Brucella antigens (BhuA, 7α-HSDH, FliC) in poly I:C adjuvant as potential vaccine candidates against brucellosis.

A promising strategy for controlling animal brucellosis is vaccination with commercial vaccine strains (Brucella melitensis Rev.1 and Brucella abortus RB51). Owing to safety concerns associated with these vaccines, developing a more effective and safe vaccine is essential. In this study, we examined the capacity of BhuA, 7α-HSDH or FliC antigens in the presence or absence of adjuvant in eliciting immune responses against brucellosis. After cloning, expression and purification, these proteins were used to examine immunologic responses. All immunized mice induced a vigorous IgG, with a predominant IgG2a response. Moreover, splenocytes of immunized mice proliferated and produced IL-2 and IFN-γ, suggesting the induction of cellular immunity. The high IgG2a/IgG1 ratio and IL-2 and IFN-γ indicated a Th1-oriented immune response in test groups. BhuA-, 7α-HSDH- or FliC- poly I:C formulations were the most effective at inducing Th1 immune response compared to groups immunized with naked proteins. Immunization with proteins protected mice against B. melitensis 16M and B. abortus 544. The proteins in adjuvant induced higher levels of protection than proteins only and exhibited similar degree of protection to live attenuated vaccines. Our results, for first time, introduced five potential candidates for subunit vaccine development against B. melitensis and B. abortus infection.

Activity of Zearalenone in the Porcine Intestinal Tract.

This study demonstrates that low doses (somewhat above the No Observed Adverse Effect Level, NOAEL) of the mycoestrogen zearalenone (ZEN) and its metabolites display multispecificity towards various biological targets in gilts. The observed responses in gilts were surprising. The presence of ZEN and zearalenols (ZELs) did not evoke a response in the porcine gastrointestinal tract, which was attributed to dietary tolerance. Lymphocyte proliferation was intensified in jejunal mesenteric lymph nodes, and lymphocyte counts increased in the jejunal epithelium with time of exposure. In the distal digestive tract, fecal bacterial counts decreased, the activity of fecal bacterial enzymes and lactic acid bacteria increased, and cecal water was characterized by higher genotoxicity. The accompanying hyperestrogenism led to changes in mRNA activity of selected enzymes (cytochrome P450, hydroxysteroid dehydrogenases, nitric oxide synthases) and receptors (estrogen and progesterone receptors), and it stimulated post-translational modifications which play an important role in non-genomic mechanisms of signal transmission. Hyperestrogenism influences the regulation of the host's steroid hormones (estron, estradiol and progesteron), it affects the virulence of bacterial genes encoding bacterial hydroxysteroid dehydrogenases (HSDs), and it participates in detoxification processes by slowing down intestinal activity, provoking energy deficits and promoting antiporter activity at the level of enterocytes. In most cases, hyperestrogenism fulfils all of the above roles. The results of this study indicate that low doses of ZEN alleviate inflammatory processes in the digestive system, in particular in the proximal and distal intestinal tract, and increase body weight gains in gilts.

Localization of thiazide-sensitive Na(+)-Cl(-) cotransport and associated gene products in mouse DCT.

The mammalian distal nephron develops a complex assembly of specialized cell types to accomplish the fine adjustment of urinary electrolyte composition. The epithelia of the distal convoluted tubule (DCT), the connecting tubule (CNT), and the cortical collecting duct (CCD) show an axial structural heterogeneity that has been functionally elucidated by the localization of proteins involved in transepithelial ion transport. We compared the distribution of the thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC), basolateral Na(+)/Ca(2+) exchanger (Na/Ca), cytosolic calcium-binding proteins calbindin D(28K) and parvalbumin, and the key enzyme for selective aldosterone actions, 11 beta-hydroxysteroid-dehydrogenase 2 (11HSD2), in the distal convolutions of the mouse. In the mouse, as opposed to the rat, we found no clear subsegmentation of the DCT into a proximal (DCT1) and a distal (DCT2) portion. The TSC was expressed along the entire DCT. Na/Ca and calbindin D(28K) were similarly expressed along most of the DCT, with minor exceptions in the initial portion of the DCT. Both were also present in the CNT. Parvalbumin was found in the entire DCT, with an occasional absence from short end portions of the DCT, and was not present in CNT. 11HSD2 was predominantly located in the CNT and CCD. Short end portions of DCT only occasionally showed the 11HSD2 signal. We also observed an overlap of 11HSD2 immunoreactivity and mRNA staining. Our observations will have implications in understanding the physiological effects of gene disruption and targeting experiments in the mouse.

11Beta-hydroxysteroid dehydrogenase type II and mineralocorticoid receptor in human placenta.

In mineralocorticoid target organs, 11beta-hydroxysteroid dehydrogenase type II (11beta-HSD2) confers specificity on the mineralocorticoid receptor (MR) by converting biologically active glucocorticoids to inactive metabolites. Placental 11beta-HSD2 is also thought to protect the fetus from high levels of circulating maternal glucocorticoid. In this study, we examined the immunoreactivity of 11beta-HSD2 and MR in human placenta from 5 weeks gestation to full term using immunohistochemistry, 11beta-HSD2 messenger RNA (mRNA) expression using Northern blot analysis, and MR mRNA expression using RT-PCR analysis. Marked 11beta-HSD2 immunoreactivity was detected in placental syncytiotrophoblasts at all gestational stages. MR immunoreactivity was moderately detected in syncytiotrophoblasts, some cytotrophoblasts, and interstitial cells of the villous core. Marked mRNA expression of 11beta-HSD2 was detected in placenta by Northern analysis. RT-PCR analysis of MR in placental tissues showed an amplified product consistent in length with the primers selected. These results suggest that placental 11beta-HSD2 is involved in not only regulating the passage of maternal active glucocorticoids into the fetal circulation but also in regulation of maternal-fetal electrolyte and water transport in the placenta, as in other mineralocorticoid target organs.

High dietary potassium chloride intake augments rat renal mineralocorticoid receptor selectivity via 11beta-hydroxysteroid dehydrogenase.

Glucocorticoid access to renal corticosteroid receptors is regulated by 11beta-hydroxysteroid dehydrogenases (11beta-HSDs), converting 11beta-hydroxyglucocorticoids into inactive 11-ketones. This mechanism plays a key role in maintaining normal salt-water homeostasis and blood pressure. To study whether renal cortical proximal and distal tubular 11beta-HSDs are modulated, upon shifting the electrolyte status (and may thereby contribute to adjusting the salt-water homeostasis), rats were treated for 14 days with diets with low (0.058 w/w%), normal (0.58%, which is the KCl content of standard European laboratory rat food) or high (5.8%) potassium chloride content. In proximal tubules, dietary KCl had no effect regarding corticosterone 11beta-oxidation in intact cells as well as 11beta-HSD1 and 11beta-HSD2 protein (Western blotting) and mRNA levels (semi-quantitative RT-PCR). In distal tubules, the low KCl diet also had no effect. However, distal tubules of rats fed the high KCl diet showed increased corticosterone 11beta-oxidation rates (1.6-fold, P<0.01) and 11beta-HSD2 protein (4-fold, P<0.01), whereas 11beta-HSD1 protein was decreased (no longer detected, P<0.05). Distal tubular 11beta-HSD mRNA levels were not changed upon dietary treatment. Our results suggest that upon dietary KCl loading distal tubular mineralocorticoid receptor selectivity for aldosterone is increased because of enhanced corticosterone 11beta-oxidation. This may contribute to the fine-tuning of salt-water homeostasis by the kidney.

11Beta-hydroxysteroid dehydrogenase responsible for carbonyl reduction of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mouse lung microsomes.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen in laboratory animals and is most likely involved in the etiology of tobacco smoke-induced lung cancer. To exert its carcinogenic potential, NNK must be metabolically activated by alpha-hydroxylation at either the methyl or methylene carbons adjacent to the N-nitroso group. The main detoxification pathway of NNK involves carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol followed by glucuronosylation at the hydroxy moiety produced by carbonyl reduction. Whereas there has been great success in the identification of cytochrome P450 species catalyzing NNK activation, the enzyme responsible for NNK carbonyl reduction has been searched for since 1980. In previous investigations, we succeeded in identifying the NNK carbonyl reducing enzyme in mouse liver microsomes as being 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD 1; EC 1.1.1.146), an enzyme that is physiologically involved in glucocorticoid oxidoreduction. In this study, the expression of 11beta-HSD 1 was established on the mRNA (reverse transcription-PCR) and protein (immunoblot) levels. Kinetics of glucocorticoid oxidoreduction were determined with corticosterone and dehydrocorticosterone as substrates for oxidation and reduction, respectively. The apparent Vmax (135.8 versus 48.1 pmol/min/mg of protein) and Km (6.8 versus 35.8 microM) values were much in favor for corticosterone oxidation compared to dehydrocorticosterone reduction. NNK carbonyl reduction displayed an apparent Vmax of 655 pmol/min/mg of protein and a Km of 629 microM. Interestingly, the intrinsic clearance (Vmax/Km ratio) of NNK carbonyl reduction (1.04) corresponds roughly to that of glucocorticoid reduction (1.34). The physiological glucocorticoid substrates of 11beta-HSD 1 (corticosterone and dehydrocorticosterone) and the selective 11beta-HSD 1 inhibitor glycyrrhetinic acid turned out to be strong inhibitors of NNK carbonyl reduction, displaying Ki values of 37.8, 21.3, and 10.9 microM, respectively. Affinity-purified antibodies specific for mouse liver 11beta-HSD 1 inhibited NNK carbonyl reduction in a concentration-dependent manner. For example, at the highest antibody concentration (5 microg of protein), 11beta-HSD 1 activity was decreased to a residual 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol formation of only 7.9% compared to the uninhibited control, thus conclusively demonstrating NNK carbonyl reduction to be mediated by 11beta-HSD 1 in mouse lung microsomes. Evidence is provided in the present study that 11beta-HSD 1 is expressed in mouse lung and that it functions as NNK carbonyl reductase in mouse lung microsomes. These findings may have potentially important implications for smokers who express low levels of 11beta-HSD 1/NNK carbonyl reductase and/or are concurrently being exposed to 11beta-HSD 1 modulators.

Immunolocalization of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in rat uterus: variation across the estrous cycle and regulation by estrogen and progesterone.

Glucocorticoid hormone action in several target tissues is dependent not only on the expression of the glucocorticoid receptor, but also on that of the 11beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes, 11betaHSD-1 and -2. In the uterus, glucocorticoids can exert inhibitory effects on a range of important functions, particularly in relation to the effects of estrogen. Therefore, the present study examined immunolocalization of the two 11betaHSD enzymes in the rat uterus at each stage of the estrous cycle and after ovariectomy with or without estrogen/progesterone replacement. In cycling rats 11betaHSD-1 was localized to luminal and glandular epithelial cells and to eosinophils in both the endometrial stroma and myometrium. In contrast, 11betaHSD-2 immunostaining was localized to endometrial stromal cells and myometrial cells, with no staining evident in epithelial cells or eosinophils. Immunostaining for both enzymes was cycle dependent, being maximal at proestrus and minimal at diestrus. Western blot analysis of whole uterus at proestrus showed the presence of 34- and 40-kDa immunoreactive species for 11betaHSD-1 and -2, respectively. These immunoreactive signals were almost abolished by ovariectomy, but this effect was reversed for both enzymes by estrogen replacement with or without progesterone. These effects of ovariectomy and steroid replacement were confirmed by immunocytochemical analysis, with the exception that progesterone appeared to enhance the stimulatory effects of estrogen on 11betaHSD-2 specifically within the endometrial stroma. In conclusion, these results establish the presence of both 11betaHSD-1 and -2 in the nonpregnant rat uterus and show distinct distributions for the two enzymes and cyclic variation related to positive regulation by ovarian steroids. The physiological implications of these patterns of 11betaHSD expression will ultimately depend on the reaction direction for each enzyme, but 11betaHSD-2 is likely to limit disruptive effects of glucocorticoids on the endometrial stroma, and 11betaHSD-1 may then serve to selectively reactivate glucocorticoids in epithelial cells.

An NADPH-dependent reductase in neonatal pig testes that metabolizes androgens and xenobiotics.

We have isolated an NADPH-dependent reductase from neonatal pig testes that metabolizes androgens and a variety of xenobiotics. This enzyme is distinct from 3alpha/beta,20beta-hydroxysteroid dehydrogenase or its homologue, carbonyl reductase, as judged by its immunological and molecular properties and its much narrower specificity for steroids. This reductase and 3alpha/beta,20beta-hydroxysteroid dehydrogenase may be part of a mechanism for regulating androgen levels the neonatal pig testes. Interestingly, we could not find multiple isoforms of 3alpha/beta,20beta-hydroxysteroid dehydrogenase/carbonyl reductase in pig testes unlike human and rat testes and other organs in which multiple isoforms are expressed.

Human 11 beta-hydroxysteroid dehydrogenase: studies on the stably transfected isoforms and localization of the type 2 isozyme within renal tissue.

The type 1 and type 2 isoforms of human 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) play a crucial role, respectively, in modulating glucocorticoid and mineralocorticoid hormone action. Deficiency of the 11 beta-HSD2 isoform, as described in the syndrome of apparent mineralocorticoid excess and following liquorice (glycyrrhetinic acid) or carbenoxolone ingestion, results in hypertension in which cortisol acts as a potent mineralocorticoid. Several studies have addressed the effects of progesterone, glycyrrhetinic acid, and their derivatives on 11 beta-HSD activity, but these were largely undertaken before the characterization of the 11 beta-HSD isoforms. The aim of this study was to evaluate the localization of 11 beta-HSD2 in human kidney and to study the effects of progesterone, glycyrrhetinic acid, and their related compounds on stable transfectants of the human 11 beta-HSD isoforms. Using an in-house sheep antibody against human 11 beta-HSD2, immunoperoxidase studies localized 11 beta-HSD2 to renal cortical and medullary collecting ducts. Glomeruli, vascular structures, loops of Henle, and proximal tubules were all negative. Confocal laser microscopy studies indicated both a cytoplasmic and nuclear localization for the enzyme within renal collecting ducts. The nuclear staining, which was intranuclear and was not associated with the nuclear membrane, accounted for 40% of the total cellular 11 beta-HSD2 immunoreactivity. Kinetic analysis of 11 beta-HSD activity in fetal kidney 293 cells stably transfected with h11 beta-HSD1/pcDNA3 or 11 beta-HSD2/pCR3, indicated, respectively, low-affinity dehydrogenase/oxoreductase activity (Km for F, 1.8 microM; Km for E, 270 nM) and high-affinity dehydrogenase activity (Km for F, 190 nM). The reductase activity of 11 beta-HSD1 was inhibited by 11 alpha-hydroxyprogesterone > carbenoxolone = glycyrrhetinic acid = progesterone > 11 beta-hydroxyprogesterone. The dehydrogenase activity of 11 beta-HSD2 was inhibited 11 alpha-hydroxyprogesterone = 11 beta-hydroxyprogesterone > glycyrrhetinic acid > carbenoxolone = progesterone. 11 beta-HSD2, expressed in the renal collecting duct, serves to protect the mineralocorticoid receptor (MR) in an autocrine fashion. The demonstration of a nuclear localization for what was thought to be principally a microsomal enzyme suggests that interaction between the MR and its ligand (either aldosterone or cortisol) may be a nuclear rather than a cytoplasmic event. The inhibitory effects of progesterone, glycyrrhetinic acid, and related compounds on 11 beta-HSD1 and 2 were similar, and it remains to be seen what implication these findings have for 11 beta-HSD1 action in tissues such as the liver and gonad and renal 11 beta-HSD2 activity in relation to sodium homeostasis and blood pressure control.

Cloning and production of antisera to human placental 11 beta-hydroxysteroid dehydrogenase type 2.

By inactivating potent glucocorticoid hormones (cortisol and corticosterone), 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) plays an important role in the placenta by controlling fetal exposure to maternal glucocorticoids, and in aldosterone target tissues by controlling ligand access to co-localized glucocorticoid and mineralocorticoid receptors. Amino acid sequence from homogeneous human placental 11 beta-HSD2 was used to isolate a 1897 bp cDNA encoding this enzyme (predicted M(r) 44126; predicted pI 9.9). Transfection into mammalian (CHO) cells produces 11 beta-HSD2 activity which is NAD(+)-dependent, is without reductase activity, avidly metabolizes glucocorticoids (Km values for corticosterone, cortisol and dexamethasone of 12.4 +/- 1.5, 43.9 +/- 8.5 and 119 +/- 15 nM respectively) and is inhibited by glycyrrhetinic acid and carbenoxolone (IC50 values 10-20 nM). Rabbit antisera recognizing 11 beta-HSD2 have been raised to an 11 beta-HSD2-(370--383)-peptide-carrier conjugate. Recombinant 11 beta-HSD2, like native human placental 11 beta-HSD2, is detectable with affinity labelling and anti-11 beta-HSD2 antisera, and appears to require little post-translational processing for activity. 11 beta-HSD2 mRNA (approximately 1.9 kb transcript) is expressed in placenta, aldosterone target tissues (kidney, parotid, colon and skin) and pancreas. In situ hybridization and immunohistochemistry localize abundant 11 beta-HSD2 expression to the distal nephron in human adult kidney and to the trophoblast in the placenta. 11 beta-HSD2 transcripts are expressed in fetal kidney (but not lung, liver or brain) at 21-26 weeks, suggesting that an 11 beta-HSD2 distribution resembling that in the adult is established by this stage in human development.

Comparison of 11 beta-hydroxysteroid dehydrogenase in spontaneously hypertensive and Wistar-Kyoto rats.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) modulates glucocorticoid interactions with mineralocorticoid and glucocorticoid receptors in vivo, by converting 11 beta-hydroxyglucocorticoids to their inactive 11-ketone derivatives. Defective 11 beta-oxidation of glucocorticoids has been associated with hypertension. The objective of this study was to investigate whether 11 beta-HSD contributes to the occurrence of hypertension in spontaneously hypertensive rats (SHRs). The liver and kidney microsomal oxidations of corticosterone (the physiological glucocorticoid in rats) in organs from juvenile (3 weeks old) and adult (3 months old) SHR and Wistar-Kyoto (WKY) rats, with NAD and NADP, show no differences between rat strains. For cortisol, with NADP, adult SHRs show (1.3-3 times; P < 0.05) lower kidney microsomal oxidation rates. The liver microsomal reduction of cortisone shows remarkable interstrain differences; with NADH, reduction is conducted only by adult WKY rats, whereas with NADPH, juvenile animals show similar reduction rates, but at adulthood, only WKYs reduce cortisone. Using Western blot analysis with antibodies against 11 beta-HSD1, positive signals are obtained only for liver microsomes, appearing somewhat lower in SHRs for juvenile but not adult animals. Urinary corticosterone/11-dehydrocorticosterone ratios (measured in adult animals) are not different between rat strains, but are elevated after administration of corticosterone in both strains (although significant only in SHRs). The data provide no indications for exaggerated stimulation of renal corticosteroid receptors, due to modified 11 beta-HSD, in SHRs. However, the experiments suggest the existence of multiple 11 beta-HSDs, in addition to 11 beta-HSD1 and 11 beta-HSD2, some of which may be modified in SHR, but the nature and physiological role of these 11 beta-HSDs is unclear.

11 beta-hydroxysteroid dehydrogenase activity in human lung cells and transcription regulation by glucocorticoids.

Selectivity to aldosterone (Aldo) in mineralocorticoid target tissues has been suggested to be due to the activity of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). This enzyme inactivates the endogenous glucocorticoid cortisol, thus permitting the unhindered access of Aldo to the mineralocorticoid receptor. The 11 beta-HSD activity was measured by the conversion of cortisol to cortisone and vice versa. Concomitant treatment of the cells with either cortisone or cortisol in the presence of the glycyrrhetinic acid derivative carbenoxolone (CBX) blocked both activities of 11 beta-HSD. Dexamethasone and Aldo activated the transcription of transiently transfected mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase chimeric gene in LU-19 cells. The transcription activation by cortisol was synergized by concomitant treatment of the transfectants with CBX. Transactivation with Aldo was inhibited by spironolactone. The enzyme 11 beta-HSD in LU-19 cells is similar to the cloned liver isoform and catalyzes both reduction and dehydrogenation.

Ontogenic pattern of carbonyl reductase activity of 11 beta-hydroxysteroid dehydrogenase in mouse liver and kidney.

1. The ontogenic pattern of xenobiotic carbonyl reducing activity and glucocorticoid 11 beta-oxidoreducing activity of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) in mouse liver and kidney was examined. In addition, the expression of this enzyme was investigated by means of immunoblot analysis in the same tissues. 2. In liver, the foetus shows low or no enzyme activities. After birth both activities increase dramatically with age and remain then on a high plateau until the time of sexual maturity (4 weeks). After maturity, the enzyme activities decline to intermediate values. The developmental pattern of immunological expression of the liver enzyme corresponds well with that of the enzyme activity. 3. Considerable activities of xenobiotic carbonyl reduction and glucocorticoid 11 beta-oxidoreduction are also present after birth in all developmental stages of the kidney. However, no immunological crossreaction was found in any stages with the antibody against the liver 11 beta-HSD suggesting the presence of a structurally different isozyme form in the kidney. 4. The dramatic increase of both activities during the peri- and postnatal developmental periods suggest a potentially biological significance of the liver 11 beta-HSD isozyme in early animal life. 5. Besides being involved in 11 beta-glucocorticoid metabolism in particular the liver enzyme seems to play an additional role as xenobiotic carbonyl reductase.

Detection of multiple antigenically related proteins from various rat tissues by monoclonal antibodies against 3 alpha-hydroxysteroid dehydrogenase.

Mouse hybridomas were prepared by fusing myelomas and spleen cells from mice immunized with purified rat 3 alpha-hydroxysteroid dehydrogenase. Hybridomas secreting monoclonal antibodies against 3 alpha-hydroxysteroid dehydrogenase were selected by indirect enzyme-linked immunoassay and then subcloned by limiting dilution. From two mice we have obtained four positive hybridomas, three secreting high affinity immunoglobulin (Ig) G1 and one secreting IgM. Only two of these monoclonal antibodies (MAbs 3G6 and 7D3, both IgG1) recognized denatured enzyme and, therefore, were used for further immunoblotting experiments. MAb 7D3 recognized a structurally related mouse enzyme, but not the human enzyme, whereas monoclonal antibody 3G6 recognized a human enzyme, but not the mouse enzyme. When these two monoclonal antibodies were used in immunoblotting to survey the expression of 3 alpha-hydroxysteroid dehydrogenase in rat liver and a number of other tissues, striking differences were found in the protein band patterns in kidney, lung, and testis. Both MAbs 7D3 and 3G6 recognized 3 alpha-hydroxysteroid dehydrogenase, a 34-kDa 7D3 recognized a protein of the same size as the liver protein, whereas MAb 3G6 recognized a 34-kDa protein plus another protein of 36 kDa. In kidney only MAb 3G6, but not MAb 7D3, recognized a 34-kDa protein. Conversely, the 34-kDa protein in testis was recognized by MAb 7D3, but not by MAb 3G6. These findings suggest the existence of multiple antigenically related proteins in different tissues.

Heterogeneity of 11 beta-hydroxysteroid dehydrogenase in rat tissues.

Using specific antisera to purified rat liver 11 beta-hydroxysteroid dehydrogenase (11-HSD), we showed that the antigen is widely distributed in rat organs. Enzyme activity and immunoreactivity generally corresponded. Highest by both criteria were liver, testis, kidney and lung. In some tissues (epididymis, pancreas and duodenum) activity was found, but antigen corresponding to 11-HSD at a Mw of 34 kDa was absent. It is suggested that these tissues have alternate enzyme forms. The 11-HSD of brain and liver were compared. Brain enzyme may control selective binding of aldosterone to Type I receptors in the hippocampus and other regions. Rat brain 11-HSD resembled that of liver or kidney in most characteristics. It differed in (a) its steroid specificity: cortisol was a good substrate for liver 11-HSD, and a poor substrate for brain enzyme; (b) stability of 11-oxoreductase (11-OR) component. Brain 11-OR was not readily inactivated; 11-OR from other tissues lost activity rapidly and spontaneously. The variations in properties of 11-HSD in specific tissues may reflect aspects of its various specific functions.

Corticosteroid 11 beta-dehydrogenase of rat tissues: immunological studies.

Monospecific polyclonal antibodies to purified homogeneous rat liver corticosteroid 11 beta-dehydrogenase were generated in rabbits. The antibodies were immunoprecipitins, but enzyme activity was not completely suppressed in the antigen-antibody complex. Two antibody preparations, 56-125 and 56-126, used to detect 11 beta-dehydrogenase antigen in Western blots, generated different staining patterns for kidney, liver, brain, and heart. Using the two antibodies together, the total number of antibody-reacting components in kidney was three, and that in liver was two. Based on rates of digestion with proteases, the two prominent immunoreactive proteins in kidney appeared to be structurally or conformationally different. A prominent immunostaining component was present in stomach. Tissues that showed immunochemical evidence of 11 beta-dehydrogenase antigen showed corresponding levels of 11 beta-dehydrogenase activity. Most active were liver, testis, kidney, and lung. Lower levels of activity were found in prostate and epididymis, brain, and reproductive tract. We conclude that 11 beta-dehydrogenase is widely distributed in rat organs and is present at low levels with significant exceptions. The data indicate that 11 beta-dehydrogenase may occur in several enzyme forms, and that the distribution of these forms is to some extent tissue specific.

17 beta-hydroxysteroid oxidoreductases of human fetal and adult tissues: immunological cross-reactivity with an anti-human placental cytosolic 17 beta-hydroxysteroid oxidoreductase antibody.

To determine whether the immunological determinants of human placental 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) were present in 17 beta-HSORs of tissues of the human fetus and adult and of various non-human cells maintained in culture, western immunoblot analysis was conducted by use of a polyclonal antibody directed against determinants of the placental cytosolic enzyme. Tissues and cells were evaluated for the presence of immunocross-reactive proteins with a relative molecular mass (Mr) similar to that of placental 17 beta-HSOR (approximately 34,000). By use of homogenates of human fetal tissues, immunostaining of 17 beta-HSORs of Mr approximately 34 kDa was detected in trophoblast, fetal adrenal neocortex, fetal zone of the adrenal gland, liver, intestine, kidney, brain, lung, skin, heart, spleen, pancreas, chorion laeve, and, occasionally, amnion. Immunostaining at Mr approximately 34 kDa also was demonstrated by use of cytosolic preparations of fetal tissues and, in some cases, by use of unwashed microsomal fractions; this protein was either absent or present in almost undetectable amounts in washed microsomes, except for placenta and fetal brain. Immunostaining at approximately 34 kDa was demonstrated occasionally in decidua of pregnant women by use of homogenates, but was not detected in endometrium or myometrium of non-pregnant women, testis of an adult man, mouse and rat Leydig tumour cells, mouse and rat adrenal tumour cells, and normal bovine adrenocortical cells.

Purification of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli strain 080.

Purification studies of 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) (EC 1.1.1.159) from Escherichia coli 080 showed that 1.59-fold purification could be achieved by heating (60 degrees C for 10 min) the ultracentrifuged enzyme preparation, and 6.46-fold purification was achieved by subsequent precipitation with ammonium sulfate. Further purification on Sephadex G-100 gel gave 10.1-fold purification. After pooling and concentrating the active fractions obtained from the Sephadex G-100 filtration, an 11.1-fold purification was achieved using DEAE-cellulose chromatography. The purified enzyme produced a single band on polyacrylamide gel electrophoresis and its molecular weight was determined to be 54,000. The enzyme was immunogenic and showed immunoprecipitation with homologus antisera.

Immunohistochemical localization of 11-hydroxysteroid dehydrogenase in rat kidney with monoclonal antibody.

Monoclonal antibody (MAb) against 11-hydroxysteroid dehydrogenase (11-HSD) has been raised by immunization of female balb/c mice. 11-HSD from solubilized rat renal microsomal protein could be bound in a modified ELISA using antimouse IgG and MAb against 11-HSD. On Western blots of solubilized rat renal microsomes the MAb recognized a single protein band of an approximate molecular weight of 35 kD. Immunohistochemical staining of rat renal tissue with the above MAb and the APAAP staining technique displayed a heterogenous reginal and subcellular distribution: glomeruli and arterioles were practically devoid of specific staining, as were epithelial cells in inner and outer medulla. Intense immunostaining was observed in PCT and particularly in PST, appearing granular with highest density around the nuclei. Here the enzyme bound to intracellular membranes may exert an autocrine function such as signal inactivation. In contrast to cortex, staining of interstitial cells was observed in renal medulla. The latter localization is compatible with the concept of a paracrine function of 11-HSD which might prevent corticosterone from gaining access to collecting duct cells.

Localisation of 11 beta-hydroxysteroid dehydrogenase--tissue specific protector of the mineralocorticoid receptor.

In vitro the mineralocorticoid receptor is non-specific and does not distinguish between aldosterone and cortisol. In vivo certain tissues with this receptor are aldosterone selective (eg, kidney and parotid) whereas others with the same receptor are not (eg, hippocampus and heart). Experiments in rats showed that 11 beta-hydroxysteroid dehydrogenase (which converts cortisol to cortisone in man and corticosterone to 11-dehydrocorticosterone in the rat) was much more highly concentrated in aldosterone-selective tissues than in non-selective tissues. The localisation in the selective tissues was such that the enzyme could act as a paracrine or possibly an autocrine mechanism protecting the receptor from exposure to corticosterone. Autoradiographic studies showed that protection is lost when the enzyme is inhibited; 3H-corticosterone and 3H-aldosterone were bound to similar sites. These findings seem to explain why sodium retention, hypokalaemia, and hypertension develop in subjects with congenital deficiency of 11 beta-OHSD and those in whom the enzyme has been inhibited by liquorice.