Twin‐arginine translocation system (tat) mutants of Salmonella are attenuated due to envelope defects,not respiratory defects

The twin‐arginine translocation system (Tat) transports folded proteins across the cytoplasmic membrane and is critical to virulence in Salmonella and other pathogens. Experimental and bioinformatic data indicate that 30 proteins are exported via Tat in Salmonella Typhimurium. However, there are no data linking specific Tat substrates with virulence. We inactivated every Tat‐exported protein and determined the virulence phenotype of mutant strains. Although a tat mutant is highly attenuated, no single Tat‐exported substrate accounts for this virulence phenotype. Rather, the attenuation is due primarily to envelope defects caused by failure to translocate three Tat substrates, the N‐acetylmuramoyl‐l ‐alanine amidases, AmiA and AmiC, and the cell division protein, SufI. Strikingly, neither the amiA amiC nor the sufI mutations alone conferred any virulence defect. Although AmiC and SufI have previously been localized to the divisome, the synthetic phenotypes observed are the first to suggest functional overlap. Many Tat substrates are involved in anaerobic respiration, but we show that a mutant completely deficient in anaerobic respiration retains full virulence in both the oral and systemic phases of infection. Similarly, an obligately aerobic mutant is fully virulent. These results suggest that in the classic mouse model of infection, S. Typhimurium is replicating only in aerobic environments.     

Effect of fish oil supplementation on plasma oxidant/antioxidant status in rats

The aim of this study was to investigate effect of dietary omega-3 fatty acid supplementation on the indices of in vivo lipid peroxidation and oxidant/antioxidant status of plasma in rats. The plasma thiobarbituric acid reactive substances (TBARS) and nitric oxide (NO) levels, and activities of xanthine oxidase (XO), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were studied in male Wistar Albino rats after ingestion of 0.4 g/kg fish oil (rich in omega-3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid) for 30 days and compared to untreated control rats. The rats in the treated group had significantly higher SOD activity (P < 0.001), NO levels (P < 0.01) and decreased TBARS levels (P < 0.05) with respect to controls whereas GSH-Px and XO activities were not significantly different between the groups. None of the measured parameters had significant correlation with each other in both groups. We conclude that dietary supplementation of omega-3 fatty acids may enhance resistance to free radical attack and reduce lipid peroxidation. These results support the notion that omega-3 fatty acids may be effective dietary supplements in the management of various diseases in which oxidant/antioxidant defence mechanisms are decelerated.     

Gender influence on jejunal migrating motor complex

The role of gender and the menstrual cycle in small bowel motility has not been clearly elucidated. Jejunal motility was recorded with a nasojejunal catheter incorporating five solid-state pressure transducers in ambulatory menstruating women and men of comparable age over 24 h. All women were studied twice, in the early follicular (early-F) and midluteal (mid-L) phases of the menstrual cycle, verified by determining serum levels of gonadal steroids and gonadotropins. The propagation velocity of phase III was slow and the contraction amplitude was high in both menstrual cycle phases compared with men, and these parameters were correlated with serum estrogen levels in the mid-L phase. In the early-F phase, migrating motor complex (MMC) cycle duration during sleep was long compared with other groups and positively correlated with estrogen concentrations, whereas in the mid-L phase MMC cycle duration during sleep was negatively correlated with serum progesterone levels. In all groups, the frequency of phase III contractions was low and the intercontractile interval measured from pressure peak to peak was long during sleep compared with the awake state. Postprandial motility did not display gender difference in any parameter examined. The results demonstrate that the majority of patterns of motility are similar in menstruating women and men, whereas certain aspects of the MMC, most conspicuously propagation velocity and phase III contraction amplitude, differ. We have also documented circadian variation of phase III contraction frequency in both women and men.     

Optimization of nutrient parameters for lovastatin production by <Emphasis Type="Italic">Monascus purpureus</Emphasis> MTCC 369 under submerged fermentation using response surface methodology

Lovastatin, an inhibitor of HMG-CoA reductase, was produced by submerged fermentation using Monascus purpureus MTCC 369. Five nutritional parameters screened using Plackett–Burman experimental design were optimized by Box–Behnken factorial design of response surface methodology for lovastatin production in shake flask cultures. Maximum lovastatin production of 351 mg/l were predicted in medium containing 29.59 g/l dextrose, 3.86 g/l NH₄Cl, 1.73 g/l KH₂PO₄, 0.86 g/l MgSO₄·7H₂O, and 0.19 g/l MnSO₄·H₂O using response surface plots and point prediction tool of DESIGN EXPERT 7.0 (Statease, USA) software.     

Electrochemical sensors, MTT and immunofluorescence assays for monitoring the proliferation effects of cissus populnea extracts on Sertoli cells

Background

The mechanism of theca cell layer formation in mammalian ovaries has not been elucidated; one reason is that there is no follicle culture system that can reproduce theca cell layer formation in vitro. Therefore, a three-dimensional follicle culture system that can reproduce theca cell layer formation is required.

Methods

A collagen gel was used in the follicle culture system. To determine the optimum conditions for follicle culture that can reproduce theca cell layer formation, the effects of hormonal treatment and cell types co-cultured with follicles were examined. In addition, immunohistochemistry was used to examine the properties of the cell layers formed in the outermost part of follicles.

Results

Follicles maintained a three-dimensional shape and grew in collagen gel. By adding follicle-stimulating hormone (FSH) and co-culturing with interstitial cells, the follicles grew well, and cell layers were formed in the outermost part of follicles. Immunohistochemistry confirmed that the cells forming the outermost layers of the follicles were theca cells.

Conclusion

In this study, follicle culture system that can reproduce theca cell layer formation in vitro was established. In our opinion, this system is suitable for the analysis of theca cell layer formation and contributes to our understanding of the mechanisms of folliculogenesis.     

Protective agent, erdosteine, against cisplatin-induced hepatic oxidant injury in rats

Cisplatin, one of the most active cytotoxic agents against cancer, has several toxicities. Hepatotoxicity is one of them occurred during high doses treatment. The aim of this study was to determine the effects of erdosteine against cisplatin-induced liver injury through tissue oxidant/antioxidant parameters and light microscopic evaluation. The rats were randomly divided into three groups: control (n=5), cisplatin (10 mg/kg, n=6) and cisplatin+erdosteine (50 mg/kg/day oral erdosteine, n=8) groups. The rats were sacrificed at the 5th day of cisplatin treatment. The liver tissues were examined with light microscopy and oxidant/antioxidant biochemical parameters. The malondialdehyde (MDA) and nitric oxide (NO) levels were increased in the cisplatin group in comparison with the control and cisplatin+erdosteine groups (p<0.05). There was no significant difference in MDA and NO levels between control and cisplatin+erdosteine groups. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were higher in cisplatin+erdosteine group than cisplatin group (p<0.05). However, the CAT and GSH-Px activities were significantly lower in cisplatin group than in control group (p<0.05). The light microscopic examination revealed that cytoplasmic changes especially around cells of central vein were observed in cisplatin group. Hepatocellular vacuolization was seen in these cells. In the cisplatin plus erdosteine group, a decrease in cytoplasmic changes with the hepatocytes and sinusoidal dilatations around cells of central vein were noticed in as compared to cisplatin group. In the light of microscopic and biochemical results, it was concluded that cisplatin-induced liver damage in high dose and erdosteine prevented this toxic side effect by the way of its antioxidant and radical scavenging effects. (Mol Cell Biochem 278: 79–84, 2005)     

Homocysteine levels in acromegaly patients

Acromegaly is associated with a two to three-fold increase in mortality related predominantly to cardiovascular disease. The excess mortality is associated most closely with higher levels of growth hormone (GH). Survival in acromegaly may be normalized to a control age-matched rate by controlling GH levels; in particular, GH levels less than 2.5 ng/mL are associated with survival rates equal to those of the general population. Hyperhomocysteinemia has also been recognized as a risk factor for cardiovascular disease, yet there are limited data on the prevalence of hyperhomocysteinemia in patients with acromegaly. Eighteen acromegaly patients (7 male, 11 female, mean age 42.8 +/- 11.0 years) in our endocrine clinic consented to having the following tests performed: complete blood count (CBC), thyroid hormones, folic acid, vitamin B12, plasma homocysteine levels, uric acid, fibrinogen, CRP, fasting glucose, insulin, C-peptide, total serum cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, GH, insulin-like growth factor-1 (IGF-1) and GH levels after an oral glucose tolerance test (OGTT). By history, fourteen had macroadenomas and four had microadenomas; eight had hypertension; two had glucose intolerance, and four had diabetes. Fifteen had had transsphenoidal or transfrontal surgery: two had been cured, but 13 others were taking long-acting octreotide. Five patients had undergone radiotherapy and the acromegaly in two was treated primarily with long-acting octreotide. CBC, thyroid hormone, folic acid, and vit B12 levels were normal in all patients. We divided the patients into two groups according to mean GH levels after an OGTT: Group 1 (GH<2.5 ng/mL, n=10), and Group 2 (GH<2.5 ng/mL, n=8). Comparison of the two groups using Mann-Whitney U testing revealed statistically significant lower levels in Group 1 of the following parameters: GH (1.91 +/- 0.90 vs. 8.58 +/- 5.55 ng/mL, p=0.002), IGF-1 (338.30 +/- 217.90 vs. 509.60 +/- 293.58 ng/dL, p=0.06), GH after an OGTT (1.42 +/- 0.81 vs. 9.01 +/- 4.53 ng/mL, p=0.001), plasma homocysteine (12.85 +/- 4.47 vs. 18.20 +/- 4.99 micromol/L, p=0.05), total cholesterol (164.0 +/- 20.81 vs. 188.0 +/- 22.26 mg/dL, p=0.05) and LDL cholesterol (81.0 +/- 9.64 vs. 116.70 +/- 13.03 mg/dl, p=0.01). Differences between the other parameters were not significantly different. Acromegaly patients with high GH levels after an OGTT have much higher levels of homocysteine than patients with lower GH levels. The role of elevated homocysteine levels as an independent cardiovascular risk factor in the mortality of acromegaly patients should be determined in future studies.     

Anaesthesia and the acute phase protein response in children undergoing circumcision

Concentrations of acute phase proteins (CRP: C-reactive protein, albumin) change during surgery. We investigated the acute phase response to circumcision and the effects of anaesthesia on this response. The children were divided into four groups; group 1 (intratracheal general anaesthesia, n=40), group 2 (general anaesthesia with mask, n=20), group 3 (ketamine, n=20), group 4 (local anaesthesia, n=35). Blood samples were obtained, 24 hours before circumcision, after premedication, and 24 hours after circumcision. CRP and albumin before circumcision were comparable for all groups. There was no increase in CRP, and albumin remained steady throughout the study. No difference was observed among the groups, and related to anaesthesia. No responsiveness may be explained with the size of injured tissue or anatomical and histological type of preputium.     

Phenotypic analysis of pneumococcal polysaccharide-specific B cells

The phenotype of B cells responsible for the production of anti-pneumococcal polysaccharide Ab has been unclear. Although individuals that respond poorly to the 23-valent pneumococcal polysaccharide (PPS) vaccine, Pneumovax, such as children <2 y, the asplenic, and a subset of common variable immunodeficiency patients, are profoundly deficient or lack IgM memory cells (CD27(+)IgM(+)), they are also deficient in the switched memory (CD27(+)IgM(-)) compartment. Direct characterization of PPS-specific B cells has not been performed. In this study, we labeled PPS14 and PPS23F with fluorescent markers. Fluorescently labeled PPS were used in FACSAria flow cytometry to characterize the phenotype of PPS-specific B cells obtained from 18 young adults pre- and postimmunization with Pneumovax. The labeled PPS were capable of inhibiting binding of Ab to the native PPS. Similarly, the native PPS were able to inhibit binding of PPS-specific B cells in a flow cytometric assay demonstrating specificity and functionality. Phenotypic analysis of unselected B cells, pre- and postimmunization, demonstrated a predominance of naive CD27(-)IgM(+) cells accounting for 61.5% of B cells. Likewise, the PPS-specific B cells obtained preimmunization consisted primarily of naive, CD27(-) B cells, 55.4-63.8%. In contrast, the PPS-specific B cells obtained postimmunization were predominantly IgM memory cells displaying the CD27(+)IgM(+), 54.2% for PPS14 and 66% for PPS23F, significantly higher than both unselected B cells and PPS-specific B cells. There was no significant difference in switched memory B cell populations (CD27(+)IgM(-)) between groups. These results suggest a dominant role of IgM memory cells in the immune response to pneumococcal polysaccharides.     

Effect of short-term interferon-β treatment on cytokines in multiple sclerosis: significant modulation of IL-17 and IL-23

Therapeutic effect of interferon-β (IFN-β) treatment has been associated with modulation of the balance between Th1, Th17, Th2 and regulatory T (Treg) cells, whereas the impact of disease modifying drugs on Th9-immunity in multiple sclerosis (MS) has not been studied. To investigate the short-term effects of IFN-β treatment on cytokines in MS, we determined serum levels of IL-17, IL-23, IL-10, IL-4, IFN-γ, IL-9 and TGF-β in relapsing remitting MS patients before and 2 months after IFN-β treatment by ELISA. MS patients showed increased IL-17, IL-23 and IL-4 levels and decreased IL-9 levels as compared to healthy controls. IFN-β treatment only reduced IL-17 and IL-23 levels, whereas the levels of other cytokines remained unchanged. IFN-β treatment appears to exert its earliest therapeutic effect on Th17-immunity. The influence of IL-9 on MS pathogenesis needs to be further studied.