Expression Levels of Vascular Endothelial Growth Factors A and C in Patients with Peptic Ulcers and Gastric Cancer

PURPOSE: Vascular endothelial growth factor (VEGF) is one of the most important growth factors for metastatic tumors. To clarify the role of VEGF-A and C in patients with peptic ulcer disease (PUD) or gastric cancer (GC), we evaluated the expression levels of these two molecules. We also analyzed the effect of Helicobacter pylori infection on VEGF-A and C expression levels. MATERIALS AND METHODS: Patients with dyspepsia who needed diagnostic endoscopy were selected and divided into three groups: non-ulcer dyspepsia (NUD), PUD, and GC, according to their endoscopic and histopathological results. Fifty-two patients with NUD, 50 with PUD, and 38 with GC were enrolled in this study. H. pylori infection was diagnosed by the rapid urease test. After RNA extraction and synthesis of cDNA, the expression levels of VEGF-A and C were determined by quantitative reverse transcriptase polymerase chain reaction. RESULTS: The VEGF-C expression level in the PUD and GC groups was significantly higher than that in the NUD group. Moreover, the VEGF-A expression level in the PUD and GC groups was higher than in the NUD group, although the differences were not statistically significant. Significant positive correlations were also observed between the expression levels of these two molecules in the PUD and GC groups. In addition, the expression levels of these two molecules were higher in H. pylori positive patients with PUD or GC than in H. pylori negative patients of the same groups; however, these differences did not reach statistical significance. CONCLUSIONS: Up-regulation of VEGF-C expression during gastric mucosal inflammation may play a role in the development of peptic ulcers or GC.

Effects of Vitamin-E treatment on CatSper genes expression and sperm quality in the testis of the aging mouse

CatSper genes are a novel family of four sperm-specific calcium channels, which indicate testis-specific expression patterns. Despite the crucial role of CatSper genes in the male reproduction, very little is known about the factors that regulate their expression. The objective of this study was to investigate the effects of vitamin E treatment on the expression of CatSper 1 and CatSper 2 genes as well as sperm quality in the aged male mice. Twenty four 11-12 months old aged male mice and twenty four 2-3-months old young male mice were randomly divided into four groups. Control groups received no injection. The experimental groups of male mice were received intraperitoneal injection of 106 mg/kg vitamin E daily for 35 days. Left testis and cauda epididymides from each mouse were collected on the days 21, 28 and 35 following vitamin E treatment and were used for Real-Time PCR and immunohistochemistry. Also, sperm analysis was performed according to the WHO guidelines given for human sperm examination. Data were analyzed using SPSS software Administration of vitamin E improved sperm parameters in the aged as well as young adult male mice. In addition, the expression of CatSper genes increased following vitamin E treatment. Also, intensity of signal for CatSper1 and CatSper2 increased in the head and middle piece of sperm in experimental group as compared to those of control ones. The vitamin E treatment significantly improved the sperm quality, especially in terms of sperm motility, count and morphology rate. Furthermore, CatSper genes expression could be up-regulated by the vitamin E treatment

Molecular cloning, expression and purification of protein TB10.4 secreted by Mycobacterium tuberculosis

Tuberculosis [TB] is the leading cause of mortality among the infectious diseases, especially in developing countries. One of the main goals in tuberculosis research is to identify antigens which have the ability of inducing cellular and/or humoral immunity in order to use them in diagnostic reagents or vaccine design. The aim of this study was to clone and express the TB10.4 protein in Escherichia coli expression system. DNA was extracted from Mycobacerium tuberculosis H37Rv. Gene specific primers were designed using Gene Runner software according to sanger sequence database. Gene tb 10.4 fragment was amplified by PCR method and purified tb 10.4 gene was cloned into pET 102/D vector. Plasmid containing pET 102/D-10.4 was transformed into competence E. coli TOP10. A positive transformant was chosen and plasmids DNA was isolated and subsequently transformed into competence E. coli BL21 [DE3]. The bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE. Purified recombinant protein was achieved using metal affinity chromatography [Ni-nitrilotriacetic acid]. TB10.4 molecule was successfully cloned, expressed, and purified. An approximately 26.4 kDa exogenous protein was observed on the SDS-PAGE. The recombinant protein was confirmed by DNA sequencing of correct insert. The success of expressing the TB10.4 protein could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of submit vaccine and diagnostic method

Production and characterization of monoclonal antibody against saffron pollen profilin, Cro s 2

Allergy to Saffron [Crocus sativus] pollen has been described in people involved in processing of saffron flower stamens. Profilins have been identified as a pan-allergen in different plant pollens and foods. This molecule is an actin-binding protein with a molecular weight of 12-16 kDa found in eukaryotic species. The aim of this study was to generate monoclonal antibody against Cro s 2 in order to characterize this major allergen of saffron pollen. BALB/c mice were immunized to obtain adequate humoral response. Splenocytes were prepared from the immunized animals, mixed with the P3-X63-Ag8.653 myeloma cells and fused by means of PEG 1500. After two weeks of culturing in HAT-containing media, the supernatant from those wells growing hybridomas were screened by ELISA using plates coated with Cro s 2. Cells from positive wells were cloned at least 3 times by limiting dilution. Specificity and cross-reactivity of the mAbs were determined by Western blot analysis and sandwich ELISA. Two stable hybridoma clones secreting mAbs against Cro s 2 were obtained and expanded. The anti-Cro s 2 mAbs were also found to cross-react with other plant profilins. Isotype of this mAb was identified as micro heavy chain and k light chain. The anti-Cro s 2 mAb could be a useful tool for characterization and standardization of many pollen and fruit-derived profilins

Specific IgG antibodies [total and subclasses] against saffron pollen: a study of their correlation with specific IgE and immediate skin reactions

Saffron [Zaaferan], botanical name Crocus sativus, is the most expensive spice in the world. It is derived from the dried stigma and pistil of the purple saffron crocus flowers. Iran is the largest saffron producer accounting for more than 80% of the world's production. Saffron contains an aeroallergen that causes reactive respiratory allergic reactions in atopic subjects. IgG antibody to allergens in the serum of allergic patients is not routinely measured. In this study in order to find out more about mechanism of allergy against saffron pollen, specific antibodies [IgE and IgG, total and subclasses] in atopic subjects were assayed. We used an ELISA assay for measuring specific IgE and IgG against saffron pollen extract in the sera of 38 atopic subjects [test group] and 20 non allergic subjects [control group]. The optical densities were compared between allergic subjects and non-allergic individuals. The prick test with saffron pollen extract was used to evaluate the cutaneous and specific antibody responses in the allergic subjects. The correlation was determined by statistical analysis. Specific saffron pollen IgE and IgG subclasses were found significantly higher in the allergic subjects than the control group. The immediate skin reaction was found positive in 70% of the test group. We report here, the existence of a positive correlation between specific IgE and skin reaction by prick test in atopic subjects [R=0.433]. A negative correlation between specific IgE and IgG4 subclass was also found [R=-0.576]. These data may be useful to understand the mechanism of allergy to saffron and may help in clarifying clinical manifestations and to prevent IgE production as well as therapeutic application