Site-directed mutagenesis of five conserved residues of subunit i of the cytochrome cbb3 oxidase in Rhodobacter capsulatus

Cytochrome cbb(3) oxidase is a member of the heme-copper oxidase superfamily that catalyses the reduction of molecular oxygen to the water and conserves the liberated energy in the form of a proton gradient. Comparison of the amino acid sequences of subunit I from different classes of heme-copper oxidases showed that transmembrane helix VIII and the loop between transmembrane helices IX and X contain five highly conserved polar residues; Ser333, Ser340, Thr350, Asn390 and Thr394. To determine the relationship between these conserved amino acids and the activity and assembly of the cbb(3) oxidase in Rhodobacter capsulatus, each of these five conserved amino acids was substituted for alanine by site-directed mutagenesis. The effects of these mutations on catalytic activity were determined using a NADI plate assay and by measurements of the rate of oxygen consumption. The consequence of these mutations for the structural integrity of the cbb(3) oxidase was determined by SDS-PAGE analysis of chromatophore membranes followed by TMBZ staining. The results indicate that the Asn390Ala mutation led to a complete loss of enzyme activity and that the Ser333Ala mutation decreased the activity significantly. The remaining mutants cause a partial loss of catalytic activity. All of the mutant enzymes, except Asn390Ala, were apparently correctly assembled and stable in the membrane of the R. capsulatus.     

The determination of the relationship between p97/VCP,small VCP-interacting protein,two ERAD proteins and steroidogenesis in Leydig cell lines

Background

In recent studies, it was shown that Endoplasmic reticulum-associated degradation (ERAD) is regulated by androgens and small VCP-interacting protein (SVIP) is an ERAD inhibitor. There is no data available about the interactions of ERAD proteins with proteins involved in steroidogenesis. The aim of the study was to investigate the expressions of SVIP, p97/VCP, StAR, CYP17A1 and 3β-HSD in human and mouse.

Methods and results

HLC, TM3 and MA-10 Leydig cell lines were used to determine roles of ERAD proteins in steroidogenesis based on immunofluorescence, Western blot, qRT-PCR, ELISA. Findings showed that StAR, CYP17A1 and 3β-HSD were colocalized with SVIP and p97/VCP in Leydig cells. A decrease in CYP17A1, 3β-HSD and StAR expressions was observed as a result of suppression of SVIP siRNAs and p97/VCP siRNAs expressions in MA10, TM3 and HLC. When siSVIP transfected cells were compared with siSVIP transfected with hCG-exposed cells, SVIP protein expression was significantly increased as compared to the SVIP transfected group in human Leydig cells.

Conclusion

We suggest that the suppression of protein expressions by p97/VCP and SVIP siRNAs in Leydig cells, the effects of proteins involved in steroidogenesis (StAR, CYP17A1 and 3β-HSD) have proven to be originating from p97/VCP and SVIP which were playing a role in the steroidogenesis process. Additionally, it was demonstrated that testosterone levels decreased after transfection with p97/VCP siRNA and SVIP siRNA, p97/VCP and SVIP created an effect on testosterone synthesis while taking place in the steps of testosterone synthesis. Further, it was determined in the study that the SVIP was affected by hCG stimulations.

     

3-Pyridinylboronic Acid Ameliorates Rotenone-Induced Oxidative Stress Through Nrf2 Target Genes in Zebrafish Embryos

Neurochemical Research - Parkinson’s disease (PD) is one of the most common forms of neurodegenerative diseases and research on potential therapeutic agents for PD continues. Rotenone is a...     

Evaluation of insulin resistance and plasma levels for visfatin and resistin in obese and non-obese patients with polycystic ovary syndrome

This study was designed to evaluate insulin resistance and plasma levels of visfatin and resistin in obese and non-obese patients with polycystic ovary syndrome (PCOS).A total of 37 premenopausal PCOS patients with (n = 18, mean (SD) age: 27.5 (5.7 years) or without obesity (n = 19, mean (SD) age: 23.7 (3.1) years) and healthy volunteers (n = 18, mean (SD) age:29.8 (4.1) years) were included in this study. Data on clinical characteristics, glycemic parameters and lipid parameters were recorded for each subject as were plasma visfatin and resistin levels. Mean (SD) HOMA-IR values were significantly higher in obese PCOS patients (3.4 (1.7)) compared with non-obese PCOS patients (2.0 (1.2), p<0.01) and controls (1.6 (0.8), p<0.01). No significant difference was noted between study groups in terms of plasma resistin (ng/mL) or visfatin (ng/mL) levels. There was no correlation between serum plasma visfatin (r = 0.127, p = 0.407) and resistin (r = -0.096, p = 0.544) levels and HOMA-IR. In conclusion, our findings revealed increased likelihood of metabolic and dyslipidemic manifestations in obese compared to non-obese PCOS patients, while no significant difference was noted in visfatin and resistin levels among PCOS patients in terms of co-morbid obesity and in comparison to controls.     

Purification,biochemical characterization and gene sequencing of a thermostable raw starch digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov.

This study reports the purification and biochemical characterization of a raw starch-digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov. (strain Pizzo^T). The molecular weight was estimated to be 58 kDa by SDS–PAGE. The enzyme was highly active over a wide range of pH from 4.0–10.0. The optimum temperature of the enzyme was 70°C. It showed extreme thermostability in the presence of Ca²⁺, retaining 50% of its initial activity after 90 h at 70°C. The enzyme efficiently hydrolyzed 20% (w/v) of raw starches, concentration normally used in starch industries. The α-amylase showed an high stability in presence of many organic solvents. In particular the residual activity was of 73% in presence of 15% (v/v) ethyl alcohol, which corresponds to ethanol yield in yeast fermentation process. By analyzing its complete amyA gene sequence (1,542 bp), the enzyme was proposed to be a new α-amylase.     

Flocculating performances of exopolysaccharides produced by a halophilic bacterial strain cultivated on agro-industrial waste

This study reports the first systematic investigation of the flocculation dynamics of exopolysaccharides (EPSs) produced by a halophilic bacterial strain grown on pretreated molasses as fermentation substrate. The potential use of these EPSs as an easily biodegradable, natural alternative for synthetic polyelectrolytes which are widely used and contain toxic and carcinogenic monomers was investigated. Flocculating activities of the EPS samples in synthetic water, synthetic sea water and natural sea water media which were used as model raw waters were monitored via the Photometric Dispersion Analyser (PDA 2000) instrument and removals were determined by measuring residual turbidities. One of the six EPS specimens, which formed the largest flocs thus performed highest turbidity removal, exhibited flocculation performance and particle removal efficiency comparable with commercial cationic, nonionic and anionic synthetic polyelectrolytes.     

Inhibition of human breast cancer Matrigel invasion by Streptolysin O activation of the EGF receptor ErbB1

Streptolysin O (SLO) is a protein cytotoxin derived from Group A beta-hemolytic streptococci that associates with membranes and permeabilizes cells. Oxidation inactivates SLO, eliminating the characteristic hemolytic and cytotoxic activities. However, oxidized SLO produces beneficial therapeutic effects in vivo on scleroderma, scar formation and wound healing. Here we report that oxidized SLO also significantly inhibited invasion by human metastatic breast cancer MDA-MB-231 cells through Matrigel in an in vitro model of metastatic disease. This dose-dependent response corresponded to selective SLO activation of epidermal growth factor receptor (EGFR) ErbB1. SLO and EGF were equally selective in activation of EGFR, but EGF elicited larger relative increases in phosphorylation at various sites, especially pronounced for Tyr845. Addition of SLO did not affect either ERK1/2 or Akt kinases and altered the expression of only 10 of 84 metastasis-related genes in MDA-MB-231 cells. Neither SLO nor EGF promoted growth of several human breast cancer cell lines. Knockdown of EGFR by siRNA ablated the inhibitory effect of SLO on cancer cell invasion, showing SLO selectively activated ErbB1 kinase to reduce invasion without increasing cell growth. The results suggest SLO might have promise as a new therapy to inhibit metastasis.     

The efficiency of CAPE on retardation of hepatic fibrosis in biliary obstructed rats

The aim of this study was to evaluate the possible protective effects of caffeic acid phenethyl ester (CAPE) against cholestatic oxidative stress and liver damage in the common bile duct ligated rats. A total of 18 male Sprague–Dawley rats were divided into three groups: control, bile duct ligation (BDL) and BDL + received CAPE; each group contain 6 animals. The rats in CAPE treated groups were given CAPE (10 μmol/kg) once a day intraperitoneally (i.p) for 2 weeks starting just after BDL operation. The changes demonstrating the bile duct proliferation and fibrosis in expanded portal tracts include the extension of proliferated bile ducts into lobules, inflammatory cell infiltration into the widened portal areas were observed in BDL group. Treatment of BDL with CAPE attenuated alterations in liver histology. The proliferating cell nuclear antigen and the activity of TUNEL in the BDL were observed to be reduced with the QE treatment. The application of BDL clearly increased the tissue hydroxyproline (HP) content, malondialdehyde (MDA) levels and decreased the antioxidant enzyme (superoxide dismutase (SOD), glutathione peroxidase (GPx)) activities. CAPE treatment significantly decreased the elevated tissue HP content, and MDA levels and raised the reduced of SOD, and GPx enzymes in the tissues. The data indicate that CAPE attenuates BDL-induced cholestatic liver injury, bile duct proliferation, and fibrosis. The hepatoprotective effect of CAPE is associated with antioxidative potential.