Effect of obesity and troglitazone on expression of two glutathione peroxidases: cellular and extracellular types in serum, kidney and adipose tissue

To determine the effect of obesity on expression of cellular- (C-) and extracellular (EC-) glutathione peroxidase (GPX) in serum, kidney and adipose tissue, we measured GPX in serum, kidneys and adipose tissue of the obese Otsuka-Long-Evans-Tokushima Fatty (OLETF) rat and its lean counterpart (LETO). We also investigated the effect of troglitazone. Five each of OLETF and LETO rats were fed diet with or without 0.2% troglitazone for 10 days. Final body weight, kidney weight, blood glucose and serum tumor necrosis factor-alpha (TNF-alpha) level were higher in OLETF rats than in LETO rats. Serum and kidney GPX activities were higher, but adipose tissue GPX activity was lower, in OLETF rats than in LETO rats. Troglitazone treatment decreased adipose tissue GPX activity and abolished overproduction of TNF-alpha in OLETF rats. Immunoblot analysis, for the first time, revealed that both obesity and troglitazone suppressed the protein signals for C-GPX and EC-GPX in adipose tissue. Serum protein carbonyl groups were increased in OLETF rats and troglitazone completely blocked this increase. Increased serum GPX activity in obese rat was due to the increased secretion of EC-GPX from the kidney. Troglitazone protected against the enhanced oxidative stress induced by obesity independently of the serum GPX concentration.     

A-350619: a novel activator of soluble guanylyl cyclase

Nitric oxide (NO) is a key mediator in many physiological processes and one of the major receptors through which NO exerts its effects is soluble guanylyl cyclase. Guanylyl cyclase converts GTP to cyclic GMP as part of the cascade that results in physiological processes such as smooth muscle relaxation, neurotransmission, inhibition of platelet aggregation and immune response. The properties of A-350619, a novel soluble guanylyl cyclase activator, were examined to determine the modulatory effect on the catalytic properties of soluble guanylyl cyclase. A-350619 increased V(max) from 0.1 to 14.5 micromol/min/mg (145 fold increase), and lowered K(m) from 300 to 50 microM (6 fold decrease). When YC-1 (another sGC activator) and A-350619 were combined, a 156 fold increase in V(max) and a 5 fold decrease in Km were observed, indicating that the modulation of the enzyme brought about by YC-1 and A-350619 are not additive, suggesting a common binding site. Activation of soluble guanylyl cyclase by A-350619 was partially inhibited by ODQ, a specific inhibitor of soluble guanylyl cyclase by oxidation of the enzyme heme. YC-1 and A-350619 after pre-treatment with N-omega-nitro-L-arginine, an NO-synthase inhibitor, relaxed cavernosum tissue strips in a dose-dependent manner with EC(50) of 50 microM and 80 microM, respectively. Addition of SNP potentiated the relaxation effect of YC-1 and A-350619, shifting the dose-response curve to the left to 3 microM and 10 microM, respectively. Consistent with its biochemical activity, A-350619 (1 micromol/kg) alone induced penile erection in a conscious rat model. Activation of soluble guanylyl cyclase in cavernosum tissue as an alternate method of enhancing the effect of NO may provide a novel treatment of sexual dysfunction.     

Growth hormone-releasing factor releases ACTH from an AtT-20 mouse pituitary tumor cell line but not from normal pituitary cells

Corticotropin-releasing factor (CRF) and both human pancreatic growth hormone-releasing factor (hp-GRF) and rat hypothalamic GRF (rh-GRF) stimulated ACTH release from neoplastic AtT-20 mouse pituitary tumor cells in a dose-dependent fashion, with CRF inducing a 10-fold increase and GRF a maximal increment of approximately one-half that of CRF. Neither rh-GRF nor hp-GRF induced ACTH release in normal anterior pituitary cells. Pretreatment with either dexamethasone or somatostatin prior to the addition of rh-GRF inhibited the increase in ACTH release. Both ovine CRF and rh-GRF stimulated adenosine 3,5-monophosphate production in AtT-20 cells. The weak but clearly discernible effect of GRF on ACTH release from AtT-20 cells may be due to an abnormality in the AtT-20 cell receptor.     

Immunohistochemical localization and quantitative analysis of cellular glutathione peroxidase in foetal and neonatal rat tissues: Fluorescence microscopy image analysis

Summary To quantitate the developmental changes in selenium-dependent cellular glutathione peroxidase during the perinatal period, tissue sections from foetal (day 12 to day 22) and neonatal (day 6) rats were stained immunohistochemically using specific polyclonal antiserum. The intensity of the staining was quantified by fluorescence microscopy image analysis. There was a general trend of enriched glutathione peroxidase in the epithelial linings and metabolically active sites. Significant fluorescence was detected in cardiomyocytes, hepatocytes, renal tubular epithelium, bronchiolar epithelium and intestinal epithelium at day 15. The intensity increased in a stepwise manner therafter. The overall increase in the intensity of staining in the heart, liver, kidneys, lungs and intestine was 1.5-, 2.3-, 1.6-, 1.7- and 3.0-fold, respectively. The phase of most rapid increase occurred during the foetal period in the liver, intestine and heart. In the kidneys and lungs, glutathione peroxidase increased significantly during foetal life, and to a similar extent postnatally. These results suggest that the intracellular H₂O₂-scavenging system develops during the foetal period as an essential mechanism for living under atmospheric oxygen conditions. The late development observed in the kidneys and lungs is consistent with the relative biological immaturity of these organs in full-term neonates.     

Fibronectin‐binding protein,FbpA, is the adhesin responsible for pathogenesis of Listeria monocytogenes infection

The role of fibronectin binding protein A (FbpA) in Listeria monocytogenes infection and its pathogenesis were studied in vivo and in vitro by constructing a fbpA‐deficient mutant of L. monocytogenes (ΔfbpA). In vivo, ΔfbpA was less pathogenic in mutant mice than was wild‐type L. monocytogenes. FbpA did not affect the amounts of various virulence‐determining factors, including internalin B and listeriolysin O. However, adherence to, and invasion of, mouse hepatocytes by the ΔfbpA mutant were reduced. In contrast, adherence to, but not invasion of, the ΔfbpA mutant to macrophages was attenuated. Fibronectin contributed to the efficient adherence and invasion of wild‐type L. monocytogenes, but not to those of the ΔfbpA mutant. Attenuation of adhesion and uptake of the ΔfbpA mutant were reversed by overexpression of FbpA in it. FbpA was not involved in intracellular growth, autophagy induction or actin tail formation. Thus, the present findings clearly show that FbpA acts as an important adhesion molecule of L. monocytogenes, especially regarding hepatocytes, without modulating the expression of other virulence factors that have been implicated in the pathogenesis of L. monocytogenes infection.     

In vitro culture technique for Cryptocaryon irritans, a parasitic ciliate of marine teleosts

A medium for the in vitro culture of Cryptocaryon irritans, which is an obligatorily parasitic ciliate of marine teleosts and causes 'white spot disease', was developed. The medium consisted of a layer of cultured fish cells (FHM), with an agarose gel layer covering the cell layer. The agarose gel contained 0.22% agarose, 10% fetal calf serum, 100 I.U. ml(-1) Penicillin G potassium and 100 microg ml(-1) streptomycin sulphate. Theronts of C. irritans transformed to trophonts and grew to 180 microm in mean length in the medium, although they gradually decreased in number. When trophonts fully developed in medium were transferred into seawater 4 d after inoculation, approximately 70% of them transformed to encysted tomonts and released theronts. When fish were challenged with theronts obtained from in vitro-raised parasites, approximately 40% of the theronts were recovered from fish, indicating comparative infectivity of in vitro-raised theronts to those of in vivo-raised theronts. This is the first report that C. irritans fully developed in vitro and its entire life cycle was completed without a host fish.     

Positive and negative modulation of Bombyx mori adenylate cyclase by 5-phenyloxazoles: identification of octopamine and tyramine receptor agonists

Nineteen 5-phenyloxazoles (5POs) were examined for their ability to modulate adenylate cyclase by measuring cAMP produced in head membrane homogenates of fifth instar larvae of the silkworm Bombyx mori. Among the compounds tested, 5-(4-methoxyphenyl)oxazole (9) and the 2,6-dichlorophenyl congener showed the highest activation of adenylate cyclase; both compounds produced approximately half the level of cAMP produced by the action of octopamine (OCT). The OCT receptor antagonists chlorpromazine, mianserin, and metoclopramide attenuated 9-stimulated cAMP production. In contrast, 5-(4-hydroxyphenyl)oxazole (8) and the 4-cyanophenyl congener attenuated both OCT-stimulated and basal cAMP production. The tyramine (TYR) receptor antagonist yohimbine inhibited the negative effect of 8. These findings indicate that the 5PO class of compounds includes both positive and negative modulators of adenylate cyclase in the heads of B. mori larvae, and that 9 and 8 are OCT and TYR receptor agonists, respectively. These compounds might prove useful for a pharmacological dissection of biogenic amine receptors.     

A novel antiinflammatory maintains glucocorticoid efficacy with reduced side effects

Glucocorticoids (GCs) are commonly used to treat inflammatory disease; unfortunately, the long-term use of these steroids leads to a large number of debilitating side effects. The antiinflammatory effects of GCs are a result of GC receptor (GR)-mediated inhibition of expression of proinflammatory genes as well as GR-mediated activation of antiinflammatory genes. Similarly, side effects are most likely due to both activated and repressed GR target genes in affected tissues. An as yet unachieved pharmaceutical goal is the development of a compound capable of separating detrimental side effects from antiinflammatory activity. We describe the discovery and characterization of AL-438, a GR ligand that exhibits an altered gene regulation profile, able to repress and activate only a subset of the genes normally regulated by GCs. When tested in vivo, AL-438 retains full antiinflammatory efficacy and potency comparable to steroids but its negative effects on bone metabolism and glucose control are reduced at equivalently antiinflammatory doses. The mechanism underlying this selective in vitro and in vivo activity may be the result of differential cofactor recruitment in response to ligand. AL-438 reduces the interaction between GR and peroxisomal proliferator-activated receptor gamma coactivator-1, a cofactor critical for steroid-mediated glucose up-regulation, while maintaining normal interactions with GR-interacting protein 1. This compound serves as a prototype for a unique, nonsteroidal alternative to conventional GCs in treating inflammatory disease.     

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H(2)O(2) were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H(2)O(2) in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H(2)O(2) via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.