Synergistic induction of rat hepatic ornithine decarboxylase by multiple doses of cobalt chloride

The level of rat hepatic ornithine decarboxylase (ODC) induced by repetitive administration of Co2+ was determined by affinity labeling with [3H]difluoromethylornithine. Such a treatment with Co2+ ion induced ODC level to a 10-fold greater extent than single dose of the metal ion or well-known inducers of the enzyme, such as thioacetamide or carbon tetrachloride. The half life of ODC activity induced by repetitive treatment with Co2+ (95 min) was substantially increased to about 10-fold over the value obtained from the enzyme induced by single treatment with the metal ion (10 min). ODC activity induced by repetitive treatment with Co2+ was separated into two peaks by DEAE-Sepharose column chromatography. The two independently collected fractions of ODC peaks exhibited different affinity for pyridoxal 5'-phosphate in vitro and sensitivity to cycloheximide in vivo.     

Aspartate aminotransferase from Panicum maximum Jacq. var. trichoglume Eyles, a C4 plant: purification, molecular properties, and preparation of antibody

Extracts of the leaf tissue of Panicum maximum Jacq. var. trichoglume Eyles (a phosphoenolpyruvate carboxykinase type of C4 plant) were examined and at least two isoforms of aspartate aminotransferase (EC 2.6.1.1), with different electrophoretic mobilities, were detected. The predominant isoform was purified to homogeneity from mesophyll cells. The purification procedure included fractionation with ammonium sulfate followed by chromatography on diethylaminoethyl-cellulose, Sephacryl S-300, and hydroxyapatite. The purified enzyme had specific activities of 182 and 165 mumol/min/mg protein, measured in terms of the synthesis of oxaloacetate and aspartate, respectively, at pH 8.0. The enzyme, with an apparent molecular size of 100 kDa, appears to be a dimer of a single polypeptide with a molecular size of 42 kDa. Mono specific polyclonal antibodies were raised against the 42-kDa polypeptide. Only a single stained band was detected in extracts of whole leaves by immunoblot analysis with this antibody after two-dimensional polyacrylamide electrophoresis. Furthermore, no difference in mobility was observed between the enzymes extracted from mesophyll and bundle sheath cells on native polyacrylamide gels. These findings are discussed in relation to the other isoform in the leaves of this species.     

19-Hydroxy-4-androsten-17-one: potential competitive inhibitor of estrogen biosynthesis

3-Deoxy steroids having a 4-ene system were found to be competitive inhibitors of human placental aromatase. 19-Hydroxy-4-androsten-17-one (2) potently inhibits the enzyme with an apparent Ki of 12.5 nM, but does not produce a time-dependent inactivation of the enzyme.     

Comparative studies of the effects of stilbene compounds on hepatic ornithine decarboxylase and S-adenosylmethionine decarboxylase induction in rats

Trans-Stilbene oxide (TSO, 2 mmol/kg, ip.) induced ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) to 60-fold and 5-fold of the controls, respectively, in the liver of rats. Parallel to ODC induction, there was a marked increase in putrescine content to 50-fold of the control levels. Cis-Stilbene oxide (CSO), a stereoisomer of TSO, also produced the induction of ODC and SAMDC and the increase in putrescine content. There was no difference in the ability to induce ODC and SAMDC between TSO and CSO with respect to the extents of induction and the time needed to reach maximal levels. Trans-Stilbene (TS), a mother compound of TSO, did not show such an effect on ODC, while cis-stilbene (CS) induced both ODC and SAMDC. Treatment with glutathione inhibited TSO- and CSO-mediated induction of ODC and SAMDC. These findings add new information concerning the abilities of TSO, CSO and CS on hepatic polyamine metabolism.     

Synthesis of 3 beta,16 beta,19-trihydroxyandrost-5-en-17-one and 16 beta,19-dihydroxyandrost-4-ene-3,17-dione and their 19-oxo derivatives

3 beta,16 beta,19-Trihydroxyandrost-5-en-17-one (12) was synthesized from 5 alpha-bromo-3 beta-acetoxy-6 beta,19-epoxyandrostan-17-one (2) through acetoxylation at C-16 beta of the enol acetate 4 with lead tetraacetate and reductive cleavage of the epoxide ring with zinc dust yielding the 3 beta,16 beta-diacetoxy-19-hydroxy steroid 11, followed by hydrolysis of the acetoxy groups with sulfuric acid. Jones oxidation of compound 11 followed by the acid hydrolysis gave the 19-oxo steroid 15. 5 alpha-Bromo-3 beta-hydroxy-16 beta-acetoxy-6 beta,19-epoxyandrostan-17-one (8), obtained by selective hydrolysis of the 3-formate 5 with ammonium hydroxide, was oxidized with Jones reagent to afford the 3-oxo steroid 16, which was converted into the 19-hydroxy derivative 17 by treatment with zinc dust. 16 beta,19-Dihydroxyandrost-4-ene-3,17-dione (18) and its 19-oxo derivative 21 were obtained from compound 17 through a similar reaction sequence.     

Improved synthesis of 16alpha-hydroxylated androgens: intermediates of estriol formation in pregnancy.

16alpha-Hydroxyandrostenedione (16alpha-hydroxyandrost-4-ene-3,17-dione), 16alpha-hydroxytestosterone (16alpha,17beta-dihydroxyandrost-4-en-3-one) and 16alpha-hydroxydehydroepiandrosterone 3-sulfate (3beta, 16alpha-dihydroxyandrost-5-en-17-one 3-monosulfate) were synthesized by a new chemical approach with much improved yield. 16alpha-Bromoandrostendione was converted to the hydrazone of 16alpha-hydroxyandrostenedione which gave 16alpha-hydroxyandrostenedione on acid hydrolysis in total 63% yield. Oxidation of 16alpha-hydroxydehydroepiandrosterone with Jones' reagent also selectively afforded 16alpha-hydroxyandrostenedione. 16alpha-Hydroxytestosterone was observed by selective reduction of 16alpha-hydroxyandrostenedione with sodium borohydride. Reaction of 16alpha-hydroxydehydroepiandrosterone with chlorosulfonic acid in pyridine selectively gave the 3-monosulfate. The structure of the sulfate was deduced from its solvolysis to the starting material, and its acetylation and subsequent solvolysis to 16alpha-hydroxydehydroepiandrosterone 16-acetate. All procedures are suitable for large scale synthesis without the use of microorganisms.     

Inhibitors of sterol synthesis. Characterization of trimethylsilyl dienol ethers of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one. Applications in the analysis of mitochondrial metabolites of the 15-ketosterol by gas chromatography-mass spectrometry.

Derivatization of delta 8(14)-15-ketosterols as bis-TMS dienol ethers facilitates their analysis by gas chromatography-mass spectrometry (GC-MS). Conditions are presented for the preparation of each of the three possible bis-TMS dienol ethers of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (1), a potent regulator of cholesterol metabolism. Treatment of 1 with N,O-bis(tri-methylsilyl)-trifluoroacetamide (BSTFA) and pyridine for 20 h at 100 degrees C produced primarily 3 beta,15-bis(trimethylsilyloxy)-5 alpha-cholesta-7,14-diene. Treatment of 1 with BSTFA-pyridine in the presence of 0.1% perchloric acid for 20 h at 22 degrees C gave mainly the delta 8(14)15, dienol ether. Heating this reaction mixture at 100 degrees C for 4 days gave mainly the delta 8,14 ether. The structures of the three dienol ethers were established by GC-MS and 1H and 13C nuclear magnetic resonance spectroscopy. The three TMS dienol ethers of 1 were resolved by capillary GC and gave very simple mass spectra upon electron impact with fragmentation limited almost exclusively to the elimination of trimethylsilanol, methyl, and the side chain. The TMS dienol ethers showed reduced artefact formation, improved chromatographic resolution, and increased sensitivity relative to the delta 8(14)-15-ketosterols, properties that improve the detection and identification of minor components in analyses of complex biological mixtures. The utility of this derivatization is illustrated for the delta 7,14 TMS dienol ethers of the 3-deoxy, 3-keto, 3 alpha-hydroxy, and 3 beta-methoxy analogs of 1 and for delta 8(14)-15-ketosterols in mixtures obtained from incubations of 1 with rat liver mitochondria in the presence of NADPH.     

Controlled alkaline hydrolysis of steroidal α-bromoketones: New conditions and synthesis of 2α-hydroxy-3-ones

Controlled alkaline hydrolysis of 16α-bromo-17-keto steroids $\text{1}$, $\text{5}$ and $\text{7}$ with potassium carbonate and tetra-n-butylammonium hydroxide (n-Bu₄NOH) and synthesis of 2α-hydroxy-3-ones $\text{11}$, $\text{13}$ and $\text{16}$ by the controlled hydrolysis of the corresponding 2α-bromo-3-ones $\text{9}$, $\text{12}$ and $\text{15}$ are described. Treatm carbonate in aqueous acetone or with n-Bu₄NOH in aqueous dimethylformamide (DMF) gave 16α-hydroxy-17-ones $\text{3}$, $\text{6}$ and $\text{8}$ in 85–90% yield, respectively. 2α-Hydroxy-3-ones $\text{11}$, $\text{13}$ and $\text{16}$ were obtained by hydrolysis of the corresponding bromoketones $\text{9}$, $\text{12}$ and $\text{15}$ in high yields using the above conditions or sodium hydroxide in pyridine or DMF, respectively. Deuterium labeling experiments suggested that equilibration between the 2α-bromoketone $\text{9}$ and the 2β-bromo isomer $\text{10}$ precedes the formation of the ketol $\text{11}$ in which the true intermediate might be the 2β-isomer $\text{10}$. However, rearranged androstane derivatives, 3β-hydroxy-2-ones $\text{18}$ and $\text{20}$, were stereoselectively obtained by treatment of the bromoketones $\text{12}$ and $\text{15}$ with an excess amount of sodium hydroxide.     

Biological aromatization of delta4,6- and delta1,4,6-androgens and their 6-alkyl analogs, potent inhibitors of aromatase

Enzymic aromatization of Δ⁶- and Δ^1,6-derivatives of the natural substrate androstenedione with human placental aromatase was first studied using gas-chromatography-mass spectrometry. The two steroids were aromatized with apparent K_m and V_max values of 62 nM and 32 pmol/min/mg protein for the Δ⁶-steroid and 167 nM and 10 pmol/min/mg protein for the Δ^1,6-steroid, respectively. We next explored the aromatization of a series of 6-alkyl (methyl, ethyl, n-propyl, and n-pentyl)-substituted Δ⁶-androstenediones and their Δ^1,6-analogs, potent competitive inhibitors of aromatase, to gain insight into the relationships between the inhibitory activity of the 6-alkyl-C₁₉ steroids and their ability to serve as a substrate of aromatase. In a series of the Δ^1,6-androstenediones, all the 6-alkyl steroids were more efficient substrates than the parent Δ^1,6-steroid in which the aromatization rates of the alkyl steroids were about 2-fold that of the parent steroid, in contrast, all of the 6-alkyl-substituted Δ⁶-androstenediones were converted into the corresponding 6-alkyl-Δ⁶-estrogens with the rates of less than about a half that of the parent steroid. These results indicate that the 6-alkyl function decreases the aromatization rate of the Δ⁶-steroid but enhances that of the Δ^1,6-steroid. The relative apparent K_m values for the C₁₉ steroids obtained in this study are different from the relative K_i values obtained previously, indicating that a good inhibitor is not essentially a good substrate in the 6-alkyl-substituted Δ⁶- and Δ^1,6-androstenedione series.     

Influence of interleukin-12 receptor β1 polymorphisms on tuberculosis

Host genetic factors may be important determinants of susceptibility to tuberculosis, and several candidate gene polymorphisms have been shown to date. A series of recent reports concerning rare human deficiencies in the type-1 cytokine pathway suggest that more subtle variants of relevant genes may also contribute to susceptibility to tuberculosis at the general population level. To investigate whether polymorphisms in the interleukin-12 receptor (IL-12R) gene predispose individuals to tuberculosis, we studied these genes by single-strand conformational polymorphism analysis and direct sequencing. Although no common polymorphisms could be identified in the IL-12R beta 2 gene ( IL-12RB2), we confirmed four single nucleotide polymorphisms (SNPs; 641A-->G, 684C-->T, 1094T-->C, and 1132G-->C) causing three missense variants (Q214R, M365T, G378R) and one synonymous substitution in the extracellular domain of the IL-12R beta 1 gene ( IL12RB1). All SNPs were in almost perfect linkage disequilibrium (D'=0.98), and two common haplotypes of IL12RB1(allele 1: Q214-M365-G378; allele 2: R214-T365-R378) were revealed. Polymerase chain reaction/restriction fragment length polymorphism and sequence analyses were used to type IL12RB1polymorphisms in 98 patients with tuberculosis and 197 healthy controls in Japanese populations. In our case-control association study of tuberculosis, the R214-T365-R378 allele (allele 2) was over-represented in patients with tuberculosis, and homozygosity for R214-T365-R378 (the 2/2 genotype) was significantly associated with tuberculosis (odds ratio: 2.45; 95% CI: 1.20-4.99; P=0.013). In healthy subjects, homozygotes for R214-T365-R378 had lower levels of IL-12-induced signaling, according to differences in cellular responses to IL-12 between two haplotypes. These data suggest that the R214-T365-R378 allele, i.e., variation in IL12RB1, contribute to tuberculosis susceptibility in the Japanese population. This genetic variation may predispose individuals to tuberculosis infection by diminishing receptor responsiveness to IL-12 and to IL-23, leading to partial dysfunction of interferon-gamma-mediated immunity.