Characterisation of the beta-tubulin gene of Cylicocyclus nassatus?

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

Regulation of sex steroid formation by interleukin-4 and interleukin-6 in breast cancer cells

Sex steroids play a predominant role in the development and differentiation of normal mammary gland as well as in the regulation of hormone-sensitive breast cancer growth. There is evidence suggesting that local intracrine formation of sex steroids from inactive precursors secreted by the adrenals namely, dehydroepiandrosterone (DHEA) and 4-androstenedione (4-dione) play an important role in the regulation of growth and function of peripheral target tissues, including the breast. Moreover, human breast carcinomas are often infiltrated by stromal/immune cells secreting a wide spectra of cytokines. These might in turn regulate the activity of both immune and neoplastic cells. The present study was designed to examine the action of cytokines on 17β-hydroxysteroid dehydrogenase (17β-HSD) and 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) activities in human breast cancer cells. The various types of human 17β-HSD (five types) and 3β-HSD (two types), because of their tissue- and cell-specific expression and substrate specificity, provide each cell with necessary mechanisms to control the level of intracellular active androgens and estrogens. We first investigated the effect of exposure to IL-4 and IL-6 on reductive and oxidative 17β-HSD activities in both intact ZR-75-1 and T-47D human breast cancer cells. In ZR-75-1 cells, a 6 d exposure to IL-4 and IL-6 decreased E₂-induced cell proliferation, the half maximal inhibitory effect being exerted at 88 and 26 pM, respectively. In parallel, incubation with IL-4 and IL-6 increased oxidative 17β-HSD activity by 4.4- and 1.9-fold, respectively, this potent activity being observed at ₅₀ values of 22.8 and 11.3 pM, respectively. Simultaneously, reductive 17β-HSD activity leading to E₂ formation was decreased by 70 and 40% by IL-4 and IL-6, respectively. Moreover, IL-4 and IL-6 exerted the same regulatory effects on 17β-HSD activities when testosterone and 4-dione were used as substrates, thus strongly suggesting the expression of the type 2 17β-HSD ZR-75-1 cells. In contrast, in T-47D cells, IL-4 increased the formation of E₂, whereas IL-6 exerts no effect on this parameter. However, we found that T-47D cells failed to convert testosterone efficiently into 4-DIONE, thus suggesting that there is little or no expression of type 2 17β-HSD in this cell line. The present findings demonstrate that the potent regulatory effects of IL-4 and IL-6 on 17β-HSD activities depend on the cell-specific gene expression of various types of 17β-HSD enzymes. We have also studied the effect of cytokines on the regulation of the 3β-HSD expression in both ZR-75-1 and T-47D human breast cancer cells. Under basal culture conditions, there is no 3β-HSD activity detectable in these cells. However, exposure to IL-4 caused a rapid and potent induction of 3β-HSD activity, whereas IL-6 failed to induce 3β-HSD expression. Our data thus demonstrate that cytokines may play a crucial role in sex steroid biosynthesis from inactive adrenal precursors in human breast cancer cells.     

Two non-reactive ternary complexes of estrogenic 17beta-hydroxysteroid dehydrogenase: crystallization and preliminary structural analysis.

Human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1, EC1.1.1.62) is an important enzyme that catalyses the last step of active estrogen formation. 17β-HSD1 plays a key role in the proliferation of breast cancer cells. The three-dimensional structures of this enzyme and of the enzyme-estradiol complex have been solved (Zhu et al., 1993, J. Mol. Biol. 234:242; Ghosh et al., 1995, Structure 3:503; Azzi et al., 1996, Nature Struct. Biol. 3:665). The determination of the non-reactive ternary complex structure, which could mimic the transition state, constitutes a further critical step toward the rational design of inhibitors for this enzyme (Ghosh et al. 1995, Structure 3:503; Penning, 1996, Endocrine-Related Cancer, 3:41).

To further study the transition state, two non-reactive ternary complexes, 17β-HSD1–EM519-NADP⁺ and 17β-HSD1–EM553-NADP⁺ were crystallized using combined methods of soaking and co-crystallization. Although they belong to the same C2 space group, they have different unit cells, with a=155.59 Å, b=42.82 Å, c=121.15 Å, β=128.5° for 17β-HSD1–EM519-NADP⁺, and a=124.01 Å, b=45.16 Å, c=61.40 Å, β=99.2° for 17β-HSD1–EM553-NADP⁺, respectively. Our preliminary results revealed that the inhibitors interact differently with the enzyme than do the natural substrates.     

The role of 11β-hydroxysteroid dehydrogenase in steroid hormone specificity

11β-hydroxysteroid dehydrogenase (11β-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. In aldosterone target cells the type 2 11β-HSD is present, which, in contrast to the type 1 11β-HSD, has very high affinity for its substrate, is unidirectional and prefers NAD as cofactor. cDNAs encoding 11β-HSD2 have been recently cloned from different species, and the cell-specific expression of its mRNA and protein were determined. 11β-HSD2 is expressed in every aldosterone target tissue. Northern analysis revealed that the rabbit 11β-HSD2 is expressed at high levels in the renal collecting duct and at much lower levels in the colon. RT-PCR experiments demonstrated that 11β-HSD2 mRNA is present only in aldosterone target cells within the kidney. We determined the subcellular localization of the rabbit 11β-HSD2 using a chimera encoding 11β-HSD2 and the green fluorescent protein (GFP). This construct was stably transfected into CHO and MDCK cells. The expressed 11β-HSD2/GFP protein retained high enzymatic activity, and its characteristics were undistinguishable from those of the native enzyme. The intracellular localization of this protein was determined by fluorescence microscopy. 11β-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Co-staining with markers for ER proteins, the Golgi membrane, mitochondria and nucleus confirmed that 11β-HSD2 is localized exclusively to the ER. To determine what structural motifs are responsible for the ER localization, we generated deletion mutants missing the C-terminal 42 and 118 amino acids, and fused them to GFP. Similarly as with the intact 11β-HSD2, these mutants localized exclusively to the ER. Both C-terminal deletion mutants completely lost dehydrogenase activity, independently whether activity was determined in intact cells or homogenates. These results indicate that 11β-HSD2 has a novel ER retrieval signal which is not localized to the C-terminal region. In addition, the C-terminal 118 amino acids are essential for NAD-dependent 11β-HSD activity.     

Immunoassay of 7-hydroxysteroids: 1. Radioimmunoassay of 7beta-hydroxy dehydroepiandrosterone

High sensitivity radioimmunoassay of 3β,7β-dihydroxy-5-androsten-17-one (7β-OH-DHEA) has been developed and evaluated. The method is based on polyclonal rabbit antisera raised against its 19-O-(carboxymethyl)oxime bovine serum albumin conjugate and bridge- and position homologous [¹²⁵I]iodotyrosine methyl ester as a tracer. Alternatively, [³H]tracer has been prepared, which was recognized by the antiserum as well, but the assay sensitivity was lower. The identity of measured immunoreactive material was confirmed by high performance liquid chromatography which separated 7β-OH-DHEA from its 7-isomer. Using radioiodinated tracer, the sensitivity of the method was 3.48 fmol (1.06 pg) per tube, the mean recovery of standard added to steroid-free serum was 98.5%. Free (unconjugated and not-esterified) 7β-OH-DHEA amounted in average 5.8% of the total 7β-OH-DHEA present in human serum. It was measured in 42 normal subjects (28 females and 14 males) and in 92 randomly selected patients with various endocrinopathies. The mean values±SD in normals were 2.05±1.02 nmol l⁻¹, the broad range of values from undetectable levels to 10.3 nmol l⁻¹ was found in the patients. Serum levels of free 7β-OH-DHEA in the patients significantly correlated with DHEA and its sulfate.     

Tissue- and temporal-specific regulation of 11beta-hydroxysteroid dehydrogenase type 1 by glucocorticoids in vivo.

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) catalyses the interconversion of active corticosterone and inert 11-dehydrocorticosterone. Short-term glucocorticoid excess upregulates 11β-HSD-1 in liver and hippocampus leading to suggestions that 11β-HSD-1 ameliorates the deleterious effects of glucocorticoid excess by its 11β-dehydrogenase activity. However the predominant activity of 11β-HSD-1 in vivo is 11β-reduction, thus generating active glucocorticoid. We have re-examined the time-course of glucocorticoid regulation of 11β-HSD-1 in the liver, hippocampus and kidney of adult male rats in vivo.

Sham operation markedly reduced 11β-HSD-1 mRNA expression in all tissues, and reduced 11β-HSD bioactivity in liver and hippocampus when compared to untouched controls. Adrenalectomy reduced 11β-HSD-1 expression in all tissues in the short-term (7 days), followed by subsequent recovery of enzyme activity by 21 days in liver and hippocampus. Dexamethasone replacement of adrenalectomised rats attenuated the initial decrease in hepatic 11β-HSD-1 activity, but by 21 days dexamethasone reduced activity compared to control levels.

Thus glucocorticoids regulate 11β-HSD-1 in a complex tissue- and temporal-specific manner. This pattern of regulation suggests glucocorticoids repress 11β-HSD-1 at least in the liver, a pattern of regulation more consistent with the evidence that 11β-HSD-1 is an 11β-reductase in vivo. Operational stress per se down-regulates 11β-HSD-1 which has implications for interpretation and design of in vivo studies of 11β-HSD-1.     

Cortisol as a mineralocorticoid in human disease

The type 2 isozyme of 11β-hydroxysteroid dehydrogenase inactivates cortisol to cortisone and enables aldosterone to bind to the MR. Congenital deficiency of the enzyme results in cortisol-mediated mineralocorticoid excess and arises because of inactivating mutations in the HSD11B2 gene. Inhibition of the enzyme following licorice or carbenoxolone ingestion results in a similar, though milder phenotype and the enzyme is overwhelmed in ectopic ACTH syndrome. Loss of 11β-HSD2 expression may be important in sodium balance and blood pressure control in some patients with renal disease. Finally, while some studies demonstrate impaired 11β-HSD activity in broader populations of patients with hypertension, further studies are required to clarify the role of 11β-HSD2 in ‘essential’ hypertension.     

Glutamine transaminase K and cysteine S-conjugate beta-lyase activity stains.

An activity stain to detect glutamine transaminase K subjected to nondenaturing polyacrylamide gel electrophoresis (ND-PAGE) was developed. The gel is incubated with a reaction mixture containing L-phenyl-alanine, alpha-keto-gamma-methiolbutyrate (alpha KMB), glutamate dehydrogenase, phenazine methosulfate (PMS) and nitroblue tetrazolium (NBT). Glutamine transaminase K catalyzes a transamination reaction between phenylalanine and alpha KMB. The resultant methionine is a substrate of glutamate dehydrogenase. The NADH formed in the oxidative deamination of methionine reacts with PMS and NBT to form a blue band on the surface of the gel coincident with glutamine transaminase K activity. Cysteine S-conjugate beta-lyase activity is detected in the gel by incubating the gel with a reaction mixture containing alpha KMB (to ensure maintenance of the enzyme in the pyridoxal 5'-phosphate form), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), PMS, and NBT. The products of the lyase reaction interact with PMS and NBT to form a blue dye coincident with the lyase activity. In addition, a new assay procedure for measuring cysteine S-conjugate beta-lyase activity was devised. This procedure couples pyruvate formation from DCVC to the alanine dehydrogenase reaction. Preparations of purified rat kidney glutamine transaminase K yield a single protein band on ND-PAGE (apparent Mr approximately 95,000). This band coincides with both the cysteine S-conjugate beta-lyase and glutamine transaminase K activities. Activity staining showed that homogenates of rat kidney, liver, skeletal muscle, and heart possess a glutamine transaminase K/cysteine S-conjugate beta-lyase activity with an Rf value on ND-PAGE identical to that of purified rat kidney glutamine transaminase K.(ABSTRACT TRUNCATED AT 250 WORDS)     

3D-structure of human estrogenic 17beta-HSD1: binding with various steroids.

Human estrogenic dehydrogenase (17β-HSD1) catalyses the last step in the biosynthesis of the active estrogens that stimulate the proliferation of breast cancer cells. While the primary substrate for the enzyme is estrone, the enzyme has some activity for the non-estrogenic substrates. To better understand the structure–function relationships of 17β-HSD1 and to provide a better ground for the design of inhibitors, we have determined the crystal structures of 17β-HSD1 in complex with different steroids.

The structure of the complex of estradiol with the enzyme determined previously (Azzi et al., Nature Structural Biology 3, 665–668) showed that the narrow active site was highly complementary to the substrate. The substrate specificity is due to a combination of hydrogen bonding and hydrophobic interactions between the steroid and the enzyme binding pocket. We have now determined structures of 17β-HSD1 in complex with dihydrotestosterone and 20-OH-progesterone. In the case of the C19 androgen, several residues within the enzyme active site make some small adjustments to accommodate the increased bulk of the substrate. In addition, the C19 steroids bind in a slightly different position from estradiol with shifts in positions of up to 1.4 Å. The altered binding position avoids unfavorable steric interactions between Leu 149 and the C19 methyl group (Han et al., unpublished). The known kinetic parameters for these substrates can be rationalized in light of the structures presented. These results give evidence for the structural basis of steroid recognition by 17β-HSD1 and throw light on the design of new inhibitors for this pivotal steroid enzyme.     

The type I and type II 11beta-hydroxysteroid dehydrogenase enzymes.

Local tissue concentrations of glucocorticoids are modulated by the enzyme 11β-hydroxysteroid dehydrogenase which interconverts cortisol and the inactive glucocorticoid cortisone in man, and corticosterone and 11-dehydrocorticosterone in rodents. The type I isoform (11β-HSD1) is a bidirectional enzyme but acts predominantly as a oxidoreductase to form the active glucocorticoids cortisol or corticosterone, while the type II enzyme (11β-HSD2) acts unidirectionally producing inactive 11-keto metabolites. There are no known clinical conditions associated with 11β-HSD1 deficiency, but gene deletion experiments in the mouse indicate that this enzyme is important both for the maintenance of normal serum glucocorticoid levels, and in the activation of key hepatic gluconeogenic enzymes. Other important sites of action include omental fat, the ovary, brain and vasculature. Congenital defects in the 11β-HSD2 enzyme have been shown to account for the syndrome of apparent mineralocorticoid excess (AME), a low renin severe form of hypertension resulting from the overstimulation of the non-selective mineralocorticoid receptor by cortisol in the distal tubule of the kidney. Inactivation of the 11β-HSD2 gene in mice results in a phenotype with similar features to AME. In addition, these mice show high neonatal mortality associated with marked colonic distention, and remarkable hypertrophy and hyperplasia of the distal tubule epithelia. 11β-HSD2 also plays an important role in decreasing the exposure of the fetus to the high levels of maternal glucocorticoids. Recent work suggests a role for 11β-HSD2 in non-mineralocorticoid target tissues where it would modulate glucocorticoid access to the glucocorticoid receptor, in invasive breast cancer and as a mechanism providing ligand for the putative 11-dehydrocorticosterone receptor. While previous homologies between members of the SCAD superfamily have been of the order of 20–30% phylogenetic analysis of a new branch of retinol dehydrogenases indicates identities of >60% and overlapping substrate specificities. The availability of crystal structures of family members has allowed the mapping of conserved 11β-HSD domains A–D to a cleft in the protein structure (cofactor binding domain), two parallel β-sheets, and an -helix (active site), respectively.     

Inhibitory effects of post low dose gamma-ray irradiation on ferric-nitrilotriacetate-induced mice liver damage

We studied the effects of a single post whole-body low-dose irradiation (50 cGy of γ-ray) on mice with ferric nitrilotriacetate (Fe³⁺-NTA)-induced transient hepatopathy. As a result, low-dose irradiation accelerated the rate of recovery. Based on the changes in glutamic oxaloacetic transaminase (GOT) activities, glutamic pyruvic transaminase (GPT) activities and lipid peroxide levels, it was shown that hepatopathy was improved by low-dose irradiation 3 h after Fe³⁺-NTA administration. This may be because of the enhancement of antioxidant agents such as total glutathione (GSH + GSSG), glutathione peroxidase (GPX), glutathione reductase (GR) and γ-glutamylcysteine synthetase (γ-GCS) by low-dose irradiation. These findings suggest that low-dose irradiation relieved functional disorders at least in the livers of mice with active oxygen species related diseases.     

Up-regulation of nitric oxide synthase by estradiol in human aorticendothelial cells

We have examined the effects of sex hormones on calcium-dependent NO production and protein levels of NO synthase in cultured human aortic endothelial cells, which were treated with various doses of 17β-estradiol and testosterone for 8–48 h. Treatment with 17β-estradiol enhanced calcium-dependent NO production, but testosterone had exerted no effect. Western blot using monoclonal anti-human endothelial NO synthase antibody clarified that increased NO production by 17β-estradiol treatment was accompanied by increased NO synthase protein. Our results provide evidence that human endothelial NO synthase can be regulated by estrogens.     

Cloning and Characterization of a Soluble Kynurenine Aminotransferase from Rat Brain: Identity with Kidney Cysteine Conjugate β-Lyase

Abstract: In this study, we describe the cloning and characterization of a soluble form of kynurenine aminotransferase (KAT, EC 2.6.1.7) present in rat brain. Soluble KAT was purified from rat kidney and the amino acid sequences of four tryptic peptides determined. These peptides were found to belong to the amino acid sequence reported for rat kidney soluble cysteine conjugate β-lyase, indicating that rat kidney KAT and β-lyase represent the same molecular entity. Oligonucleotide probes derived from the β-lyase cDNA were then used as primers for PCR of reverse-transcribed rat brain poly(A)⁺ RNA. After subcloning of the resulting PCR fragment and sequencing of the isolated rat brain clone, its oligonucleotide sequence was found to be identical to that reported for the β-lyase cDNA. Further evidence that the isolated rat brain clone encoded for KAT was obtained by transfecting HEK-293 cells with a construct containing the coding sequence for the enzyme. The transfected cells exhibited KAT activity and, in the presence of 2 m M pyruvate and 2-oxoglutarate, the K _m values for l -kynurenine were 1.2 m M and 86.3 µ M , respectively. Northern blot analysis of rat kidney, liver, and brain RNA revealed a single species of KAT/β-lyase mRNA of ∼2.1 kb.     

Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes that have Cysteine S-Conjugate β-Lyase Activity and Can Transaminate l-Selenomethionine

Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-l-selenocysteine (MSC) and l-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH₃SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH₃SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH₃SeH and/or seleno-keto acid metabolites.     

Immobilization of β-galactosidase from Kluyveromyces lactis on silica and agarose: comparison of different methods

The covalent immobilization of β-galactosidase from Kluyveromyces lactis (β-gal) on to two different porous carriers, CPC-silica and agarose, is reported. CPC-silica was silanizated and activated with glutaraldehyde. The activation of agarose via a cyanylating agent (CDAP) was optimized. Gel-bound protein and gel-bound activity were both measured directly, allowing the determination of apparent specific activities (S.A.). Higher amounts of β-gal were immobilized on the activated CPC-silica (maximum capacity, 23 mg ml⁻¹ of packed support) than on the CDAP-activated agarose. For the lower enzyme loading assayed (12.6 mg ml⁻¹ packed support), 100% of the enzyme was immobilized but only 34% of its activity was expressed. This inactivation during immobilization was confirmed by the S.A. values (22–29 EU mg⁻¹ for the CPC-derivatives and 80 EU mg⁻¹ for soluble β-gal). The K_app (3.4 mM) for the CDAP-derivative with ONPG as substrate was higher than the K_M value for soluble β-gal (2 mM). When the enzyme loading was increased five-fold, the K_app increased four-fold, to 13 mM. The V_app values for the CPC-derivatives were remarkably lower than the V_max for soluble β-galactosidase. CDAP-derivatives showed better thermal stabilities than CPC-derivatives but neither of them enhanced the stability of the soluble enzyme. When stored at 4°C, the activity of both derivatives remained stable for at least 2 months. Both derivatives displayed high percentages of lactose conversion (90%) in packed bed mini-reactors. Glucose production was 3.3-fold higher for the CPC-derivative than for the CDAP-derivative, as a consequence of the higher flow rates achieved.     

Ergosterol peroxides as phospholipase A2 inhibitors from the fungus Lactarius hatsudake

Four ergosterol derivatives (1–4) have been isolated for the first time from the fruiting bodies of a basidiomycete fungus, Lactarius hatsudake, through activity-guided fractionation. Their structures were determined, using spectroscopic analysis, as: (22E,24R)-ergosta-5,7,22-dien-3β-ol (ergosterol, 1); 5,8-epidioxy-(22E,24R)-ergosta-6,22-dien-3β-ol (ergosterol peroxide, 2); 5,8-epidioxy-(24S)-ergosta-6-en-3β-ol (3); and (22E,24R)-ergosta-7,22-dien-3β,5,6β-triol (cerevisterol, 4). Compounds 2 and 3 showed selective inhibitory activity against Crotalus adamenteus venom phospholipase A₂ (PLA₂) enzyme, but not against Apis mellifcra bee venom PLA₂. The antiphospholipase A₂ activity of compounds 2 and 3 are reported here for the first time.