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Prostate cancer (PCa) is one of the most diagnosed cancers in men worldwide. Several studies have identified that circular RNAs (circRNAs) have a crucial impact on the biological processes in PCa. Therefore, it is necessary to study the molecular mechanism of circRNAs in tumor progression and metastasis.RNA interference was used to decrease circHIPK3 and MTDH expression. Overexpression vector was used to increase circHIPK3 and MTDH expression. Luciferase reporter assay were used to detect the relationship between circHIPK3 and miR-448 or between miR-448 and MTDH. MTT assay, colony formation assay and transwell assay were used to measure proliferation and migration of PCa cells.Circular RNA circHIPK3 was significantly increased in PCa tissues and cell lines. And overexpression of circHIPK3 promoted the migration, proliferation, and invasion of PC-3 and 22Rv1 cells, while knockdown of circHIPK3 markedly repressed the above-mentioned series of biological processes. Furthermore, circHIPK3 promoted metadherin (MTDH) expression by sponging miR-448. In vivo experiments, it was also found that overexpression of circHIPK3 significantly promoted tumor growth.Our research shows that circHIPK3 plays a carcinogenic effect in PCa by regulating the miR-448/MTDH axis, indicating that circHIPK3 may be a potential therapeutic target for PCa.

To explore the effect of miR-543-3p on the recovery of locomotor function after spinal cord injury (SCI) by regulating tumor necrosis factor superfamily member 15 (TNFSF15) mediated inflammation and apoptosis.Macrophages were isolated from the abdominal cavity of 2-3 months old Sprague-Dawley (SD) rats and cultured. The levels of miR-543-3p, tumor necrosis factor superfamily member 15 (TNFSF15), TNF-like molecule 1A (TL1A) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) after transfection of miR-92b-5p into activated macrophages were detected by quantitative Real-time polymerase chain reaction (qRT-PCR). Moreover, the mRNA expressions of miR-543-3p, TNFSF15, TL1A and NF-κB after SCI in rats were detected by qRT-PCR. Meanwhile, the protein expressions of tumor necrosis factor (TNF-α), interleukin-1 β (IL-1β) and Caspase8 were detected by Western blot. After intrathecal injection of miR-543-3p mimics, the mRNA expressions of miR-543-3p, TNFSF15, TL1A and NF-κB and the protein expressions of TNF-α, IL-1β and Caspase8 in spinal cord injured area of mice were measured by qRT-PCR and Western blot, respectively. Basso Beattie Bresnahan (BBB) locomotor rating scale was used to determine the recovery of locomotor function after spinal cord injury and injection of miR-543-3p mimics.Compared with inactivated cells, the expression of miR-543-3p in activated macrophages was significantly declined. However, the levels of TNFSF15, TL1A and NF-κB were significantly elevated. The expressions of TNFSF15, TL1A and NF-κB were remarkably downregulated after transfection of miR-543-3p. In addition, the level of miR-543-3p was significantly downregulated, accompanied by an increase in TNFSF15, TL1A and NF-κB in SCI rats. Accordingly, the protein levels of TNF-α and IL-1β and Caspase8 were also significantly increased. However, the expressions of TNFSF15, TL1A and NF-κB were significantly down-regulated in rats injected with miR-543-3p mimics, whereas the protein levels of TNF-α and IL-1β and Caspase8 were significantly suppressed. Finally, compared with SCI group, the recovery of locomotor function in miR-543-3p mimics administration group was significantly improved.After SCI, miR-543-3p can inhibit the activity of NF-κB by suppressing the inflammatory aggravation of TNFSF15 and decreasing its product TL1A. MiR-543-3p leads to the improvement of neuron protection and locomotor function via attenuating inflammatory reaction and cell apoptosis.

The presented study aimed to investigate the antitumor efficacy of combination of oxaliplatin with rapamycin, an mTOR inhibitor, in hepatocellular carcinoma (HCC). The activation status of mTOR pathway was first examined in HCC cell lines HepG2, BEL7402, and HuH7 using Western blotting. Effects of rapamycin, oxaliplatin, and their combination on the proliferation of HCC cells were determined in vitro using MTT assay and in vivo using a nude mice model bearing HepG2 xenografts. Drug-induced cell apoptosis was examined by flow cytometry. Expression of apoptosis-related protein was determined by Western blotting. We observed that mTOR pathway was activated in all three cell lines used in the current study. MTT assay demonstrated that oxaliplatin in combination with rapamycin synergistically inhibited the proliferation of HCC cells. The combination regimen reduced terminal tumor burden more efficiently than the corresponding monotherapy. The percentages of apoptotic cells and the expression levels of apoptosis-related proteins including cleaved caspase-9, -3, and PARP were significantly higher in combination-treatment groups than those in mono-drug-treatment groups. The ratios of Bax/Bcl-2 in cells exposed to both oxaliplatin and rapamycin were significantly increased compared to those in cells subjected to oxaliplatin or rapamycin alone treatment. Results obtained in the presented study suggested that combination of oxaliplatin and rapamycin was superior to mono-drug and may have a potential value in treatment of HCC.

To determine the cytotoxic effect of lymphocytes activated by melanoma-associated antigen 3 (MAGE-3)-sensitized dendritic cells (DCs) on BIU-87 tumor cells, and to evaluate the possibility of MAGE-3-peptide-pulsed DCs as a vaccine in bladder cancer immunotherapy, the proliferation of T cells and the activity of cytotoxic T lymphocytes (CTLs) were examined by the MTT method. CTLs were induced by MAGE-3-sensitized DCs, or by ovalbumin (OVA) peptide and non-sensitized DCs as controls, respectively. The results indicated that MAGE-3-sensitized DCs have the ability to promote the proliferation of T cells as well as the cytotoxic activity of CTLs on bladder cancer cells in comparison with OVA peptide and non-sensitized DCs. In other words, DCs sensitized by the MAGE-3 antigen peptide could obviously upregulate the proliferation of T cells, which resulted in the growth inhibition of bladder cancer BIU-87 cells. In addition, MAGE-3-sensitized DCs played an important role in inhibiting the growth of human BIU-87 tumor xenografts in nude mice.

Supplementary dietary plant oils have the potential to alter milk fatty acid composition in ruminants as a result of changes in the amount and kind of fatty acid precursors. We hypothesized that linseed oil in combination with malate (a key propionate precursor in the rumen) would increase ∆9 unsaturated fatty acids and specific gene expression in somatic cells and mammary glands of lactating goats. Twelve lactating goats were used in a 3 × 3 Latin square design. Treatments included the basal diet (CON), the CON plus 4% linseed oil (LO), and the CON plus 4% linseed oil and 2% -malate (LOM). Relative to CON, the LO and LOM supplements increased the daily intake of palmitic (16:0), stearic (18:0), oleic (18:1-9), linoleic (18:2-6), α-linolenic (18:3-3), and γ-linolenic acids (18:2-6); α-linolenic acid intake was increased over 9-fold, from 6.77 to over 51 g/d (0.02). The LO and LOM supplements increased daily milk yield, milk fat yield, and milk fat percentage (0.05). The LOM supplement also increased milk lactose percentage and daily yield ( = 0.03). Both the LO and LOM supplements increased plasma glucose and total cholesterol and decreased plasma β-hydroxbutyrate concentrations ( = 0.03). The LO and LOM supplements increased concentrations of stearic acid; -vaccenic acid (TVA; 18:1-11); -9, -11 CLA; -10 -12 CLA; and α-linolenic acid in rumen fluid and increased the concentrations of oleic acid; TVA; -9, -11 CLA; -10, -12 CLA; and α-linolenic acid in plasma lipids and milk fat (0.05). Conversely, the LO and LOM supplements decreased short- and medium-chain SFA, including lauric (12:0), myristic (14:0), and palmitic acids, in plasma and milk fat (0.05). Relative mRNA levels for and () gene expression were increased in somatic cells and mammary gland tissue by LO and LOM (0.05). We conclude that the higher intake and ruminal production of stearic acid promoted SCD gene expression in somatic cells and mammary tissue. Furthermore, milk somatic cells are a suitable substitute for documenting treatment effects of dietary oils on gene expression in goat mammary tissue.

Biodetoxification of mycotoxins is a novel strategy to control mycotoxicoses in animals. Bacillus sp. LS100, which transforms deoxynivalenol (DON) to a less toxic chemical de-epoxy DON (DOM-1), was evaluated for its efficacy in reducing the adverse effects of DON on swine growth performance. A feeding trial was conducted in growing pigs with four treatments: (1) corn meal without detectable DON served as control (Non-toxic Corn); (2) Fusarium-infected corn giving a toxic diet containing 5 µg DON g(-1) (Toxic Corn); (3) Toxic Corn detoxified with Bacillus sp. LS100 giving a detoxified diet containing 5 µg DOM-1 g(-1) (LS100-De-toxic Corn); (4) Non-toxic Corn treated with Bacillus sp. LS100 serving as bacterial control (LS100-Non-toxic Corn). During 9 days of exposure to the treatments, pigs on Toxic Corn showed a significant reduction in daily feed consumption, daily weight gain and feed efficiency by 29, 48 and 29%, respectively, compared to pigs on Non-toxic Corn. These parameters of the pigs fed LS100-De-toxic Corn diet were 45, 82 and 32% greater, respectively, than those of pigs fed Toxic Corn diet, and were similar to those pigs fed Non-toxic Corn and LS100-Non-toxic Corn diets. There were no significant differences between the treatments of LS100-Non-toxic Corn and Non-toxic Corn diets, implying that the bacterial isolate might not have significantly affected nutrition and palatability of the feed or had negative effects on the pig's feeding performance. The results have proved that microbial detoxification of DON in contaminated feed can eliminate negative effects of the mycotoxin, and the pre-feeding detoxification approach may be applied in the livestock industry.

International audience; Antimicrobial resistance is a global challenge that impacts both human and veterinary health care. The resilience of microbes is reflected in their ability to adapt and survive in spite of our best efforts to constrain their infectious capabilities. As science advances, many of the mechanisms for microbial survival and resistance element transfer have been identified. During the 2012 meeting of Antimicrobial Agents in Veterinary Medicine (AAVM), experts provided insights on such issues as use vs. resistance, the available tools for supporting appropriate drug use, the importance of meeting the therapeutic needs within the domestic animal health care, and the requirements associated with food safety and food security. This report aims to provide a summary of the presentations and discussions occurring during the 2012 AAVM with the goal of stimulating future discussions and enhancing the opportunity to establish creative and sustainable solutions that will guarantee the availability of an effective therapeutic arsenal for veterinary species.

To evaluate the potential roles that both receptors and enzymes play in corticosteroid regulation of intestinal function, we have determined glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11β-hydroxysteroid dehydrogenase (11β-HSD) expression in intestinal epithelial cells. GR and MR mRNA and receptor binding were ubiquitously expressed in epithelial cells, with receptor levels higher in ileum and colon than jejunum and duodenum. RNase protection analysis showed that 11β-HSD1 was not expressed in intestinal epithelial cells, and enzyme activity studies detected no 11-reductase activity. 11β-HSD2 mRNA and protein were demonstrated in ileal and colonic epithelia; both MR and GR binding increased when enzyme activity was inhibited with carbenoxolone. Duodenal and jejunal epithelial cells showed very little 11β-HSD2 mRNA and undetectable 11β-HSD2 protein; despite minor (

Local tissue concentrations of glucocorticoids are modulated by the enzyme 11 β -hydroxysteroid dehydrogenase which interconverts cortisol and the inactive glucocorticoid cortisone in man, and corticosterone and 11-dehydrocorticosterone in rodents. The type I isoform (11 β -HSD1) is a bidirectional enzyme but acts predominantly as a oxidoreductase to form the active glucocorticoids cortisol or corticosterone, while the type II enzyme (11 β -HSD2) acts unidirectionally producing inactive 11-keto metabolites. There are no known clinical conditions associated with 11 β -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 11 β -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 11 β -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. 11 β -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 11 β -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 11 β -HSD domains A–D to a cleft in the protein structure (cofactor binding domain), two parallel β -sheets, and an α -helix (active site), respectively.

Recently, we identified a novel putative nuclear receptor in colonic crypt cells distinct from both mineralocorticoid receptor and glucocorticoid receptor, with high affinity for 11-dehydrocorticosterone (11-DHB) (33). In the present study, competitive nuclear binding assays demonstrated that this site has a unique steroid binding specificity that distinguishes it from other steroid receptors. Western blot analysis showed the presence of 11β-hydroxysteroid dehydrogenase-2 (11βHSD2) but not 11βHSD1in colonic crypt cells and showed that 11βHSD2was present in the nuclear pellet. Differences in steroid specificity between the putative DHB receptor and inhibition of 11βHSD activity indicate that binding is not to the enzyme. Furthermore, modified Chinese hamster ovary cells transfected with the 11βHSD2gene express nuclear 11βHSD2but not a nuclear DHB binding site. In conclusion, these data support the existence of a novel nuclear DHB receptor in rat colon that is distinct from the classic steroid receptors and from both 11βHSD1and 11βHSD2.

Background Apparent mineralocorticoid excess (AME) is a cause of low-renin, low-aldosterone hypertension in which cortisol acts as a mineralocorticoid. The condition reflects an inability to inactivate cortisol to cortisone due to defective activity of the type 2 isozyme of 11β-hydroxysteroid dehydrogenase (11β-HSD2). Homozygous mutations in 11β-HSD2 gene in patients with AME have been described. A 7-year-old Brazilian girl had previously been found to have AME. Her father recently presented with mineralocorticoid hypertension at age 38 years. Objective To describe the clinical details, to perform steroid analyses and to assess the molecular basis for the hypertension in this kindred. Methods The 11βHSD2 gene was amplified from genomic DNA by the polymerase chain reaction and sequenced by direct chain-termination sequencing on an automatic DNA sequencer. The sequencing results were validated by restriction-site polymorphism. The mutant 11β-HSD2 protein was expressed in Chinese hamster ovary polyoma cells and enzymatic activity was assessed by metabolizing cortisol in vitro. Results Sequence analysis of genomic DNA revealed a novel C1061T point mutation in exon V of the human 11β-HSD2 gene, resulting in an amino acid substitution of alanine by valine at codon 328 of the enzyme protein (A328V). Expression studies confirmed that the mutant protein was devoid of 11β-HSD2 activity. A Hhal restriction-site polymorphism confirmed that the proband was homozygous for the mutation whereas both parents were heterozygotes. The father of the proband had hypertension, a normal serum potassium level, suppressed plasma renin activity and plasma aldosterone level and a moderately elevated urinary cortisol: cortisone metabolite ratio. Conclusions AME in this kindred is caused by a novel mutation in the 11β-HSD2 gene. Detection of hypokalaemia, at least in this kindred, is an insensitive screening test for mineralocorticoid-based hypertension. In contrast to results from previously investigated kindreds, we have demonstrated that this kindred has an abnormal phenotype in the heterozygote state. Further studies are now required in order to evaluate the role of 11β-HSD2 activity in the pathophysiology of 'essential' hypertension.

The 11beta-hydroxysteroid dehydrogenase type II enzyme (11betaHSD2) endows specificity on the mineralocorticoid receptor by metabolising glucocorticoids. Sequence comparisons with other microsomal proteins showed the strongly preferred topology of a lumenal pentapeptide followed by three transmembrane helices with residues beyond Ala73 on the cytoplasmic side of the membrane, suggesting that 11betaHSD2 is anchored to the endoplasmic reticulum by the N-terminal region. However, deletion of the N-terminus (11betaHSD2 deltaN) and expression of the construct in mammalian cells showed that the enzyme remained bound to the microsomal fraction, indicating that other regions are also involved in membrane anchoring. Crosslinking studies and nonreducing SDS-PAGE demonstrated that 11betaHSD2 is a non-covalently linked dimer. Deletion of the non-conserved C-terminal region (11betaHSD2 deltaC) resulted in an enzyme with a Km of 215 nM for cortisol in whole cell assays, while 11betaHSD2 and 11betaHSD2 deltaN displayed a Km of 62 and 74 nM, respectively. In homogenates 11betaHSD2 and 11betaHSD2 deltaC displayed maximal activity at 140 mM NaCl or KCl, but showed a marked decrease in enzyme activity with increasing salt. 11BetaHSD2 was more stable than 11betaHSD2 deltaC in the presence of NaSCN, suggesting that the C-terminal region plays a role in enzyme stability. There was no detectable activity in homogenates containing 11betaHSD2 deltaN, while 11betaHSD2 deltaC and 11betaHSD2 displayed a Km of 135 and 46 nM, respectively. Although 11betaHSD2 is conventionally considered a unidirectional dehydrogenase all constructs converted 11-dehydrodexamethasone to dexamethasone in whole cell assays, providing an explanation for the potency of the synthetic glucocorticoid in the face of a powerful inactivator of natural glucocorticoids.

This study investigated the effects of a mixture of steam-flaked corn and extruded soybeans on performance, ruminal development, ruminal fermentation variables, and intestinal absorptive capability in Holstein male calves (n = 39). Calves were assigned to 1 of 3 treatments (13 calves per treatment): 1) milk replacer (MR), 2) one-half of the amount of MR in treatment 1, plus a mixture of 62.1% steam-flaked corn and 30.5% extruded soybeans provided ad libitum (HMCS), or 3) a mixture of 62.1% steam-flaked corn and 30.5% extruded soybeans provided ad libitum (CS). All the calves were started at 2 ± 1 d of age and studied for 150 d. Each 30 d was defined as 1 period. Dry matter intake and growth were measured daily and monthly, respectively. All calves were harvested at 150 d of age, after which rumen fluid was collected. Rumen and intestine samples were gathered. Calves fed MR exhibited greater BW (P = 0.001) and ADG (P0.001), compared with calves fed HMCS and CS from period 2 to 3; however, from period 4 to 5, CS calves had greater (P0.04) ADG than MR calves. The treatments did not differ in final BW (P = 0.72) and ADG (P = 0.20) from period 2 to 5. Compared with HMCS and MR calves, CS calves had the greatest DMI (P0.001) and the least feed efficiency (P0.001) from period 2 to 5. For ruminal fermentation parameters, CS calves had decreased (P = 0.04) rumen pH than MR calves. The NH3 concentrations were greater (P = 0.03) in calves fed HMCS than calves fed MR and CS. Total VFA concentrations were greatest in CS calves (P = 0.02). Calves fed CS had the greatest molar concentrations of propionate, butyrate, and valerate (P0.002), and calves fed HMCS had the greatest molar concentrations of isobutyrate (P = 0.001) and isovalerate (P = 0.001). The CS calves exhibited greater empty rumen weight (P = 0.001), papillae length (P0.001), papillae width (P0.001), rumen wall thickness (P = 0.012), and papillae density (P = 0.003). The greatest villus heights in the jejunum (P = 0.04) and ileum (P = 0.005) were observed in CS calves. Compared with HMCS calves, calves fed CS had greater villus:crypt ratios in the duodenum (P = 0.001) and jejunum (P = 0.001). Results indicate that CS improves ADG in period 4 to 5 and positively contributes to ruminal development, ruminal fermentation, and intestinal absorptive capability in veal calves.

The 11 beta-hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) converts cortisol to cortisone, allowing the non-selective mineralocorticoid receptor to bind aldosterone. When the activity of this enzyme is compromised, as occurs in licorice intoxication or in the congenital syndrome of apparent mineralocorticoid excess (AME), there is marked sodium retention, hypokalemia, and hypertension. The first proof that this enzyme was defective in AME came from the identification of the R337C mutation in a number of siblings with the syndrome. Subsequent expression studies showed that the mutant had a Km one order of magnitude higher than the wild-type enzyme while in the cell-free system it was without detectable activity. In the present work we have extended our studies on this mutant and provide evidence that the mutant protein may also partially inhibit the wild-type enzyme in heterozygotes. Furthermore, experiments incorporating the protein synthesis inhibitor cycloheximide show that the mutant enzyme is less stable than the wild-type activity in intact cells. These results suggest that mutations in the 11 beta HSD2 enzyme may have multiple consequences for the mineralocorticoid target cell.

We examined the underlying neural-endocrine mechanisms of asthma associated with respiratory syncytial virus infection. Thirty Sprague-Dawley rats were randomly divided into control group, respiratory syncytial virus (RSV) group, and anti-nerve growth factor (NGF) IgG group. An RSV infection model was established by nasal drip once a week. In the anti-NGF antibody intervention group, each rat was given an intraperitoneal injection of anti-NGF IgG 3 h before RSV infection. Optical microscopy and transmission electron microscopy were used to observe the structural changes in adrenal medulla cells. Changes in adrenaline and norepinephrine in serum were detected by ELISA. NGF expression was assayed by immunohistochemistry. Expression differences in synaptophysin mRNA were detected by RT-PCR. Transmission electron microscopy displayed widened adrenal medulla intercellular spaces, reduced chromaffin particle concentration, and increased mitochondria in the RSV infection group. At the same time, NGF expression was increased in the RSV infection group significantly. In addition, the adrenaline concentration was significantly decreased compared with the control and anti-NGF antibody groups. Synaptophysin mRNA expression was significantly increased in the RSV infection and anti-NGF antibody groups. However, compared with the RSV infection group, synaptophysin mRNA expression was significantly decreased in the anti-NGF antibody group. We conclude that RSV infection could induce adrenal medulla cell differentiation to nerve cells by over-expression of NGF, resulting in the decreased endocrine function found in asthma progression.

The paper discusses mainly about the modelling process and related problems with examples from Chinese and French cases. Five practical problems must be solved for modelling the functioning of any landscape: (1) The field data are necessarily taken with a sampling procedure that implies a spatial (and often temporal) scale. (2) Every landscape modelled has to be identified, delimited and characterised before application of the hierarchical theory. (3) The functioning of a landscape involves data of multiple types (climate, soil, vegetation, fauna, buildings, communications, economy, aesthetics, etc.) which must be integrated in a holistic approach. (4) Every landscape is spatially heterogeneous, and the structure of the model must be more or less isomorphic with its heterogeneity. (5) The evolution of the landscape must be modelled on a rather long period of time. For all these reasons, it is necessary to build ad hoc models. Object-oriented computing languages may be useful for this purpose.

When small intestinal epithelial cells are incubated with [3H]corticosterone, nuclear binding is displaced neither by aldosterone nor RU-28362, suggesting that [3H]corticosterone is binding to a site distinct from mineralocorticoid receptor and glucocorticoid receptor. Saturation and Scatchard analysis of nuclear [3H]corticosterone binding demonstrate a single saturable binding site with a relatively low affinity (49 nM) and high capacity (5 fmol/μg DNA). Competitive binding assays indicate that this site has a unique steroid binding specificity, which distinguishes it from other steroid receptors. Steroid specificity of nuclear binding mirrors inhibition of the low 11β-dehydrogenase activity, suggesting that binding may be to an 11β-hydroxysteroid dehydrogenase (11βHSD) isoform, although 11βHSD1 is not present in small intestinal epithelia and 11βHSD2 does not colocalize intracellularly with the binding site. In summary, a nuclear [3H]corticosterone binding site is present in small intestinal epithelia that is distinct from other steroid receptors and shares steroid specificity characteristics with 11βHSD2 but is distinguishable from the latter by its distinct intracellular localization.

Short chain alcohol dehydrogenases have an invariant YXXXK motif at the active site. Database analysis of 116 superfamily members showed that 92% also contain a Serine or Threonine residue at the Y + 1 or Y + 3 positions, a pattern we previously described as the ST rule. In the present study we have mutated Serines in the active site, YSASK, motif of 11 beta-hydroxysteroid dehydrogenase type I (11 beta HSD1). These studies were facilitated by the generation of a new specific polyclonal antibody (RAH113) raised against a synthetic peptide derived from the rat 11 beta-HSD1 sequence. Immunopurified RAH113 recognized a single band at 34 kDa in liver homogenates. Kinetic analysis of equivalent amounts of wild type and mutant proteins showed that mutagenesis of active site Serines resulted in modest increases of Km values for corticosterone from 325 nM for 11 beta HSD1 to 512 nM-588 nM for the S1 (YAASK), S2 (YSAGK) and S3 (YAAGK) mutants in homogenates of transfected CHOP cells. However, far greater effects were observed on the first order rate constants with mutants displaying 10%, 1% and 1% of the wild type activity, respectively. When the oxidoreductase reaction was studied in whole cells mutagenesis again had a minimal effect on the Km value but dramatically lowered first order rate constants to 34%, 5% and 6%, respectively, of the wild type. These data show that Serines at the active site of 11 beta HSD1 play an important role in determining the rate of catalysis. Coexpression of wild type and mutant enzymes did not lower wild type activity suggesting that the active site of the multimeric enzyme is not a composite of active site Serines from different subunits.

The 11beta-hydroxysteroid dehydrogenase type 2 enzyme (11betaHSD2) metabolizes glucocorticoids into their inactive 11-keto metabolites. Although the type 1 enzyme (11betaHSD1) displays both oxidative and reductive activity, to date 11betaHSD2 has been shown to have dehydrogenase activity only. In this study we compared both dehydrogenase and reductase characteristics of the cloned rat 11betaHSD1 and rat and human 11betaHSD2 for three different 11-hydroxysteroid substrates, cortisol (F), corticosterone (B), and dexamethasone (Dex), and the corresponding 11-keto metabolites, cortisone (E), 11-dehydrocorticosterone (A), and 11-dehydrodexamethasone (DH-Dex), respectively. In cell homogenates expressing either the rat or the human 11betaHSD2, the relative potency for the dehydrogenase reaction was BFDex. Although there was no reduction of A or E, DH-Dex was readily converted to Dex with an equilibrium far on the side of the 11-hydroxy metabolite. DH-Dex reduction in homogenates was inhibited by both glycyrrhetinic acid and carbenoxolone, with a 50% inhibition at 80 and 100 nM, respectively. In intact cells transfected with rat 11betaHSD1, the equilibrium was on the reductase side for all substrates. Dehydrogenation of B or F was more potent with rat 11betaHSD2 than with rat 11betaHSD1. There was no detectable 11betaHSD1 oxidation of Dex. These data indicate that both the cloned human and rat 11betaHSD2 reduce DH-Dex and do this more readily than they oxidize Dex. Thus, 11betaHSD2 seems also to be a bidirectional enzyme, although no reduction of the physiological compounds A and E was observed.

Mineralocorticoid action is facilitated by 11 beta-hydroxysteroid dehydrogenase type II (11 beta HSD2), which metabolizes glucocorticoids and allows aldosterone to bind to the nonselective mineralocorticoid receptor. We have recently demonstrated the presence of the 11 beta HSD2 protein in a wide range of human epithelia, suggesting that it is the sole isoform endowing specificity in man. In the present study we have used an immunopurified polyclonal antibody (RAH23) raised against a C-terminal peptide derived from the cloned rat 11 beta HSD2 protein to perform immunohistochemical and molecular analysis in rat tissues. In frozen sections of rat kidney, strong staining was seen with the RAH23 antibody in the distal tubule; weaker staining was observed in the thick ascending loop of Henle and the medullary and papillary collecting ducts. Punctate cortical staining was observed in the fetus at 20 days gestation and in 8-day-old rats, with a noticeable increase in the staining pattern at 16 days of age. The kidney did not attain the adult pattern of staining until 28 days of age. Epithelia of ileum and colon also stained with RAH23, as did excretory ducts of the submandibular gland. Intrahepatic and excretory bile ducts displayed strong immunoreactivity in the epithelial lining. Rat adrenal glands showed evidence of the 11 beta HSD2 antigen in the zona fasciculata and zona reticularis, but not in the zona glomerulosa or medulla. Western blot analysis with the RAH23 antibody revealed strong bands in the kidney, colon, adrenal gland, and submandibular gland at 40 kDa, colinear with the migration of the cloned 11 beta HSD2 enzyme. A band of medium intensity was also seen at this size in the pancreas, whereas a band of moderate intensity was seen in the bile duct, and weaker bands were noticed in the stomach, small intestine, and liver, with a diffuse band at 36-42 kDa in the prostate. Strong bands were seen in the pancreas and prostate at 78 kDa, with weaker signals in the colon, adrenal, stomach, and bile duct. A number of tissues also displayed multiple bands at about 30 kDa. Enzymatic assays on tissue homogenates showed extensive conversion of corticosterone to its 11-dehydro product in an NAD-dependent manner in the submandibular gland, adrenal gland, and kidney, but not in the pancreas or prostate. This study confirms the ubiquitous presence of 11 beta HSD2 in sodium-transporting epithelia, demonstrates the high level of 11 beta HSD2 protein and enzyme activity in the rat adrenal, and suggests a possible role for the enzyme in the biliary system. Further studies are required to determine the relevance of the various molecular species to the activity, latency, and processing of the enzyme.

11 beta-hydroxysteroid dehydrogenase (11-HSD) activity allows aldosterone occupancy of mineralocorticoid receptors by inactivating endogenous glucocorticoids. The expression of the 11-HSD2 gene, a low Km, NAD+ dependent species of 11-HSD, was found in several discrete areas of the rat brain by in situ hybridization. Cells strongly positive for 11-HSD2 mRNA were found in the commissural portion of the nucleus tractus solitarius, subcommissural organ and ventrolateral ventromedial hypothalamus. Scattered labeled cells were also seen in the medial vestibular nucleus. The expression of 11-HSD2 mRNA in the brain is quite distinct from that of 11-HSD1 mRNA and allows for diverse roles in modulating corticosteroid receptor involvement in control of salt appetite, blood pressure and the hypothalamo-pituitary-adrenal axis.

Human Pro-Urokinase (Pro-UK) is expressed in CHO-DHFR- cells at high efficiency by co-transfecting the mouse dhfr gene and Pro-UK cDNA under the control of the SV40 late promoter. After gene co-amplification, the product level could reach 2-3 micrograms/10(6) cells/24 h in the presence of 5 x 10(-6) mol/L MTX, and the levels can be further raised to 3.5-4 micrograms/10(6) cells/24 h by PMA superinduction. The copy number of Pro-UK cDNA in the genomes of host cells is about 200-300 copies/cell. The Western blot analysis shows that the recombinant Pro-UK has similar molecular weight to its natural counterpart, and also the amidolytic activity of the product is determined by S-2444 assay.

Expression vectors containing the pro-urokinase (pro-UK) cDNA (pSV2-proUK) and a dihydrofolate reductase cDNA (pSV2-dhfr or MMTV-dhfr) were cotransfected into CHO-dhfr- cells by the calcium phosphate precipitation technique. The dhfr+ transformants were selected by fibrinolytic agarose plate assay. Two colonies, named CLF-14 and CLF-8, exhibited significantly high expression levels of the biological activity of urokinase-type plasminogen activator (mu-Pa). They reached more than 24 IU/10(6) cells/48 h and 16 IU/10(6) cells/48 h, respectively. Examination of the cell supernatants for mu-Pa antigenicity using ELISA method also showed strong positive results, and the quantities of expression were about 0.14-0.22 micrograms/10(6) cells/48 h and 0.08-0.14 micrograms/10(6) cells/48 h, respectively. The mu-Pa secreted by stable transformed cells could be completely inhibited by UK anti-serum, but not by tissue-type plasminogen activator (t-PA) antiserum nor by normal rabbit serum.

The differences of serum lipid and lipoprotein (LP, profiles of animals susceptible (rabbit) and nonsusceptible (Beijing duck) to atherosclerosis as well as the distribution of apolipoprotein (apo) A-I and its catabolism in vivo were studied. Eight items, i.e. total cholesterol (TC), high density lipoprotein (HDL) cholesterol, triglyceride, percentage of alpha-LP and beta-LP, beta-LP concentration, lecithin cholesterol acyl transferase (LCAT), and agarose and polyacrylamide gel electrophoresis of both species were assayed before and after feeding a high fat, high cholesterol diet. Results indicate that the exogenous cholesterol consumed by Beijing ducks was carried and transported by HDL, while that in rabbits was transported by low density lipoprotein (LDL). The biological half lives of apo A-I in serum and in HDL were 41.1 +/- 2.4 and 42.8 +/- 1.7 h respectively, and its distribution in different organs was in the order of liver greater than kidney greater than spleen greater than lung greater than heart greater than intestine greater than muscles greater than aorta. These results show that the liver is the major organ for metabolizing HDL apo A-I, and the kidney is also important. The results also imply that in Beijing ducks the cholesterol carried by HDL may be catabolized through apo A-I receptors in the liver and kidney. The differences in cholesterol binding and in lipoprotein and apolipoprotein metabolism of the two species provide important clues for the elucidation of the pathogenesis of atherosclerosis.

DNA was extracted from NIH 3T3 cells transformed with DNAs from human primary hepatic cancer (PHC) and Hepatoma 7402 cell line. The transformant DNA was analyzed by Southern transfer and hybridization with 32P-labeled probes of various oncogenes. The EcoRI 7.2 and 9.0 kb bands characteristic of human N-ras gene were identified in transformed NIH 3T3 cells derived both from PHC and 7402 DNA. The BamHI 6.6 kb band characteristic of human c-Ha-ras I was present only in 7402 transformants, but not in PHC transformants. Using 35S-methionine incorporation, immunoprecipitation with anti-p21 monoclonal antibodies, SDS-PAGE and autoradiography, it was demonstrated that p21 synthesis was remarkably enhanced in 7402 cells as well as in transformed cells derived from both 7402 and PHC DNA. Taking the data together, it strongly implies that N-ras is one of the transforming genes for human liver cancer.