Localization of testosterone and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase in cynomolgus monkey (Macaca fascicularis) testes

The enzyme 3β-hydroxysteroid dehydrogenase /Δ⁵-Δ⁴-isomerase (3β-HSD) is essential for the biosynthesis of all classes of steroid hormones, including androgens. We localized testosterone and 3β-HSD by light microscopic immunocytochemistry in the testes of adult cynomolgus monkeys. Immunoreactive testosterone was located as intense deposits in the labeled cytoplasm of Leydig cells, and located weakly in the interstitial tissues, basement membranes, and the regions near tubular walls within tubules. Immunoreactive 3β-HSD was located in the cytoplasm of all Sertoli cells and was especially intense in the parts near tubular walls and located weakly to intensely in the cytoplasm of some Leydig cells. This is the first immunocytochemical evidence that Sertoli cells of cynomolgus monkeys, as well as Leydig cells, are involved in biosynthesis of androgens.     

Dehydroepiandrosterone 7α-hydroxylation in human tissues: Possible interference with type 1 11β-hydroxysteroid dehydrogenase-mediated processes

Dehydroepiandrosterone (DHEA) is 7α-hydroxylated by the cytochome P450 7B1 (CYP7B1) in the human brain and liver. This produces 7α-hydroxy-DHEA that is a substrate for 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which exists in the same tissues and carries out the inter-conversion of 7α- and 7β-hydroxy-DHEA through a 7-oxo-intermediary. Since the role of 11β-HSD1 is to transform the inactive cortisone into active cortisol, its competitive inhibition by 7α-hydroxy-DHEA may support the paradigm of native anti-glucocorticoid arising from DHEA. Therefore, our objective was to use human tissues to assess the presences of both CYP7B1 and 11β-HSD1. Human skin was selected then and used to test its ability to produce 7α-hydroxy-DHEA, and to test the interference of 7α- and 7β-hydroxy-DHEA and 7-oxo-DHEA with the 11β-HSD1-mediated oxidoreduction of cortisol and cortisone. Immuno-histochemical studies showed the presence of both CYP7B1 and 11β-HSD1 in the liver, skin and tonsils. DHEA was readily 7α-hydroxylated when incubated using skin slices. A S9 fraction of dermal homogenates containing the 11β-HSD1 carried out the oxidoreduction of cortisol and cortisone. Inhibition of the cortisol oxidation by 7α-hydroxy-DHEA and 7β-hydroxy-DHEA was competitive with a K_i at 1.85 ± 0.495 and 0.255 ± 0.005 μM, respectively. Inhibition of cortisone reduction by 7-oxo-DHEA was of a mixed type with a K_i at 1.13 ± 0.15 μM. These findings may support the previously proposed native anti-glucocorticoid paradigm and suggest that the 7α-hydroxy-DHEA production is a key for the fine tuning of glucocorticoid levels in tissues.     

Selective inhibition of 11β-hydroxysteroid dehydrogenase 1 by 18α-glycyrrhetinic acid but not 18β-glycyrrhetinic acid

Elevated cortisol concentrations have been associated with metabolic diseases such as diabetes type 2 and obesity. 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1, catalyzing the conversion of inactive 11-ketoglucocorticoids into their active 11β-hydroxy forms, plays an important role in the regulation of cortisol levels within specific tissues. The selective inhibition of 11β-HSD1 is currently considered as promising therapeutic strategy for the treatment of metabolic diseases. In recent years, natural compound-derived drug design has gained considerable interest. 18β-glycyrrhetinic acid (GA), a metabolite of the natural product glycyrrhizin, is not selective and inhibits both 11β-HSD1 and 11β-HSD2. Here, we compare the biological activity of 18β-GA and its diastereomer 18α-GA against the two enzymes in lysates of transfected HEK-293 cells and show that 18α-GA selectively inhibits 11β-HSD1 but not 11β-HSD2. This is in contrast to 18β-GA, which preferentially inhibits 11β-HSD2. Using a pharmacophore model based on the crystal structure of the GA-derivative carbenoxolone in complex with human 11β-HSD1, we provide an explanation for the differences in the activities of 18α-GA and 18β-GA. This model will be used to design novel selective derivatives of GA.     

Activin-A, but not inhibin, regulates 17β-hydroxysteroid dehydrogenase type 1 activity and expression in cultured rat granulosa cells

17β-Hydroxysteroid dehydrogenase type 1 (17HSD type 1) catalyzes the reduction of estrone (E₁) to biologically more active estradiol (E₂). In the present study, the effect of activin, inhibin, and follistatin on 17HSD activity and 17HSD type 1 expression in cultured, unluteinized rat granulosa cells was examined. Furthermore, the effects of these hormones on 17HSD type 1 expression were compared with the expression of P450 aromatase (P450arom). Rat granulosa cells were pre-incubated in serum-free media for 3 days, followed by a 2-day treatment with activin, inhibin, follistatin and 8-Br-cAMP. Activin in increasing concentrations appeared to effect a dose-dependent increase in 17HSD activity. In addition, increasing concentrations of activin also increased 17HSD type 1 mRNA expression. Addition of 8-Br-cAMP at concentrations of 0.25 and 1.5 mmol/l together with activin significantly augmented the stimulatory effects of activin alone in the cultured cells. Neither inhibin, nor follistatin, either alone or in combination with 8-Br-cAMP, had any notable effects on 17HSD activity and 17HSD type 1 expression. Preincubation of activin with increasing concentrations of follistatin significantly diminished the stimulatory effect of activin. In the presence of follistatin, activin did not significantly increase the 8-Br-cAMP-induced 17HSD activity and 17HSD type 1 expression. The culturing of granulosa cells in the presence or the absence of inhibin or follistatin with or without 8-Br-cAMP did not alter the effect of these peptides on P450arom expression in rat granulosa cells as judged by Northern blot analysis of total RNA. However, cAMP-induced P450arom expression was enhanced by activin treatment, except when follistatin was present. This is in line with the suggested role of follistatin as an activin-binding protein, which limits the bioavailability of activin to its membrane receptors. Thus, the results support the notion of a paracrine/autocrine role of activin in follicular steroidogenesis of growing follicles.     

Expression and characterization of the human 3β-hydroxysteroid sulfotransferases (SULT2B1a and SULT2B1b)

The human hydroxysteroid sulfotransferase, dehydroepiandrosterone sulfotransferase (DHEA-ST), is highly expressed in liver and adrenal cortex and displays reactivity towards a broad range of hydroxysteroids including 3β-hydroxysteroids, 3-hydroxysteroids, estrogens with a 3-phenolic moiety, and 17-hydroxyl group of androgens. In contrast, characterization of the newly described human hydroxysteroid sulfotransferase SULT2B1 isoforms shows that these enzymes are selective for the sulfation of 3β-hydroxysteroids, such as pregnenolone, epiandrosterone, DHEA, and androstenediol. There was no activity detected towards testosterone, dexamethasone, β-estradiol, androsterone, or p-nitrophenol. The SULT2B1 gene encodes two isoforms, SULT2B1a and SULT2B1b, which are generated by alternate splicing of the first exon; therefore the SULT2B1 isoforms differ at their N-terminals. Northern Blot analysis detected a SULT2B1 message in RNA isolated from the human prostate and placenta. No SULT2B1 message was observed in RNA isolated from human liver, colon, lung, kidney, brain, or testis tissue. Purified SULT2B1a was used to generate a specific rabbit polyclonal anti-SULT2B1 antibody. The anti-SULT2B1 antibody did not react with expressed human EST, P-PST-1, M-PST, DHEA-ST, or ST1B2, during immunoblot analysis. The substrate specificity of the expressed SULT2B1 isoforms suggests that these enzymes are capable of regulating the activity of adrenal androgens in human tissues via their inactivation by sulfation.     

Cloning of a cDNA Coding for Active Tyrosine Hydroxylase in the Rainbow Trout (Oncorhynchus mykiss): Comparison with Other Hydroxylases and Enzymatic Expression

Abstract: Although catecholamines are of critical importance for neuroendocrine function in teleost fishes, there has been no tool to give access to pretranslational regulation of their synthesis enzymes. In this study, we undertook cloning of the cDNA coding for the tyrosine hydroxylase (TH) of the rainbow trout ( Oncorhynchus mykiss ). First, we looked for a tissue sufficiently rich in TH to make an expression library. The cDNA coding for the rainbow trout TH (rtTH) was then cloned and sequenced. The rtTH sequence encodes a protein of 489 amino acids. Several domains and amino acids required for enzyme activity, like cysteines or phosphorylation sites, are highly conserved between species. Northern blot analysis showed a single rtTH messenger RNA of 4.2 kb expressed in the anteroventral brain. The ability of rtTH to hydroxylate l -tyrosine was analyzed by transient expression of the rtTH cDNA in COS-1 cells. In vitro TH activity, using COS-1 cell extracts, demonstrated that TH activity in transfected cells was 40-fold higher than in untransfected cells. Western blot analysis revealed a single protein of ∼65 kDa in both COS-1 cells and in trout brain. This rtTH cDNA provides us with a tool for further studies on pretranslational regulation of the enzyme in salmonids.     

Immunostimulatory CpG oligodeoxynucleotides stimulate expression of IL-1β and interferon-like cytokines in rainbow trout macrophages via a chloroquine-sensitive mechanism

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs are known to stimulate immune responses and are potent adjuvants in higher vertebrates, but so far the effects in fish are poorly described. We here report that CpG ODNs induce IL-1β expression and production of interferon-like cytokines in rainbow trout head-kidney macrophages, whereas ODNs with an inverted motif (GpC) have a much less stimulatory effect. We further demonstrate that endosomal maturation is essential for CpG signalling, as chloroquine, a compound known to block endosomal acidification, inhibits cytokine expression in the macrophages.     

Biotransformations of α,β-Epoxyalcohols Catalyzed by Epoxide Hydrolases

Microsomal and cytosolic fractions of mammalian livers were screened for their capacity to resolve racemic mixtures of trans -2,3-epoxy-l-alkanols. The epoxide hydrolase activities showed some specificity for the 2S, 3S enantiomers which were attacked at the proximate carbon atom. The best resolutions were observed with guinea pig liver microsomal enzymes.     

Enzymic properties of β-mannanase from Polyporus versicolor

Substrate specificities and the kinetic parameters, K_m and V_max, of the four multiple enzyme forms of extracellular β-mannanase activity purified from Polyporus versicolor were determined. Although K_m values were significantly greater than those encountered in other β-mannanase systems V_max values were equivalent or much greater, rendering the physiological efficiencies of the β-mannanase comparable to those of other β-mannanases. All enzymes preferred glucomannan as substrate, were highly refractory at low concentrations to n-octylglucopyranoside, sodium deoxylcholate, and sodium dodecylsulfate, and were largely insensitive to methanol, ethanol, acetonitrile, and dimethylsulfoxide.     

Cloning and Functional Expression of the Mucrosobin Protein, a β-Fibrinogenase of Trimeresurus mucrosquamatus (Taiwan Habu)

A venom-specific cDNA encoding for a thrombin-like enzyme designated as mucrosobin has been cloned and sequenced from the cDNA library of the venomous gland of Trimeresurus mucrosquamatus. The full-length cDNA of mucrosobin was assembled by oligonucleotide screening and 5′-rapid amplification of cDNA ends. The amino acid sequence deduced from the cDNA consists of 257 amino acid residues with a putative signal peptide of 24 residues. It is highly homologous to the other thrombin-like enzymes (batroxobin, mucofirase, and calobin), suggesting that it is a serine proteinase with a conserved catalytic triad of His⁴¹, Asp⁸⁴ and Ser¹⁷⁹ in the deduced form of mucrosobin protein. Northern blot analysis revealed that the mucrosobin gene encodes an mRNA of 1.5 kb and suggested a tissue-specific expression in the venomous gland. In an effort to study the biological property of mocrosobin, we have expressed the 28-kDa protein as inclusion bodies in Escherichia coli. For analyzing enzymatic activity, the inclusion bodies were solubilized and the recombinant protein was refolded with a two-step dialysis protocol. The refolded recombinant protein exhibited a specific β-fibrinogenolytic activity. This study offers a possibility of using genetic engineering to acquirie a functional snake venom protein with therapeutic potential.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Temporal Changes of Acid Phosphatase, Arylsulphatase, β-Galactosidase and β-N-ACETYL-d-Glucosaminidase Activities in Subcellular Fractions of Rat Liver

In this paper circadian changes in the liver enzyme activities of rat housed under highly standardized conditions with 12:12 hour light-dark cycle are shown. Activities of acid phosphatase, arylsulphatase, β-galactosidase and β-N-acetyl-d-glucosaminidase in microsomal and lysosomal fractions and crude homogenate were estimated every 4 hr during one 24-hr period. The enzyme activities were related to 1 mg of protein, 1 mg of DNA and 1 g fresh tissue. Daily changes of enzyme activities were found. In case of activity calculated per 1 mg DNA two maxima at 0500 and at 2100 hr were observed, while activity calculated per 1 mg protein showed one maximum at 0500 hr. Activity calculated per 1 g fresh tissue showed the maximum at 0500 hr for each enzyme only in microsomal fraction. As far as acrophase table is concerned for all enzymes and fractions the acrophase occurred during the night. The obtained results are discussed in relation to lysosomal enzymes synthesis process as well as different reference values.     

Purification and properties of the β-fructofuranosidase from Kluyveromyces fragilis

The β-fructofuranosidase from Kluyveromyces fragilis was purified to one band on electrophoresis by 3 different methods. Two of the preparations were found to be impure by isoelectric focusing. This demonstrates the need for more than one criteria of homogeneity when purifying this enzyme. The enzyme was found to be a glycoprotein, stable at 50°C, with a pH optimum of 4.5. The cations Hg²⁺, Ag⁺, Cu²⁺ and Cd²⁺ exhibited a marked inhibition of the enzyme. Competitive inhibition was observed with the fructose analog 2,5-anhydro-D-mannitol suggesting that the enzyme is inhibited by the furanose form of fructose.     

Rheological properties of barley β-glucan

Health benefits of cereal β-glucan are linked to its high viscosity. Although viscosity of β-glucan gum solutions has been reported previously, there are conflicting reports about its behavior at elevated temperatures. Therefore, the viscosity behavior of barley β-glucan gum obtained in a pilot plant (PP) or in a laboratory (LAB) was determined at different shear rates (1.29–129 s⁻¹) and temperatures (0.1–75 °C) in this study. Viscosity decrease with temperature was demonstrated for both gums and activation energy E_a was calculated from the Arrhenius equation. None of the fresh gum solutions exhibited thixotropic behavior at ≤1% (w/w) concentration, but the measurement demonstrated that increased shear rate is not applicable to polymer solutions of low viscosity. Information about rheological properties of β-glucan will lead to better understanding of its behavior under physiological and processing conditions.     

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.     

Production, purification and properties of γ-glutamyltranspeptidase from a newly isolated Bacillus subtilis NX-2

Production, purification and properties of γ-glutamyltranspeptidase from a newly isolated Bacillus subtilis NX-2 was investigated. At the optimum conditions for enzyme formation, a high level, 3.2 U/ml of γ-GTP was obtained. The extracellular γ-GTP from this strain was purified 111.15-fold to homogeneity from the culture supernatant by acetone precipitation, hydrophobic interaction chromatography and ion exchange chromatography. The purified enzyme was a heterodimer consisting of one large subunit (43 kDa) and one small subunit (32 kDa), and exhibited high activity at 40–60 °C, pH 8.0. It preferred basic amino acids as γ-glutamyl acceptor in transpeptidation, and the stereochemistry of the γ-glutamyl acceptor had no influence on the enzyme activity, which was different from other γ-GTPs reported. Furthermore, it was proved that γ-GTP of this strain could catalyze the transfer of l-glutamine to glycylglycine to synthesize Gln–Gly–Gly, which was promising for the synthesis of valuable γ-glutamyl peptides.