome, based on samples from a large set of animals representing the Brahman cattle of the tropical Northern Australia. Based on putative biochemical attributes, seminal proteins act during sperm maturation, protection, capacitation and fertilization. PMID:24889044

  10. 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.

  11. 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

  12. Cytoplasmic anchoring of cAMP-dependent protein kinase (PKA) by A-kinase anchor proteins (AKAPs) is required for meiotic arrest of porcine full-grown and growing oocytes.

    PubMed

    Nishimura, Takanori; Fujii, Wataru; Sugiura, Koji; Naito, Kunihiko

    2014-03-01

    Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. We previously hypothesized that the meiotic incompetence of porcine GOs was attributed to complex spatial-temporal regulation of cAMP-dependent protein kinase (PKA) by A-kinase anchor proteins (AKAPs), but found that AKAP1 is not involved in the meiotic incompetence of porcine GOs. In the present study, we cloned porcine cDNAs of AKAP5 and AKAP7alpha, and found that inhibiting the expression of these AKAPs induced PKA translocation into the nucleus and promoted meiotic resumption of porcine GOs without affecting the total PKA activity of GOs, whereas overexpressing these AKAPs had no effect. Because AKAPs regulate PKA localization through binding with regulatory subunits of PKA (PKA-Rs), PKA-R binding with AKAPs was inhibited by AKAP-binding inhibition peptides or PKA-R expression inhibition by antisense RNAs. We found that the expression inhibition and binding inhibition of PRKAR1A, an isoform of mammalian PKA-R, promoted meiotic resumption of porcine GOs, whereas these inhibitions of PRKAR2A, another PKA-R isoform, had no effect. In contrast, the expression inhibition and binding inhibition of PRKAR2A had higher effects than those of PRKAR1A on meiotic resumption of porcine full-grown oocytes. These results suggest that cytoplasmic anchoring of PKA by AKAPs is required for meiotic arrest of oocytes and that the PKA-R isoform working for the maintenance of meiotic arrest changed from PRKAR1A to PRKAR2A during the acquisition of meiotic competence. PMID:24501172

  13. Masou salmon (Oncorhynchus masou) ethanol extract decreases 3-hydroxy-3-methylglutaryl coenzyme A reductase expression in diet-induced obese mice.

    PubMed

    Oh, Hyun-Taek; Chung, Mi Ja; Kim, Soo-Hyun; Choi, Hyun-Jin; Ham, Seung-Shi

    2009-02-01

    This study was designed to evaluate the hypocholesterolemic effects of masou salmon 70% ethanol extract (MSE) and to determine the molecular mechanism by which MSE exerts its effects in high-fat (HF) diet-induced obese mice. We hypothesize that the MSE may contain abundant n-3 fatty acids, so a diet containing MSE may also have hypolipidemic effects by assessing several key gene expressions in cholesterol metabolism such as the low-density lipoprotein (LDL) receptor, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and cholesterol 7alpha-hydroxylase (CYP7A1). To test this hypothesis, C57BL/6J mice were fed a 40% HF diet for 5 weeks, after which time the animals were fed an HF diet containing 0 mg/kg, 75 mg/kg, or 150 mg/kg MSE (HF, HF + MSE 1, and HF + MSE 2 groups, respectively) for an additional 4 weeks (n = 8 in each group, for a total of 24 mice). We found that feeding MSE with an HF diet prevented hypercholesterolemia in diet-induced obese mice; daily MSE feeding reduced total cholesterol levels in plasma and liver by 12.3% and 16.2%, respectively. Furthermore, we examined the expression of key cholesterol metabolism genes by reverse transcription-polymerase chain reaction and found that messenger RNA levels of HMG-CoA reductase were decreased by up to 5-fold, but the expression of both LDL receptor and CYP7A1 did not change. Thus, MSE may exert its hypocholesterolemic effect by altering the expression of HMG-CoA reductase. PMID:19285603

  14. Mecamylamine, dihydro-beta-erythroidine, and dextromethorphan block conditioned responding evoked by the conditional stimulus effects of nicotine.

    PubMed

    Struthers, Amanda M; Wilkinson, Jamie L; Dwoskin, Linda P; Crooks, Peter A; Bevins, Rick A

    2009-12-01

    Current smokers express the desire to quit. However, the majority find it difficult to remain abstinent. As such, research efforts continually seek to develop more effective treatment. One such area of research involves the interoceptive stimulus effects of nicotine as either a discriminative stimulus in an operant drug discrimination task, or more recently as a conditional stimulus (CS) in a discriminated goal-tracking task. The present work investigated the potential role nicotinic acetylcholine receptors play in the CS effects of nicotine (0.4mg/kg) using antagonists with differential selectivity for beta2*, alpha7*, alpha6beta2*, and alpha3beta4* receptors. Methyllycaconitine (MLA) had no effect on nicotine-evoked conditioned responding. Mecamylamine and dihydro-beta-erythroidine (DHbetaE) dose-dependently blocked responding evoked by the nicotine CS. In a time-course assessment of mecamylamine and DHbetaE, each blocked conditioned responding when given 5min before testing and still blocked conditioned responding when administered 200min before testing. Two novel bis-picolinium analogs (N, N'-(3, 3'-(dodecan-1,12-diyl)-bis-picolinium dibromide [bPiDDB], and N, N'-(decan-1,10-diyl)-bis-picolinium diiodide [bPiDI]) did not block nicotine-evoked conditioned responding. Finally, pretreatment with low dose combinations of mecamylamine, dextromethorphan, and/or bupropion was used to target alpha3beta4* receptors. No combination blocked conditioned responding evoked by the training dose of nicotine. However, a combination of mecamylamine and dextromethorphan partially blocked nicotine-evoked conditioned responding to a lower dose of nicotine (0.1mg/kg). These results indicate that beta2* and potentially alpha3beta4* nicotinic acetylcholine receptors play a role in the CS effects of nicotine and are potential targets for the development of nicotine cessation aids.

  15. 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

  16. Apparent selective bile acid malabsorption as a consequence of ileal exclusion: effects on bile acid, cholesterol, and lipoprotein metabolism.

    PubMed Central

    Akerlund, J E; Björkhem, I; Angelin, B; Liljeqvist, L; Einarsson, K

    1994-01-01

    A new model has been developed to characterise the effect of a standardised ileal exclusion on bile acid, cholesterol, and lipoprotein metabolism in humans. Twelve patients treated by colectomy and ileostomy for ulcerative colitis were studied on two occasions: firstly with a conventional ileostomy and then three months afterwards with an ileal pouch operation with an ileoanal anastomosis and a protective loop ileostomy, excluding on average 95 cm of the distal ileum. The ileostomy contents were collected during 96 hours and the excretion of bile acids and cholesterol was determined using gas chromatography-mass spectrometry. Fasting blood and duodenal bile samples were collected on two consecutive days. After the exclusion of the distal ileum, both cholic and chenodeoxycholic acid excretion in the ileostomy effluent increased four to five times without any change in cholesterol excretion. Serum concentrations of lathosterol (a marker of cholesterol biosynthesis) and 7 alpha-hydroxycholesterol (a marker for bile acid biosynthesis) were increased several fold. Plasma concentrations of total VLDL triglycerides were also increased whereas the concentrations of total and LDL cholesterol, and apolipoprotein B were decreased. There were no changes in biliary lipid composition or cholesterol saturation of bile. The results show that the exclusion of about 95 cm of distal ileum causes malabsorption of bile acids but apparently not of cholesterol. The bile acid malabsorption leads to increased synthesis of both bile acids and cholesterol in the liver. It is suggested that bile acids can regulate cholesterol synthesis by a mechanism independent of the effect of bile acids on cholesterol absorption. The enhanced demand for cholesterol also leads to a decrease in plasma LDL cholesterol and apolipoprotein B concentrations. The malabsorption of bile acids did not affect biliary lipid composition or cholesterol saturations of VLDL triglycerides. PMID:7926917

  17. 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.

  18. Nanosuspension formulations for low-soluble drugs: pharmacokinetic evaluation using spironolactone as model compound.

    PubMed

    Langguth, P; Hanafy, A; Frenzel, D; Grenier, P; Nhamias, A; Ohlig, T; Vergnault, G; Spahn-Langguth, H

    2005-03-01

    Various particle sizes of spironolactone as a model low solubility drug were formulated to yield micro-and nanosuspensions of the type solid lipid nanoparticles and DissoCubes. Seven oral and one i.v. formulations were tested in an in vivo pharmacokinetic study in rats with the aim of characterizing the bioavailability of spironolactone on the basis of its metabolites canrenone and 7-alpha-thiomethylspirolactone. In addition, a dose escalation study was carried out using nonmicronized spironolactone suspension as well as a nanosuspension type DissoCubes. On the basis of AUC as well as Cmax ratios, three groups of formulations were distinguished. The biggest improvement was seen with a solid lipid nanoparticle formulation yielding a 5.7-fold increase in AUC for canrenone and a similar improvement based on the Cmax metric, followed by a group of three formulations containing nanosized, micronized, and coarse drug material and surfactant. The DissoCubes nanosuspension yielded highly significant improvements in bioavailability averaging 3.3-fold in AUC and 3.0-fold in terms of Cmax for canrenone. The third class encompasses all other formulations, which showed very little to no improvement in bioavailability. The results show that the particle size minimization was not the major determining factor in the bioavailability improvement. Rather, the type of surfactant used as stabilizer in the formulations was of greater importance. Improvement in drug solubility in the intestine as well as in dissolution rate of spironolactone are the most likely mechanisms responsible for the observed effect, although additional mechanisms such as permeability enhancement may also be involved. PMID:15830727

  19. The Met852 residue is a key organizer of the ligand-binding cavity of the human mineralocorticoid receptor.

    PubMed

    Fagart, Jérôme; Seguin, Cendrine; Pinon, Grégory Maurice; Rafestin-Oblin, Marie-Edith

    2005-05-01

    Spirolactones harboring various C7 substituents are aldosterone antagonists, and some of them are used in the treatment of essential hypertension. They bind to the human mineralocorticoid receptor and render it transcriptionally inactive. Structural analysis using a three-dimensional homology model of the ligand-binding domain of the receptor has revealed that the Met852 residue of the ligand-binding cavity faces the C7 substituent of spirolactones. We therefore tested the binding capacities of C7-substituted spirolactones in an in vitro system expressing either the mutant receptor, in which Met852 was replaced by alanine, or the wild-type receptor. The M852A mutation had almost no effect on the binding of C7-substituted spirolactones to mineralocorticoid receptor but dramatically reduced the capacity of the receptor to bind steroids with no C7 substituent (aldosterone, cortisol, deoxycorticosterone, and canrenone). cis-trans Cotransfection assays revealed that two spirolactones characterized by having a propyl group [7 alpha-propyl-17 alpha-hydroxy-3-oxo-preg-4-ene-21-carboxylic acid gamma-lactone (RU26752)] or a thioacetyl group (spironolactone) at the C7 position acquired agonist properties when bound to the mutant receptor. In contrast, mexrenone and eplerenone, both of which harbor an acetyl group at the C7 position, retained antagonist properties when bound to the mutant receptor. Overall, these findings indicate that Met852 acts as an organizer residue that plays two major roles: 1) it allows steroids with no substituent at the C7 position to be accommodated within the ligand-binding cavity; and 2) it is involved in the steric hindrance that prevents C7-substituted spirolactones from folding the receptor in its active state. PMID:15716462

  20. 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.

  1. The Mechanisms Underlying the Hypolipidaemic Effects of Grifola frondosa in the Liver of Rats.

    PubMed

    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

  2. DOTA alpha-melanocyte-stimulating hormone analogues for imaging metastatic melanoma lesions.

    PubMed

    Froidevaux, Sylvie; Calame-Christe, Martine; Sumanovski, Lazar; Tanner, Heidi; Eberle, Alex N

    2003-06-01

    Scintigraphic imaging of metastatic melanoma lesions requires highly tumor-specific radiopharmaceuticals. Because both melanotic and amelanotic melanomas overexpress melanocortin-1 receptors (MC1R), radiolabeled analogues of alpha-melanocyte-stimulating hormone (alpha-MSH) are potential candidates for melanoma diagnosis. Here, we report the in vivo performance of a newly designed octapeptide analogue, [betaAla(3), Nle(4), Asp(5), D-Phe(7), Lys(10)]-alpha-MSH(3-10) (MSH(OCT)), which was conjugated through its N-terminal amino group to the metal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to enable incorporation of radiometals (e.g., indium-111) into the peptide. DOTA-MSH(OCT) displayed high in vitro MC1R affinity (IC(50) 9.21 nM). In vivo [(111)In]DOTA-MSH(OCT) exhibited a favorable biodistribution profile after injection in B16-F1 tumorbearing mice. The radiopeptide was rapidly cleared from blood through the kidneys and, most importantly, accumulated preferentially in the melanoma lesions. Lung and liver melanoma metastases could be clearly imaged on tissue section autoradiographs 4 h after injection of [(111)In]DOTA-MSH(OCT). A comparative study of [(111)In]DOTA-MSH(OCT) with [(111)In]DOTA-[Nle(4), D-Phe(7)]-alpha-MSH ([(111)In]-DOTA-NDP-MSH) demonstrated the superiority of the DOTA-MSH(OCT) peptide, particularly for the amount of radioactivity taken up by nonmalignant organs, including bone, the most radiosensitive tissue. These results demonstrate that [(111)In]DOTA-MSH(OCT) is a promising melanoma imaging agent.

  3. 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.

  4. 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.

  5. Fbxw5 suppresses nuclear c-Myb activity via DDB1-Cul4-Rbx1 ligase-mediated sumoylation

    SciTech Connect

    Kanei-Ishii, Chie; Nomura, Teruaki; Egoh, Ayako; Ishii, Shunsuke

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Fbxw5 enhances sumoylation of c-Myb. Black-Right-Pointing-Pointer The DDB1-Cul4A-Rbx1 complex mediates c-Myb sumoylation. Black-Right-Pointing-Pointer The Fbxw5-DDB1-Cul4A-Rdx1 complex is a dual SUMO/ubiquitin ligase. Black-Right-Pointing-Pointer Fbxw5 suppresses the c-Myb trans-activating capacity. -- Abstract: The c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling. In this process, Fbxw7{alpha}, the F-box protein of the SCF complex, binds to c-Myb via its C-terminal WD40 domain, and induces the ubiquitination of c-Myb. Here, we report that Fbxw5, another F-box protein, enhances sumoylation of nuclear c-Myb. Fbxw5 enhanced c-Myb sumoylation via the DDB1-Cul4A-Rbx1 complex. Since the Fbxw5-DDB1-Cul4A-Rbx1 complex was shown to act as a ubiquitin ligase for tumor suppressor TSC2, our results suggest that this complex can function as a dual SUMO/ubiquitin ligase. Fbxw5, which is localized to both nucleus and cytosol, enhanced sumoylation of nuclear c-Myb and induced the localization of c-Myb to nuclear dot-like domains. Co-expression of Fbxw5 suppressed the trans-activation of c-myc promoter by wild-type c-Myb, but not by v-Myb, which lacks the sumoylation sites. These results suggest that multiple E3 ligases suppress c-Myb activity through sumoylation or ubiquitination, and that v-Myb is no longer subject to these negative regulations.

  6. 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

  7. Saposin B is a human coenzyme q10-binding/transfer protein.

    PubMed

    Jin, Guangzhi; Kubo, Hiroshi; Kashiba, Misato; Horinouchi, Ryo; Hasegawa, Makoto; Suzuki, Masaru; Sagawa, Tomofumi; Oizumi, Mikiko; Fujisawa, Akio; Tsukamoto, Hideo; Yoshimura, Shinichi; Yamamoto, Yorihiro

    2008-03-01

    Coenzyme Q10 (CoQ10) is essential for ATP production in the mitochondria, and is an important antioxidant in every biomembrane and lipoprotein. Due to its hydrophobicity, a binding and transfer protein for CoQ10 is plausible, but none have yet been isolated and characterized. Here we purified a CoQ10-binding protein from human urine and identified it to be saposin B, a housekeeping protein necessary for sphingolipid hydrolysis in lysosomes. We confirmed that cellular saposin B binds CoQ10 in human sperm and the hepatoma cell line HepG2 by using saposin B monoclonal antibody. The molar ratios of CoQ10 to saposin B were estimated to be 0.22 in urine, 0.003 in HepG2, and 0.12 in sperm. We then confirmed that aqueous saposin B extracts CoQ10 from hexane to form a saposin B-CoQ10 complex. Lipid binding affinity to saposin B decreased in the following order: CoQ10>CoQ9>CoQ7>alpha-tocopherol>cholesterol (no binding). The CoQ10-binding affinity to saposin B increased with pH, with maximal binding seen at pH 7.4. On the other hand, the CoQ10-donating activity of the saposin B-CoQ10 complex to erythrocyte ghost membranes increased with decreasing pH. These results suggest that saposin B binds and transports CoQ10 in human cells.

  8. 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.

  9. Influence of recipient gender on intrasplenic fetal liver tissue transplants in rats: cytochrome P450-mediated monooxygenase functions.

    PubMed

    Lupp, Amelie; Hugenschmidt, Sabine; Rost, Michael; Müller, Dieter

    2004-05-01

    Rat livers display a sex-specific cytochrome P450 (P450) isoforms expression pattern with consecutive differences in P450-mediated monooxygenase activities, which have been shown to be due to a differential profile of growth hormone (GH) secretion. Parallel to previous investigations on P450 isoforms expression, the aim of the present study was to elucidate the influence of recipient gender on P450-mediated monooxygenase activities in intrasplenic liver tissue transplants in comparison to orthotopic liver. Fetal liver tissue suspensions of mixed gender were transplanted into the spleen of adult male or female syngenic recipients. Four months after grafting transplant-recipients and age-matched controls were treated with beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or the vehicles and sacrificed 24 or 48 h thereafter. P450-dependent monooxygenase activities were assessed by a series of model reactions for different P450 subtypes in liver and spleen 9000 g supernatants. In spleens of male and female control rats only very low monooxygenase activities were detectable, whereas with most model reactions distinct activities were observed in transplant-containing organs. Livers and transplant-containing spleens from male rats displayed higher basal ethoxycoumarin O-deethylase and testosterone 2alpha-, 2beta-, 6beta-, 14alpha-, 15alpha-, 15beta-, 16alpha-, 16beta- and 17-hydroxylase activities than those from females. On the other hand, like the respective livers, spleens from female transplant-recipients demonstrated more pronounced p-nitrophenol- and testosterone 6alpha- and 7alpha-hydroxylase activities than those from male hosts. With nearly all model reactions gender-specific differences in inducibility by BNF, PB or DEX could be demonstrated in livers as well as in transplant-containing spleens. These results further confirm that the P450 system of intrasplenic liver tissue transplants and the respective orthotopic livers is similarly influenced

  10. Bile acid signaling through FXR induces intracellular adhesion molecule-1 expression in mouse liver and human hepatocytes.

    PubMed

    Qin, Pu; Borges-Marcucci, Lisa A; Evans, Mark J; Harnish, Douglas C

    2005-08-01

    Previous studies have demonstrated a dramatic induction of inflammatory gene expression in livers from mice fed a high-fat, high-cholesterol diet containing cholate after 3-5 wk. To determine the contribution of cholate in mediating these inductions, C57BL/6 mice were fed a chow diet supplemented with increasing concentrations of cholic acid (CA) for 5 days. A dose-dependent induction in the hepatic levels of TNF-alpha, VCAM-1, ICAM-1, and SAA-2 mRNA were observed. As positive controls, a dose-dependent repression of cholesterol 7alpha-hydroxylase and a dose-dependent induction of small heterodimer partner (SHP) expression were also observed, suggesting that farnesoid X receptor (FXR) was activated. In addition, ICAM-1 and SHP mRNA levels were also induced in primary human hepatocytes when treated with chenodeoxycholic acid or GW4064, a FXR-selective agonist. The involvement of FXR in CA-induced inflammatory gene expression was further investigated in the human hepatic cell line HepG2. Both ICAM-1 and SHP expression were induced in a dose- and time-dependent manner by treatment with the FXR-selective agonist GW4064. Moreover, the induction of ICAM-1 by GW4064 was inhibited by the FXR antagonist guggulsterone or with transfection of FXR siRNA. Finally, the activity of FXR was mapped to a retinoic acid response element (RARE) site containing an imbedded farnesoid X response element (FXRE) on the human ICAM-1 promoter and FXR and retinoid X receptor were demonstrated to bind to this site. Finally, FXR-mediated activation of ICAM-1 could be further enhanced by TNF-alpha cotreatment in hepatocytes, suggesting a potential cooperation between cytokine and bile acid-signaling pathways during hepatic inflammatory events.

  11. 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

  12. 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.

  13. Tauroursodeoxycholic acid stimulates hepatocellular exocytosis and mobilizes extracellular Ca++ mechanisms defective in cholestasis.

    PubMed Central

    Beuers, U; Nathanson, M H; Isales, C M; Boyer, J L

    1993-01-01

    To assess the effects of tauroursodeoxycholic acid (TUDCA) on bile excretory function, we examined whether TUDCA modulates vesicular exocytosis in the isolated perfused liver of normal rats in the presence of high (1.9 mM) or low (0.19 mM) extracellular Ca++ and in cholestatic rats 24 h after bile duct ligation. In addition, the effects of TUDCA on Ca++ homeostasis were compared in normal and in cholestatic hepatocytes. In the isolated perfused rat liver, TUDCA (25 microM) stimulated a sustained increase in the biliary excretion of horseradish peroxidase, a marker of the vesicular pathway, in the presence of high, but not low extracellular Ca++ or in the cholestatic liver. In contrast, TUDCA stimulated bile flow to the same extent regardless of the concentration of extracellular Ca++ or the presence of cholestasis. In indo-1-loaded hepatocytes, basal cytosolic free Ca++ ([Ca++]i) levels were not different between normal and cholestatic cells. However, in cholestatic cells [Ca++]i increases induced by TUDCA (10 microM) and its 7 alpha-OH epimer taurochenodeoxycholic acid (50 microM) were reduced to 22% and 26%, respectively, compared to normal cells. The impairment of TUDCA-induced [Ca++]i increase in cholestatic cells could be mimicked by exposing normal cells to low extracellular Ca++ (21%) or to the Ca++ channel blocker NiCl2 (23%). These data indicate that (a) dihydroxy bile acid-induced Ca++ entry may be of functional importance in the regulation of hepatocellular vesicular exocytosis, and (b) this Ca++ entry mechanism across the plasma membrane is impaired in cholestatic hepatocytes. We speculate that the beneficial effect of ursodeoxycholic acid in cholestatic liver diseases may be related to the Ca+(+)-dependent stimulation of vesicular exocytosis by its conjugate. PMID:8254052

  14. 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.

  15. Raloxifene analogue LY117018 suppresses oxidative stress-induced endothelial cell apoptosis through activation of ERK1/2 signaling pathway.

    PubMed

    Yu, Jing; Eto, Masato; Kozaki, Koichi; Akishita, Masahiro; Okabe, Tetsuro; Ouchi, Yasuyoshi

    2008-07-28

    A selective estrogen receptor modulator, raloxifene, has been shown to reduce cardiovascular events in relatively high-risk postmenopausal women with osteoporosis. However, the mechanisms by which raloxifene exerts a pharmacological effect on cardiovascular organs have not been fully elucidated. The present study was designed to examine whether the raloxifene analogue, 6-hydroxy-2-(p-hydroxyphenyl)-benzo(b) thien-3-yl-p-(2-(pyrrolidinyl)ethoxy phenyl ketone (LY117018), could inhibit apoptosis and to clarify the signaling pathway in vascular endothelial cells. LY117018 significantly inhibited hydrogen peroxide-induced apoptosis in bovine carotid artery endothelial cells. The anti-apoptotic effect of LY117018 was abolished by an estrogen receptor antagonist, 7alpha,7beta-(9[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl) estra-1,3,5(10)-triene-3,17-diol (ICI 182,780). Mitogen-activated protein kinases (MAPK), including p38, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase1/2 (ERK1/2), and Akt, have been shown to act as apoptotic or anti-apoptotic signals. Phosphorylation of p38, JNK, ERK1/2 and Akt was examined. LY117018 increased ERK1/2 phosphorylation but did not enhance the phosphorylation of p38, JNK, or Akt. The anti-apoptotic effect of LY117018 was prevented by treatment with 2-[2'-amino-3'-methoxyphenyl]-oxanaphthalen-4-one (PD98059), an upstream inhibitor of ERK1/2. LY117018 stimulated an increase in ERK1/2 phosphorylation, which was diminished by ICI 182,780. The activation of ERK/1/2 by LY117018 was not inhibited by the transcription inhibitor, actinomycin D. These results suggest that estrogen receptors and the ERK1/2 signaling pathway are involved in the anti-apoptotic action of LY117018 in vascular endothelial cells. PMID:18541231

  16. 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.

  17. Hepatic microsomal metabolism of the anthelmintic benzimidazole fenbendazole: enhanced inhibition of cytochrome P450 reactions by oxidized metabolites of the drug.

    PubMed

    Murray, M; Hudson, A M; Yassa, V

    1992-01-01

    Potentiation of the anthelmintic action of benzimidazole carbamates, such as fenbendazole [methyl 5(6)-(phenylthio)-1H-benzimidazol-2-ylcarbamate], has been noted during concurrent administration of benzimidazoles that possess no intrinsic anthelmintic activity. This study investigated the possibility that inhibition of P450 enzymes by fenbendazole and its metabolites could play a role in the potentiation phenomenon. Fenbendazole underwent P450-mediated oxidation in microsomes from untreated rat liver to the sulfoxide and (4'-hydroxyphenyl)thio metabolites [2.92 and 2.87 nmol/(mg of protein.h)]. Pretreatment of rats with phenobarbital or dexamethasone enhanced sulfoxidation by 1.9- and 2.9-fold, respectively. 4'-Hydroxylation was increased slightly (by 28%) by phenobarbital and decreased slightly (by 41%) by dexamethasone. Induction also promoted further metabolism of the sulfoxide to fenbendazole sulfone. Immunoinhibition and chemical inhibition studies suggested that P450 3A proteins and the flavin-containing monooxygenase are involved in sulfoxide and sulfone formation whereas 4'-hydroxylation involved the P450s 2C11, 2C6, and 2B1, depending on the type of induction. In untreated rat liver, the sulfoxide and (4'-hydroxyphenyl)thio metabolites of fenbendazole were relatively potent inhibitors of P450-mediated androstenedione 16 alpha-, 16 beta-, and 6 beta-hydroxylation (IC50 values of 42, 36, and 74 microM, respectively); 7 alpha-hydroxylase activity was uninhibited. In contrast, fenbendazole and its sulfone metabolite were not inhibitors of these reactions. Mixed-function oxidase activities in phenobarbital-induced rat hepatic microsomes were refractory to inhibition by most compounds, but P450 1A1 mediated activities in microsomes from beta-naphthoflavone-induced rat liver were quite susceptible to inhibition by fenbendazole sulfoxide. Studies with two analogous sulfoxides yielded similar findings.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. 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. PMID:21834248

  19. Diagnosis and management of familial dyslipoproteinemias.

    PubMed

    Kwiterovich, Peter O

    2013-06-01

    The three major pathways of lipoprotein metabolism provide a superb paradigm to delineate systematically the familial dyslipoproteinemias. Such understanding leads to improved diagnosis and treatment of patients. In the exogenous (intestinal) pathway, defects in LPL, apoC-II, APOA-V, and GPIHBP1 disrupt the catabolism of chylomicrons and hepatic uptake of their remnants, producing very high TG. In the endogenous (hepatic) pathway, six disorders affect the activity of the LDLR and markedly increase LDL. These include FH, FDB, ARH, PCSK9 gain-of-function mutations, sitosterolemia and loss of 7 alpha hydroxylase. Hepatic overproduction of VLDL occurs in FCHL, hyperapoB, LDL subclass pattern B, FDH and syndrome X, often due to insulin resistance and resulting in high TG, elevated small LDL particles and low HDL-C. Defects in APOB-100 and loss-of-function mutations in PCSK9 are associated with low LDL-C, decreased CVD and longevity. An absence of MTP leads to marked reduction in chylomicrons and VLDL, causing abetalipoproteinemia. In the reverse cholesterol pathway, deletions or nonsense mutations in apoA-I or ABCA1 transporter disrupt the formation of the nascent HDL particle. Mutations in LCAT disrupt esterification of cholesterol in nascent HDL by LCAT and apoA-1, and formation of spherical HDL. Mutations in either CETP or SR-B1 and familial high HDL lead to increased large HDL particles, the effect of which on CVD is not resolved. The major goal is to prevent or ameliorate the major complications of many familial dyslipoproteinemias, namely, premature CVD or pancreatitis. Dietary and drug treatment specific for each inherited disorder is reviewed. PMID:23666884

  20. 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. PMID:19844073

  1. 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

  2. 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

  3. 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

  4. Noncovalent Interaction Energies in Covalent Complexes: TEM-1 beta-Lactamase and beta-Lactams

    SciTech Connect

    Wang, Xiaojun; Minasov, George; Shoichet, Brian K.

    2010-03-08

    The class A {beta}-lactamase TEM-1 is a key bacterial resistance enzyme against {beta}-lactam antibiotics, but little is known about the energetic bases for complementarity between TEM-1 and its inhibitors. Most inhibitors form a covalent adduct with the catalytic Ser70, making the measurement of equilibriumconstants, and hence interaction energies, technically difficult. This study evaluates noncovalent interactions withincovalent complexes by examining the differential stability of TEM-1 and its inhibitor adducts. The thermal denaturation of TEM-1 follows a two-state, reversible model with a melting temperature (T{sub m}) of 51.6 C and a van't Hoff enthalpy of unfolding ({Delta}H{sub VH}) of 146.2 kcal/mol at pH 7.0. The stability of the enzyme changes on forming an inhibitor adduct. As expected, some inhibitors stabilize TEM-1; transition-state analogues increase the T{sub m} by up to 3.7 C(1.7 kcal/mol). Surprisingly, all {beta}-lactam covalent acyl-enzyme complexes tested destabilize TEM-1 significantly relative to the apoenzyme. For instance, the clinically used inhibitor clavulanic acid and the {beta}-lactamase-resistant {beta}-lactams moxalactam and imipenem destabilize TEM-1 by over 2.6 C (1.2 kcal/mol) in their covalent adducts. Based on the structure of the TEM-1/imipenem complex (Maveyraud et al., J Am Chem Soc 1998;120:9748-52), destabilization by moxalactam and imipenem is thought to be caused by a steric clash between the side-chain of Asn132 and the 6(7)-{alpha} group of these {beta}-lactams. To test this hypothesis, the mutant enzyme N132A was made. In contrast with wild-type, the covalent complexes between N132A and both imipenem and moxalactam stabilize the enzyme, consistent with the hypothesis. To investigate the structural bases of this dramatic change instability, the structure of N132A/imipenem was determined by X-ray crystallography. In the complex with N132A, imipenemadopts a very different conformation from that observed in the wild

  5. Molecular cloning, functional expression and pharmacological characterization of a mouse melanocortin receptor gene.

    PubMed Central

    Desarnaud, F; Labbe, O; Eggerickx, D; Vassart, G; Parmentier, M

    1994-01-01

    We describe the cloning of the mouse HGMP01A gene that encodes a melanocortin receptor functionally distinct from the adrenal cortex corticotropin (adrenocorticotrophic hormone; ACTH) receptor and the melanocyte-stimulating hormone (MSH) receptor expressed in melanoma. The gene encodes a protein of 323 amino acids with a calculated molecular mass of 35,800 Da, displaying potential sites for N-linked glycosylation and phosphorylation by protein kinase C. An RNAase protection assay detected weak expression in the brain, but not in adrenal gland, skin, or any of the other tissues tested. Stable CHO cell lines expressing over 100,000 receptors per cell were generated. The recombinant receptor binds iodinated [Nle4,D-Phe7]alpha-MSH (NDP-MSH) with an apparent Kd of 700 pM. Displacement of the ligand by a variety of pro-opiomelanocortin-derived peptides revealed a pharmacological profile distinct from that of the classical ACTH and MSH receptors. NDP-MSH was the most powerful competitor (IC50 1.4 nM), followed by gamma-MSH (IC50 7 nM). alpha-MSH, beta-MSH and ACTH-(1-39) were significantly less potent, with IC50 values of 30, 19 and 21 nM respectively. ACTH-(4-10) was poorly active (IC50 2.4 microM), while corticotropin-like intermediate lobe peptide (CLIP) and beta-endorphin were totally ineffective. The recombinant receptor was found to stimulate adenylate cyclase. The potency order of the agonists in this assay was consistent with that of the binding displacement assays. This receptor represents the orthologue of the human melanocortin 3 receptor reported recently. The growing family of melanocortin receptors constitute the molecular basis for the variety of actions of melanocortins that have been described over the years. The availability of functionally expressed receptors from the melanocortin family will allow the development of a specific pharmacology, and a better understanding of the function of the pro-opiomelanocortin-derived peptides. Images Figure 6 PMID

  6. Fifty years with bile acids and steroids in health and disease.

    PubMed

    Sjövall, Jan

    2004-08-01

    , extrahepatic 7alpha-hydroxylation and 3-dehydrogenation of hydroxycholesterols, and extrahepatic formation of C27 bile acids. The final part discusses analysis of free and sulfated steroids in brain tissue by capillary liquid chromatography-electrospray MS and suggests a need for reevaluation of the function of steroid sulfates in rat brain. PMID:15638239

  7. Occurrence of estrogenic compounds in and removal by a swine farm waste treatment plant.

    PubMed

    Furuichi, Takuma; Kannan, Kurunthachalam; Suzuki, Kazuyoshi; Tanaka, Shuzo; Giesy, John P; Masunaga, Shigeki

    2006-12-15

    The total estrogenic activity of the wastewater from a swine farm in Japan was quantitatively characterized, and the compounds responsible for the estrogenic activity were identified and quantified. The wastewater treatment process consisted of a series of an up-flow anaerobic sludge blanket (UASB) and a trickling filter. Samples were collected at each treatment step, and the total estrogenic activity was determined by use of an in vitro gene expression assay (MVLN; MCF-7 human breast cancer cell stably transfected with the pVit-tk-LUC receptor plasmid). Individual estrogenic compounds were identified and quantified using liquid chromatography-mass spectrometry (LC/MS) and liquid chromatography-tandem mass spectrometry (LC/ MS/MS). To further identify the compounds contributing to the estrogenic activity in the wastewater, the sample extracts were fractionated into 12 fractions (fractions 1-12) by HPLC. The rate of removal of estrogenic activity between the effluent and the influent was greater than 97%. The trickling filter removed the majority of the estrogenic activity. The removal rates of specific estrogenic compounds ranged from 44 to 99%. Estrogenic activity was detected mainly in the fractions containing estrone (El), 17beta-estradiol (betaE2), 17alpha-estradiol (alpha E2), estriol (E3), bisphenol A (alphaPA), and equol (EQ0). The ratios of betaE2-EQc (betaE2 equivalents derived from chemical analysis) to betaE2-EQB (betaE2 equivalent derived from bioassay) in the 12 fractions collectively were contributed by El (17-30%), betaE2 (23-30%), acE2 (<1%), E3 (1-2%), BPA (<1%), and EQO (2-3%) in the influent and El (16-37%), PE2 (<1-7%), alphaE2 (<1%), E3 (<1-3%), BPA (<1%), and EQO (<1%) in the effluent. The compounds responsible for most of the estrogenic activity measured in the bioassay were natural estrogens such as El and betaE2. PMID:17256545

  8. Bioequivalence assessment of two formulations of spironolactone in Chinese healthy male volunteers.

    PubMed

    Xu, Feng-Guo; Zhang, Zun-Jian; Dong, Hai-Juan; Tian, Yuan; Liu, Ying; Chen, Yun

    2008-01-01

    The bioavailability of a new spironolactone ((7alpha,17alpha)-7-(acetylthio)-17-hydroxy-3-oxopregn-4-ene-21-carboxylic acid gamma-lactone, CAS 52-01-7) formulation (test) was compared with a commercially available original formulation (reference) of the drug in 20 Chinese healthy male volunteers, aged between 21 and 27. The trial was designed as an open, randomized, single blind two-sequence, two-period crossover study. Under fasting conditions, each subject received a single oral dose of 100 mg spironolactone as a test or reference formulation with a 7-day washout period between the two formulations. The plasma concentrations of spironolactone and its active metabolite canrenone (CAS 976-71-6) were analyzed by a sensitive liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) method. The pharmacokinetic parameters included AUC(0.t), AUC(0-infinity), C(max), t1/2, and T(max). Values of AUC(0-t) demonstrate nearly identical bioavailability of spironolactone from the examined formulations. The AUC(0.12) of spironolactone was 148.35 +/- 39.5 and 144.39 +/- 53.02 ng x h/ml for the test and reference formulation, respectively. The AUC(0-60) of the metabolite canrenone was 1873.36 +/- 318.10 and 1911.28 +/- 355.60 ng h/ml for test and reference formulation, respectively. The maximum plasma concentration (C(max)) of spironolactone was 48.34 +/- 21.16 ng/ml for the test and 47.40 +/- 23.40 ng/ml for the reference product and the C(max) of the metabolite was 122.90 +/- 27.70 and 123.35 +/- 27.29 ng/ml for the test and reference product, respectively. No statistical differences were observed for C(max) and the area under the plasma concentration-time curve for both spironolactone and its active metabolite canrenone. 90% confidence limits calculated for C(max) and AUC from zero to infinity (AUC(0-infinity)) of spironolactone and its metabolite were included in the bioequivalence range (80%-125% for AUC). This study shows that the test

  9. 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

  10. SK&F 97426-A a more potent bile acid sequestrant and hypocholesterolaemic agent than cholestyramine in the hamster.

    PubMed

    Benson, G M; Alston, D R; Bond, B C; Gee, A N; Glen, A; Haynes, C; Hickey, D M; Iqbal, S; Jackson, B; Jaxa-Chamiec, A A

    1993-06-01

    week. The activities of the liver HMG-CoA reductase and cholesterol 7 alpha-hydroxylase were increased as expected, whilst the activity of the acyl-CoA:cholesterol acyltransferase was reduced by both sequestrants at this dose. SK&F 97426-A was, therefore, 2-3-fold more potent as a bile acid sequestrant and hypocholesterolaemic agent than cholestyramine when tested in the hamster.

  11. The metabolism of primary, 7-oxo, and 7 beta-hydroxy bile acids by Clostridium absonum.

    PubMed

    Sutherland, J D; Macdonald, I A

    1982-07-01

    Clostridium absonum was shown to metabolize primary bile acids to give rise to both 7-oxo bile acids and 7 beta-hydroxy (urso) bile acids. At relatively low redox potential (Eh) values, high yields of urso bile acids were achieved (60-75%). If, however, the Eh value of the culture was allowed to rise above approximately -100 mv, the 7-oxo bile acid would tend to predominate (more than 75%) and the "death phase" was accelerated. Growth of C. absonum in sterile graduated cylinders instead of in conventional Erlenmeyer flasks was effective in delaying the rise in Eh value with time (which appears largely due to diffusion of atmospheric oxygen into the medium) and in preserving a higher viable count of organisms. It is proposed that the formation of excess amounts of 7-oxo bile acid is a manifestation of oxygen toxicity and that it could be mediated by an increasing intracellular NADP:NADPH ratio. Additionally, the reaction: primary bile acid in equilibrium oxo bile acid in equilibrium urso bile acid was shown to be partially reversible. When the organisms were grown with [24-(14)C]chenodeoxycholic, -cholic, or -7-keto-lithocholic acid, this reaction could be clearly demonstrated. The addition of an equimolar concentration of deoxycholic acid (which itself is not metabolized) effectively enhanced the rate of bioconversion of cholate and 7-keto-lithocholic, but not chenodeoxycholate (whose rate of bioconversion was the fastest of the three). When the organisms were grown with urso bile acids (ursocholic or ursodeoxycholic) or with 7-keto-deoxycholic acid, very little metabolism occurred unless deoxycholic acid was added which induced formation of primary and keto bile acids. In all cases, formation of oxo bile acid from primary or urso bile acid occurred as the Eh value of the medium rose with time and could thus be delayed by the use of a cylinder instead of a flask for growing the culture. These results were rationalized by demonstrating that induction of 7 alpha- and

  12. Sterol carrier and lipid transfer proteins.

    PubMed

    Scallen, T J; Pastuszyn, A; Noland, B J; Chanderbhan, R; Kharroubi, A; Vahouny, G V

    1985-09-01

    The discovery of the sterol carrier and lipid transfer proteins was largely a result of the findings that cells contained cytosolic factors which were required either for the microsomal synthesis of cholesterol or which could accelerate the transfer or exchange of phospholipids between membrane preparations. There are two sterol carrier proteins present in rat liver cytosol. Sterol carrier protein 1 (SCP1) (Mr 47 000) participates in the microsomal conversion of squalene to lanosterol, and sterol carrier protein 2 (SCP2) (Mr 13 500) participates in the microsomal conversion of lanosterol to cholesterol. In addition SCP2 also markedly stimulates the esterification of cholesterol by rat liver microsomes, as well as the conversion of cholesterol to 7 alpha-hydroxycholesterol - the major regulatory step in bile acid formation. Also, SCP2 is required for the intracellular transfer of cholesterol from adrenal cytoplasmic lipid inclusion droplets to mitochondria for steroid hormone production, as well as cholesterol transfer from the outer to the inner mitochondrial membrane. SCP2 is identical to the non-specific phospholipid exchange protein. While SCP2 is capable of phospholipid exchange between artificial donors/acceptors, e.g. liposomes and microsomes, it does not enhance the release of lipids other than unesterified cholesterol from natural donors/acceptors, e.g. adrenal lipid inclusion droplets, and will not enhance exchange of labeled phosphatidylcholine between lipid droplets and mitochondria. Careful comparison of SCP2 and fatty acid binding protein (FABP) using six different assay procedures demonstrates separate and distinct physiological functions for each protein, with SCP2 participating in reactions involving sterols and FABP participating in reactions involving fatty acid binding and/or transport. Furthermore, there is no overlap in substrate specificities, i.e. FABP does not possess sterol carrier protein activity and SCP2 does not specifically bind or

  13. 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.

  14. Design of potent linear alpha-melanotropin 4-10 analogues modified in positions 5 and 10.

    PubMed

    Al-Obeidi, F; Hruby, V J; Castrucci, A M; Hadley, M E

    1989-01-01

    alpha-Melanocyte stimulating hormone (alpha-MSH) is a linear tridecapeptide (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) that has diverse physiological functions in addition to its reversible darkening of amphibian skins by stimulating melanosome dispersion within melanophores. On the basis of theoretical and experimental results from our laboratory and others, we have designed a group of 1-13, 4-13, and especially 4-10 analogues related to the superpotent analogue [Nle4,D-Phe7]alpha-MSH in which the Glu5 has been replaced with Asp5, and the Gly10 has been replaced with Lys10 and other basic amino acid residues in the 4-10 analogues, and in which Gly10 and Lys11 were interchanged in the longer peptide analogues. In the 1-13 and 4-13 series the Lys10, Gly11 analogues generally retained superpotency for the D-Phe7-containing analogues. Most interestingly, synthesis of Ac-[Nle4,Xxx5,Yyy7,Zzz10]alpha-MSH4-10-NH2 analogues where Xxx = Asp or Glu, Yyy = Phe or D-Phe, and Zzz = basic amino acids (Lys, Orn, alpha,gamma-diaminobutyric acid (Dab), and alpha,beta-diaminopropionic acid (Dpr] provided melanotropins with potencies up to 10 times that of the native hormone in stimulating frog (Rana pipiens) skin darkening and 8-50 times more potent than alpha-MSH in stimulating lizard (Anolis carolinensis) skin melanophores in vitro. To our knowledge, Ac-[Nle4,Asp5,D-Phe7,Dab10]alpha-MSH4-10-NH2, the most potent analogue, is the most potent melanotropin obtained thus far for the Anolis assay system. These results provide new insights into the structural and conformational requirements for biological potency of alpha-MSH and the differential structural and conformational requirements of alpha-MSH and its analogues at two different types of pigment cell receptors. PMID:2535874

  15. Expression and function of striatal nAChRs differ in the flinders sensitive (FSL) and resistant (FRL) rat lines.

    PubMed

    Auta, J; Lecca, D; Nelson, M; Guidotti, A; Overstreet, D H; Costa, E; Javaid, J I

    2000-10-01

    Rats of Flinders Sensitive (FSL) and Flinders Resistant lines (FRL) differ in their susceptibility to physiological and associated behavioral responses elicited by nicotine. In the present study, we measured dopamine (DA) content in striatal dialysates to investigate the sensitivity of FSL and FRL rats to nicotine delivered locally through a microdialysis probe placed in the striatum. We also measured the expression density of striatal high-affinity nicotinic acetylcholine receptors (nAChRs), and that of mRNAs encoding for alpha3, alpha4, alpha7 and beta2 nAChR subunits in both lines. The DA content of dialysates was measured before and after a 1-min perfusion of nicotine (6, 10 or 20 nmoles/min) and the resulting DA increase was taken as a measure of the alkaloid's intrinsic activity for nAChRs involved in the release of DA. The nicotine-induced increase of striatal DA release was greater in FSL than in FRL rats for all concentrations of nicotine, suggesting that the intrinsic activity of nicotine was greater in the FSL than in the FRL rats. This was further supported by our finding that the density of high-affinity nAChRs in the striatum of FSL rats was 44% greater than in the FRL rats, whereas affinity (K(D)) was virtually the same in the two lines of rats. Also the expression of mRNAs encoding for alpha(4), alpha(7), and beta(2) subunits in the striatum was greater in FSL than in FRL rats (attomol/microg total RNA, alpha(4):98+/-10 vs. 77+/-7; alpha(7):279+/-16 vs. 184+/-16; beta(2):310+/-19 vs. 201+/-12). We hypothesize that the difference in nicotine-induced DA release in the striatum of FSL and FRL rats depends on the difference in nAChR subunit expression in the striatum between the two lines. The Flinders rats could be used as a model for nicotine self-administration studies to evaluate the susceptibilities of FSL and FRL rats to nicotine dependence.

  16. Atypical Renal Cysts: A Morphologic, Immunohistochemical, and Molecular Study.

    PubMed

    Matoso, Andres; Chen, Ying-Bei; Rao, Vishal; Wang, Lu; Cheng, Liang; Epstein, Jonathan I

    2016-02-01

    There is a lack of standardized nomenclature for renal cysts lined by multiple cell layers or with short papillary projections but without nests of epithelial cells within the stroma. We retrieved 29 cases (15 nephrectomies, 14 partial nephrectomies) from the surgical pathology files of Johns Hopkins Hospital from 1993 to 2014 and performed immunohistochemistry for CK7, alpha-methylacyl-CoA racemase (AMACR), CAIX, and CD10 and fluorescence in situ hybridization for trisomy 7 and 17 and 3p deletion. The mean age at excision was 58 years (range, 29 to 80 y) with 16 men and 13 women. Mean size was 2.9 cm (range, 0.3 to 10 cm). The cysts were grouped by their morphology into (1) clear cell, (2) eosinophilic stratified, and (3) eosinophilic papillary. By immunohistochemistry, 7/9 (78%) of the clear cell cases were diffusely positive for both CK7 and CAIX resembling the pattern seen in clear cell papillary renal cell carcinoma. The majority of eosinophilic stratified (4/6; 67%) and eosinophilic papillary (12/14; 86%) cases were positive for CK7 and had variable staining for AMACR, CD10, or CAIX, suggesting a differentiation more aligned with papillary renal cell carcinoma. The most common molecular alterations detected were trisomy 17 (n=6) and trisomy 7 (n=4). One case showed deletion of chromosome 3p. Clinical follow-up information was available in 23 patients; 20 were alive with no evidence of disease after a median follow-up of 20 months (range, 3 to 120 mo), 1 patient was dead due to metastatic lung cancer, 1 of sepsis, and 1 of unknown reason. Atypical renal cysts present as complex radiologic lesions, as secondary lesions in patients with a renal mass, or in a background of chronic renal disease. These atypical cysts appear heterogenous, and some follow in their morphology and immunoprofile with well-established renal tumors. The presence of 3p deletion and trisomy 7/17 suggests that in some cases they may be precursors of renal cell carcinoma. Longer follow

  17. Structural heterogeneity of the Fe(2+)-N epsilon (HisF8) bond in various hemoglobin and myoglobin derivatives probed by the Raman-active iron histidine stretching mode.

    PubMed Central

    Gilch, H.; Schweitzer-Stenner, R.; Dreybrodt, W.

    1993-01-01

    We have examined the Fe(2+)-N epsilon (HisF8) complex in hemoglobin A (HbA) by measuring the band profile of its Raman-active nu Fe-His stretching mode at pH 6.4, 7.0, and 8.0 using the 441-nm line of a HeCd laser. A line shape analysis revealed that the band can be decomposed into five different sublines at omega 1 = 195 cm-1, omega 2 = 203 cm-1, omega 3 = 212 cm-1, omega 4 = 218 cm-1, and omega 5 = 226 cm-1. To identify these to the contributions from the different subunits we have reanalyzed the nu Fe-His band of the HbA hybrids alpha(Fe)2 beta(Co)2 and alpha(Co)2 beta(Fe)2 reported earlier by Rousseau and Friedman (D. Rousseau and J. M. Friedman. 1988. In Biological Application on Raman Spectroscopy. T. G. Spiro, editor, 133-216). Moreover we have reanalyzed other Raman bands from the literature, namely the nu Fe-His band of the isolated hemoglobin subunits alpha SH- and beta SH-HbA, various hemoglobin mutants (i.e., Hb(TyrC7 alpha-->Phe), Hb(TyrC7 alpha-->His), Hb M-Boston and Hb M-Iwate), N-ethylmaleimide-des(Arg141 alpha) hemoglobin (NES-des(Arg141 alpha)HbA) and photolyzed carbonmonoxide hemoglobin (Hb*CO) measured 25 ps and 10 ns after photolysis. These molecules are known to exist in different quaternary states. All bands can be decomposed into a set of sublines exhibiting frequencies which are nearly identical to those found for deoxyhemoglobin A. Additional sublines were found to contribute to the nu Fe-His band of NES-des(Arg141 alpha) HbA and the Hb*CO species. The peak frequencies of the bands are determined by the most intensive sublines. Moreover we have measured the nu Fe-His band of deoxyHbA at 10 K in an aqueous solution and in a 80% glycerol/water mixture. Its subline composition at this temperature depends on the solvent and parallels that of more R-like hemoglobin derivatives. We have also measured the optical charge transfer band III of deoxyHbA at room temperature and found, that at least three subbands are required to fit its asymmetric

  18. Kinetic characteristics of norcocaine N-hydroxylation in mouse and human liver microsomes: involvement of CYP enzymes.

    PubMed

    Pellinen, P; Kulmala, L; Konttila, J; Auriola, S; Pasanen, M; Juvonen, R

    2000-11-01

    The first step in the oxidative metabolism of cocaine is N-demethylation to norcocaine, which is further N-hydroxylated to more toxic N-hydroxynorcocaine. In this study we examined the kinetics of norcocaine N-hydroxylation mediated by cytochrome P450 (CYP) in mouse and human liver microsomes. N-hydroxynorcocaine was identified by analytical HPLC-MS after incubation of norcocaine with mouse liver microsomes in the presence of NADPH. In mouse liver microsomes, there was no apparent difference in Km values for norcocaine N-hydroxylation between male and female microsomes, while the Vmax rate was approximately two times higher in female than in male microsomes (34+/-10 v. 16+/-4 pmol/min per mg protein). The Km value for norcocaine N-hydroxylation in human liver microsomes was approximately three times higher than that observed in comparable incubations using mouse liver microsomes, whereas the Vmax rate was ten times lower. Both cocaine and norcocaine induced type I difference spectra upon interaction with CYP in mouse liver microsomes. In contrast, in human microsomes both type I and type II spectra were recorded. In the 0.01 to 1 mM concentration range, cocaine and norcocaine inhibited mouse microsomal testosterone 6alpha-, 7alpha- and 16alpha-hydroxylation reactions by 20% to 30%. Testosterone 6beta- and 15alpha-hydroxylations were blocked by 60% and 50%, respectively, by 1 mM norcocaine, while only 40% inhibition was obtained with 1 mM cocaine. Coumarin 7-hydroxylation and pentoxyresorufin O-deethylation were inhibited by 50% by 1 and 0.4 mM norcocaine, respectively. In contrast, 10 and 2 mM cocaine, respectively, were needed to obtain the same degrees of inhibition. In human liver microsomes, 1 mM norcocaine and cocaine blocked testosterone 6beta-hydroxylase by 60% and 40%, respectively. Coumarin 7-hydroxylation was inhibited by only 30% by norcocaine (5.4 mM) and cocaine (10 mM). Norcocaine N-hydroxylation in mouse and human liver microsomes was blocked by 30

  19. A Multicenter Prospective Study to Investigate the Diagnostic Accuracy of the SeHCAT Test in Measuring Bile Acid Malabsorption: Research Protocol

    PubMed Central

    Peacock, Janet; Coker, Bola; McMillan, Viktoria; Lewis, Cornelius; Keevil, Stephen; Sherwood, Roy; Vivian, Gill; Logan, Robert; Summers, Jennifer

    2016-01-01

    Background Bile acid malabsorption (BAM) is one possible explanation for chronic diarrhea. BAM may be idiopathic, or result from ileal resection or inflammation including Crohn’s disease, or may be secondary to other conditions, including cholecystectomy, peptic ulcer surgery, and chronic pancreatitis. No “gold standard” exists for clinical diagnosis of BAM, but response to treatment with a bile acid sequestrant (BAS) is often accepted as confirmation. The SeHCAT (tauroselcholic [selenium-75] acid) test uses a radiolabeled synthetic bile acid and provides a diagnostic test for BAM, but its performance against “trial of treatment” is unknown. Fibroblast growth factor 19 (FGF-19) and 7-alpha-hydroxy-4-cholesten-3-one (C4) also offer potential new biomarkers of BAM. Objective This protocol describes a multicenter prospective study to evaluate the diagnostic accuracy of SeHCAT and 2 biomarkers in predicting BAM as assessed by trial of treatment. Methods Participating gastroenterology centers should have a minimum workload of 30 SeHCAT patients per annum. Patients should not be pregnant, on medication that could confound follow-up, or have any severe comorbidity. All eligible patients attending a gastrointestinal appointment will be invited to participate. On attending the SeHCAT test, blood and fecal samples will be collected for analysis of FGF-19 by enzyme-linked immunosorbent assay and for C4 and fractionated bile acids by liquid chromatography–mass spectrometry. A capsule containing radiolabeled SeHCAT will be administered orally and a scan performed to measure SeHCAT activity. Patients will return on day 7 to undergo a second scan to measure percentage SeHCAT retention. The test result will be concealed from clinicians and patients. BAS will be dispensed to all patients, with a follow-up gastroenterologist appointment at 2 weeks for clinical assessment of treatment response and adherence. Patients responding positively will continue treatment for a

  20. 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

  1. Kynurenic Acid Metabolism in Various Types of Brain Pathology in HIV-1 Infected Patients

    PubMed Central

    Baran, H.; Hainfellner, J.A.; Kepplinger, B.

    2012-01-01

    Kynurenic acid, an intermediate metabolite of L-kynurenine, is a competitive antagonist of inotropic excitatory amino acid (EAA) receptors as well as a non competitive antagonist of 7 alpha nicotine cholinergic receptors and its involvement in memory deficit and cognition impairment has been suggested. Alterations of kynurenic acid metabolism in the brain after HIV-1 (human immunodeficiency virus type-1) infection have been demonstrated. The present study evaluates the biosynthetic machinery of kynurenic acid e.g. the content of L-kynurenine and kynurenic acid, as well as the activity of enzymes synthesizing kynurenic acid, kynurenine aminotransferase I (KAT I) and kynurenine aminotransferase II (KAT II) in the frontal cortex and cerebellum of HIV-1 infected patients in relation to different types of pathology classified as follows: HIV in brain (HIV); opportunistic infection (OPP); infarction of brain (INF); malignant lymphoma of brain (LY); and glial dystrophy (GD) and of control (CO) subjects. Of all investigated pathologies the most frequent was OPP (65%), followed by HIV (26%), LY, INF, and GD (each 22%, respectively). Further, 68% of HIV-1 patients had bronchopneumonia, the highest incidence of which, at 60%, was seen in the OPP and LY group. Kynurenic acid was increased significantly in the frontal cortex of LY (392% of CO, P < 0.001), HIV (231% of CO, P < 0.01) and GD (193% of CO, P < 0.05), as well as in the cerebellum of GD (261% of CO, P < 0.01). A significant increase of L-kynurenine was observed in the frontal cortex of LY (385% of CO, P < 0.001) and INF (206% of CO, P < 0.01), and in the cerebellum of GD, LY, OPP and HIV (between 177% and 147% of CO). The KAT I activity increased significantly in the frontal cortex of all pathological subgroups, ie OPP = 420% > INF > LY > HIV > GD = 192% of CO. In the cerebellum, too, all pathological subgroups showed marked increase of KAT I activity (OPP = 320% > LY, HIV > GD > INF = 176% of CO). On contrary, the