Steroid bioconversion in water-insoluble organic solvents: Δ1-Dehydrogenation by free microbial cells and by cells entrapped in hydrophilic or lipophilic gels

A cell suspension in a water-insoluble organic solvent (benzene: n-heptane, 1 : 1 by volume) of Nocardia rhodocrous (previously induced to synthesize steroid Δ¹dehydrogenase) rapidly catalyzed the stoichiometric oxidation of 4-androstene-3,17-dione (4-AD) to androst-l,4-diene-3,17-dione (ADD) in the presence of phenazine methosulfate (PMS). High levels of 4-AD or PMS reduced the conversion rates. No appreciable decrease in the conversion rate was observed on adding aqueous buffer solution to the thawed ceils (up to 9.4 g water/g dry cell). The whole cells were immobilized by entrapment in a hydrophilic gel (H-gel) or a lipophilic gel (L-gel) by use of a water-soluble or water-insoluble photocrosslinkable prepolymer. The reticula of H- and L-gel matrices were impregnated with water and organic solvent, respectively. Both the H- and L-gels could convert 4-AD to ADD in the presence of PMS, the L-gel showing a slightly higher conversion rate. Various lines of evidence indicate that the limiting factor is the penetration rate of 4-AD into gel particles for the H-gel, and the penetration rate of PMS for the L-gel. The catalytic activities decreased considerably after several successive runs with the free cell suspension system, while the immobilized cells were more stable, the stability of H-gel and L-gel being almost the same.     

A novel immunosuppressant FTY720 ameliorates proteinuria and alterations of intrarenal adrenomedullin in rats with autoimmune glomerulonephritis

FTY720 has been originally developed as a new immunosuppressive agent, which prolongs graft survival in organ transplantation. Adrenomedullin (AM) participates in the regulation of sodium homeostasis and has renoprotective effects. The possible involvement of renal AM in the pathophysiology of glomerulonephritis (GN) and the effect of FTY720 has been evaluated in rats. HgCl2 (1 mg/kg body weight) was inoculated subcutaneously 3 times/week for a total of 2 weeks. FTY720 (3 or 10 mg/kg) was inoculated subcutaneously daily. The proteinuria, urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion and serum total cholesterol levels were increased and serum albumin level was reduced in rats with HgCl2-induced GN compared with controls. FTY720 reduced proteinuria (3 mg/kg: -25%; 10 mg/kg: -41%), urinary NAG excretion (-11%; -52%) and total cholesterol level (-21%; -55%) in a dose-dependent manner. Renal AM level and its mRNA expression were increased in rats with GN compared with controls (Peptide Cortex: +69%; Medulla: +82%; mRNA Cortex: +25%). Interestingly, FTY720 additionally increased these levels (Peptide Cortex: +38%; Medulla: +39%; mRNA Cortex: +20%). Renal AM levels correlated with urinary NAG excretion and creatinine clearance. These results suggest that FTY720 suppresses the renal damage in rats with GN and renal AM may participate in the pathophysiology of GN and the renoprotective effects of FTY720.     

Activation of Syk protein tyrosine kinase in response to osmotic stress requires interaction with p21-activated protein kinase Pak2/gamma-PAK

The p21-activated serine/threonine protein kinase Pak2/gamma-PAK and the nonreceptor type of protein tyrosine kinase Syk are known to be activated when the cells are exposed to osmotic stress. The purpose of the present study was to examine whether Pak2 and Syk functionally cooperate in cellular signaling. Cotransfection studies revealed that Pak2 associates with Syk in COS cells. The constitutively active form of Cdc42 increases the association of Pak2 with Syk. Pak2 coexpressed with an inactive form of Cdc42 or kinase-inactive Pak2 interacts to a lesser extent with Syk, suggesting that Pak2-Syk association is enhanced by Pak2 activation. Interaction with Pak2 enhances the intrinsic kinase activity of Syk. This is supported by in vitro studies showing that Pak2 phosphorylates and activates Syk. Treatment of cells with sorbitol to induce hyperosmolarity results in the translocation of Pak2 and Syk to the region surrounding the nucleus and in dramatic enhancement of their association. Furthermore, cotransfection of Pak2 and Syk leads to the activation of c-Jun N-terminal kinase (JNK) under hyperosmotic conditions. Pak2 short interfering RNA suppresses sorbitol-mediated activation of endogenous Syk and JNK, thus identifying a novel pathway for JNK activation by Cdc42. These results demonstrate that Pak2 and Syk positively cooperate to regulate cellular responses to stress.     

cDNA cloning and genetic polymorphism of the swine major histocompatibility complex (SLA) class II DMA gene

cDNA clones corresponding to the swine histocompatibility complex (SLA: swine leucocyte antigen)-DM alpha chain were isolated using the polymerase chain reaction (PCR) products from the third exon in the human HLA-DMA gene as a probe. Amino acid comparative analysis revealed that these clones were more closely related to the bovine and human DMA genes than to the other swine class II genes alpha chain genes, DRA, DQA and DOA. These results suggest that the SLA-DMA gene is expressed and may function, like HLA-DM, as an important modulator in class II restricted antigen processing in swine. Furthermore, based on the sequences and PCR-restriction fragment length polymorphism (PCR-RFLP) patterns in the SLA-DMA gene, no allelic variation was recognized in the second exon, but five allelic variations were recognized in the third exon in five different breeds of swine. These DMA alleles were defined by variation at four nucleotide positions. Two of these alleles resulted in an amino acid substitution. These results suggest that SLA-DMA has little polymorphism as observed in HLA-DMA and mouse H2-Ma.     

Scale-up of hydrophobic interaction chromatography for purification of antitumor antibiotic SN-07

Scale-up of hydrophobic interaction chromatography for purification of SN-07 was studied. The height equivalent to a theoretical plate of the adsorbent gel (Sepabeads FP-PH 12) was kept constant for various superficial liquid velocities and column diameters. The efficiency of purification was also constant for the Sepabeads FP-PH 12 columns ranging from 16 mm to 113 mm in inner diameter. Effects of the ligand concentration on the adsorption capacity were also studied, and the adsorption capacity increased with an increase in the ligand concentration. These effects resulted in decreasing the required amount of salt to purify SN-07.     

Diameter and flow velocity changes in small pulmonary vessels due to microembolization

The pulmonary vascular bed was embolized with glass beads in small doses that induced no significant changes in pulmonary arterial pressure in anesthetized cats. We analyzed changes in internal diameter (ID), flow velocity, and volume flow of embolized and nonembolized arteries simultaneously with ID changes of small veins. In embolized arteries, with 180-, 300-, and 500-microns beads, ID constricted maximally in just proximal portions of the plug by 22, 23, and 17%, respectively, but with 840-microns beads, no ID constriction occurred. With 50-microns beads, the maximum ID constriction occurred in arteries of 200-300 microns but not in those of 100-200 microns. The constriction decreased in the upstream larger arteries and disappeared in those greater than 800 microns ID. In the nonembolized arteries no ID change occurred. Veins constricted slightly compared with arteries. By heparin pretreatment, ID constriction was slightly attenuated in arteries and was almost abolished in veins, whereas it was not affected with hexamethonium bromide. At a branching site, volume flow to an embolized artery decreased because of a decrease in ID and flow velocity, whereas volume flow to a nonembolized artery increased because of an increase in flow velocity. We concluded that pulmonary microembolization induced a vasoconstriction chiefly in small pulmonary arteries upstream to the plug. After embolization, blood flow was locally redistributed from an embolized to a nonembolized artery at a branching site. Arterial vasoconstriction may be mediated chiefly by local mechanical factors.     

Regulation of cytosolic protein-tyrosine kinase from porcine spleen by polyamines and negative-charged polysaccharides

In vitro regulation of cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) by various positive or negative charged compounds was studied. Spermine and spermidine stimulated the activity of CPTK-40 about two-fold using (Val5)angiotensin II as a substrate. This stimulation was not specific for the peptide but was also observed in the case of tubulin phosphorylation indicating a direct effect of these compounds on the enzyme itself. On the contrary, negative-charged polysaccharides were shown to be strong inhibitors of CPTK-40. The possibility of the physiological regulation of CPTK-40 by these compounds is briefly discussed.     

Population parameters and exploitation rate of Sarotherodon galilaeus galilaeus (Cichlidae) in Lakes Doukon and Togbadji,Benin

Growth, mortality, recruitment and relative yield per recruit of Sarotherodon galilaeus galilaeus from Lakes Doukon and Togbadji were studied. Data on total length, total weight and sex were recorded on a monthly basis between January and December 2013 for S. g. galilaeus captured by local fishers. The estimated asymptotic lengths L_∞ were 26.2 and 23.6?cm for Lakes Doukon and Togbadji, respectively, while the growth rate K was 0.73 in Lake Doukon and 0.87 in Lake Togbadji. Estimates of fishing mortality, 0.27 and 0.47 y^?1 for Doukon and Togbadji, respectively, were low relative to natural mortality, 1.51 and 1.74 y^?1, respectively. Sizes at first sexual maturity were 12.8 and 13.2?cm for females and males, respectively, in Lake Doukon, and 11.5 and 12.4?cm for females and males, respectively, in Lake Togbadji. The size at first capture was estimated at 13.3 and 12.7?cm for Lakes Doukon and Togbadji, respectively, which, in the light of the size at maturity estimates, indicates that fish spawn at least once before capture. The current exploitation rates of 0.15 for Lake Doukon and 0.21 for Lake Togbadji suggest that their stocks of S. g. galilaeus are not overexploited in either lake.     

Histopathological biomarkers and genotoxicity in gill and liver tissues of Nile tilapia Oreochromis niloticus from a polluted part of the Nile River,Egypt

Fish health is affected by water pollution. Oreochromis niloticus collected during summer 2014 from El-Serw, a polluted site on the Nile River, were compared with fish from a reference site, El-Zamalek. Histopathological changes were detected in gill and liver tissue samples using light and electron microscopy. In addition, the degree of DNA damage was measured using the comet assay. To indicate the severity of water pollution at the two sites, physico-chemical properties and heavy metal concentrations were investigated. Gill damage, including lamellar cell hyperplasia and aneurysm, was observed in the fish samples from the polluted site. The livers of fish from the polluted area showed necrosis and an increase in melanomacrophage centres. Histochemical results confirmed a marked rise of gill mucopolysaccharides and a reduction of carbohydrate stored in hepatocytes. Electron microscopy revealed clear alterations in gill and liver tissue of fish from the polluted site. The comet assay showed highly significant DNA damage in tilapia collected from the polluted site, compared to those from the reference site. Histopathological biomarkers and the comet assay may therefore be sensitive indicators of exposure to mixtures of aquatic pollutants in Nile tilapia.