Neuronal nitric oxide synthase: expression in rat parietal cells

Nitric oxide synthases (NOS) are enzymes that catalyze the generation of nitric oxide (NO) from L-arginine and require nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. At least three isoforms of NOS have been identified: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS II). Recent studies implicate NO in the regulation of gastric acid secretion. The aim of the present study was to localize the cellular distribution and characterize the isoform of NOS present in oxyntic mucosa. Oxyntic mucosal segments from rat stomach were stained by the NADPH-diaphorase reaction and with isoform-specific NOS antibodies. The expression of NOS in isolated, highly enriched (>98%) rat parietal cells was examined by immunohistochemistry, Western blot analysis, and RT-PCR. In oxyntic mucosa, histochemical staining revealed NADPH-diaphorase and nNOS immunoreactivity in cells in the midportion of the glands, which were identified as parietal cells in hematoxylin and eosin-stained step sections. In isolated parietal cells, decisive evidence for nNOS expression was obtained by specific immunohistochemistry, Western blotting, and RT-PCR. Cloning and sequence analysis of the PCR product confirmed it to be nNOS (100% identity). Expression of nNOS in parietal cells suggests that endogenous NO, acting as an intracellular signaling molecule, may participate in the regulation of gastric acid secretion.     

Anti-Tumor Activity of Eurycoma longifolia Root Extracts against K-562 Cell Line: In Vitro and In Vivo Study

Eurycoma longifolia Jack has been widely used in traditional medicine for its antimalarial, aphrodisiac, anti-diabetic, antimicrobial and anti-pyretic activities. Its anticancer activity has also been recently reported on different solid tumors, however no anti-leukemic activity of this plant has been reported. Thus the present study assesses the in vitro and in vivo anti-proliferative and apoptotic potentials of E. longifolia on K-562 leukemic cell line. The K-562 cells (purchased from ATCC) were isolated from patients with chronic myelocytic leukemia (CML) were treated with the various fractions (TAF273, F3 and F4) of E. longifolia root methanolic extract at various concentrations and time intervals and the anti-proliferative activity assessed by MTS assay. Flow cytometry was used to assess the apoptosis and cell cycle arrest. Nude mice injected subcutaneously with 10⁷ K-562 cells were used to study the anti-leukemic activity of TAF273 in vivo. TAF273, F3 and F4 showed various degrees of growth inhibition with IC50 values of 19, 55 and 62 µg/ml, respectively. TAF273 induced apoptosis in a dose and time dependent manner. TAF273 arrested cell cycle at G1and S phases. Intraperitoneal administration of TAF273 (50 mg/kg) resulted in a significant growth inhibition of subcutaneous tumor in TAF273-treated mice compared with the control mice (P = 0.024). TAF273 shows potent anti-proliferative activity in vitro and in vivo models of CML and therefore, justifies further efforts to define more clearly the potential benefits of using TAF273 as a novel therapeutic strategy for CML management.     

Virulence Characterization of Salmonella enterica by a New Microarray: Detection and Evaluation of the Cytolethal Distending Toxin Gene Activity in the Unusual Host S. Typhimurium

Salmonella enterica is a zoonotic foodborne pathogen that causes acute gastroenteritis in humans. We assessed the virulence potential of one-hundred and six Salmonella strains isolated from food animals and products. A high through-put virulence genes microarray demonstrated Salmonella Pathogenicity Islands (SPI) and adherence genes were highly conserved, while prophages and virulence plasmid genes were variably present. Isolates were grouped by serotype, and virulence plasmids separated S. Typhimurium in two clusters. Atypical microarray results lead to whole genome sequencing (WGS) of S. Infantis Sal147, which identified deletion of thirty-eight SPI-1 genes. Sal147 was unable to invade HeLa cells and showed reduced mortality in Galleria mellonella infection model, in comparison to a SPI-1 harbouring S. Infantis. Microarray and WGS of S. Typhimurium Sal199, established for the first time in S. Typhimurium presence of cdtB and other Typhi-related genes. Characterization of Sal199 showed cdtB genes were upstream of transposase IS911, and co-expressed with other Typhi-related genes. Cell cycle arrest, cytoplasmic distension, and nuclear enlargement were detected in HeLa cells infected by Sal199, but not with S. Typhimurium LT2. Increased mortality of Galleria was detected on infection with Sal199 compared to LT2. Thus, Salmonella isolates were rapidly characterized using a high through-put microarray; helping to identify unusual virulence features which were corroborated by further characterisation. This work demonstrates that the use of suitable screening methods for Salmonella virulence can help assess the potential risk associated with certain Salmonella to humans. Incorporation of such methodology into surveillance could help reduce the risk of emergence of epidemic Salmonella strains.     

Effect of Physalis peruviana L. on Cadmium-Induced Testicular Toxicity in Rats

Cadmium (Cd) stimulates the production of reactive oxygen species and causes tissue damage. We investigated here the protective effect of Physalis peruviana L. (family Solanaceae) against cadmium-induced testes toxicity in rats. Twenty-eight Wistar albino rats were used. They were divided into four groups (n?=?7). Group 1 was used as control. Group 2 was intraperitoneally injected with 6.5 mg/kg body weight (bwt) of cadmium chloride for 5 days. Group 3 was orally treated with 200 mg/kg bwt of methanolic extract of physalis (MEPh). Group 4 was pretreated with MEPh before cadmium for 5 days. Changes in body and testes weights were determined. Oxidative stress markers, antioxidant enzymes, and testosterone level were measured. Histopathological changes of testes were examined, and the immunohistochemical staining for the proapoptotic (caspase-3) protein was performed. The injection of cadmium caused a significant decrease in body weight, while a significant increase in testes weight and testes weight index was observed. Pretreatment with MEPh was associated with significant reduction in the toxic effects of Cd as shown by reduced testicular levels of malondialdehyde, nitric oxide, and caspase-3 expression and increased glutathione content, and the activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and testosterone were also increased. Testicular histopathology showed that Cd produced an extensive germ cell apoptosis, and the pretreatment of MEPh in Cd-treated rats significantly reduced Cd-induced testicular damage. On the basis of the above results, it can be hypothesized that P. peruviana L. has a protective effect against cadmium-induced testicular oxidative stress and apoptosis in the rat.     

Simultaneous determination of metolazone and losartan potassium in their binary mixtures using high‐performance liquid chromatography with fluorimetric detection: application to combined tablets and spiked human plasma

A new, specific and sensitive reversed‐phase high‐performance liquid chromatography method was developed for the simultaneous determination of metolazone (MET) and losartan potassium (LOS). Good chromatographic separation was achieved within 6.0 min on a 150 × 4.6 mm i.d., 5 µm Waters, Ireland and ProDIGY 5 ODS 3 100 A column. A mobile phase containing a mixture of methanol and 0.02 M phosphate buffer (65:35, v/v) at pH 3.0 was used. The analysis was performed at a flow rate of 1 mL/min with fluorescence detection at 410 nm after excitation at 230 nm. Aspirin (ASP) was used as an internal standard. The proposed method was rectilinear over 2.0–40.0 (MET) and 40.0–800.0 ng/mL (LOS), with limits of detection of 0.22 and 4.52 ng/mL and limits of quantification of 0.68 and 13.70 ng/mL for MET and LOS, respectively. The method was successfully applied for the simultaneous analysis of the studied drugs in their laboratory‐prepared mixtures, single tablets and co‐formulated tablets. Moreover, the method was applied to an in vitro drug release (dissolution) test. The method was further extended to the determination of LOS in spiked human plasma. Statistical evaluation and comparison of data obtained using the proposed and comparison methods revealed no significant difference between the two methods in addition to good accuracy and precision for the proposed method. Copyright © 2013 John Wiley & Sons, Ltd.     

Production and characterisation of exopolysaccharide from Streptomyces carpaticus isolated from marine sediments in Egypt and its effect on breast and colon cell lines

Twenty streptomycete strains were isolated from marine sediment samples collected from Nabq area, Sharm El-Sheikh, Red Sea Coast, Egypt. Four of them produce exopolysaccharides (EPS) showing marked in vitro antitumor activities. Morphological and cultural characteristics of the most significant strain (No. 3) were shown. Moreover, the sequence of this strain showed similarity with Streptomyces carpaticus. The results reveal that EPS produced by Streptomyces carpaticus No. 3 had high cytotoxicity reaching 51.7% and 59.1% against human tumor cells of breast and colon lines respectively. A chemical analysis of EPS indicated that the composing monosaccharides were galactouronic acid, glucose, xylose, galactose, mannose, and fructose with relative ratio of 3:1:1:2:2:1 respectively, with an average molecular weight (Mw) 1.180 × 10⁵?g/mol and of a number average molecular weight (Mn) 1.052 × 10⁵?g/mol. Also the EPS contained uronic acid (0.5072%) and monosaccharide sulphates (21.753%).     

MicroRNAs 9 and 370 Association with Biochemical Markers in T2D and CAD Complication of T2D

BackgroundMicroRNAs (miRNAs) are small non coding RNAs with essential roles, of which any alteration leads to several conditions. Their roles in diabetes (DM) and its vascular complications have not been completely assessed.Aimto study the association of two miRNAs; 9 and 370, with biochemical parameters of type 2 diabetic (T2D), dyslipidemia and coronary artery disease (CAD).ResultsmiRNA 9 levels were significantly higher in T2D patients and T2D patients with CAD, (1.18±0.07, and 1.31±0.08 respectively), while miRNA 370 levels were significantly higher in T2D patients, CAD patients, and T2D patients with CAD (0.59±0.05, 1.00±0.05, and 1.20±0.06 respectively), compared to control group at p = 0.000. In addition both miRNAs were still significantly associated with each other even after conducting multiple regression analysis.ConclusionThis study associates the possible role of miRNAs in the diagnosis/prognosis of CAD complication of T2D.     

BEACON: automated tool for Bacterial GEnome Annotation ComparisON

Background

Genome annotation is one way of summarizing the existing knowledge about genomic characteristics of an organism. There has been an increased interest during the last several decades in computer-based structural and functional genome annotation. Many methods for this purpose have been developed for eukaryotes and prokaryotes. Our study focuses on comparison of functional annotations of prokaryotic genomes. To the best of our knowledge there is no fully automated system for detailed comparison of functional genome annotations generated by different annotation methods (AMs).

Results

The presence of many AMs and development of new ones introduce needs to: a/ compare different annotations for a single genome, and b/ generate annotation by combining individual ones. To address these issues we developed an Automated Tool for Bacterial GEnome Annotation ComparisON (BEACON) that benefits both AM developers and annotation analysers. BEACON provides detailed comparison of gene function annotations of prokaryotic genomes obtained by different AMs and generates extended annotations through combination of individual ones. For the illustration of BEACON’s utility, we provide a comparison analysis of multiple different annotations generated for four genomes and show on these examples that the extended annotation can increase the number of genes annotated by putative functions up to 27 %, while the number of genes without any function assignment is reduced.

Conclusions

We developed BEACON, a fast tool for an automated and a systematic comparison of different annotations of single genomes. The extended annotation assigns putative functions to many genes with unknown functions. BEACON is available under GNU General Public License version 3.0 and is accessible at: http://www.cbrc.kaust.edu.sa/BEACON/.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1826-4) contains supplementary material, which is available to authorized users.