Association of MicroRNA Polymorphisms With Hepatocellular Carcinoma in an Iranian Population

BACKGROUND: Single nucleotide polymorphisms (SNPs) can modulate various biological processes by influencing microRNA (miRNA) biogenesis and altering target selection. Common SNPs may alter the processing of miRNA and may be associated with hepatocellular carcinoma (HCC). We investigated the relationship between miR-499A>G, miR-149C>T, miR-196a2T>C, and miR-146aG>C and HCC susceptibility, examining the interaction of the miRNAs with hepatitis B virus (HBV). METHODS: We evaluated the associations of miR-499A>G (rs3746444), miR-149C>T (rs2292832), miR-196a2T>C (rs11614913), and miR-146aG>C (rs2910164) with HCC susceptibility in 100 HCC patients (70 males and 30 females) and 120 healthy controls (70 males and 50 females), using the PCR-restriction fragment length polymorphism method. RESULTS: For miR-499A>G, the frequencies of the AG genotype and G allele were higher in female HCC patients than in female controls (P=0.02 and 0.045, respectively). The frequency of the A allele was higher in HBV-positive HCC patients than in controls (P=0.019). For miR-149C>T, the frequency of the CC genotype was higher in female HCC patients than in female controls (P=0.009). For miR-196a2T>C, the frequencies of the CT and CC genotypes and the C allele were higher in HBV-positive HCC patients than in controls (P C polymorphisms did not differ between HCC patients and controls. CONCLUSIONS: miR-499A>G, miR-149C>T, and miR-196a2T>C were associated with the development of HCC in women and/or that of HBV-related HCC. They can be considered genetic risk factors for the development of HCC among Iranians.

[Designing and producing of RNA interference [RNAi] in order to reduce the expression of a candidate gene involved in cancer]

Background: cancer is one of the most important factors which affect the human health .RNA interference [RNAi] based therapies have been developed and showed significant results in this regard. One of the most important problems of siRNA i system is its low stability. In order to overcome to this weakness, venous types of hairpin RNA or shRNA was introduced Over expressed colorectal cancer gene [OCC-I], which has several transcripts is considered as a hallmark of colorectal cancer. This gene has vital role in the development and progression of cancer through the cell signaling pathways, which are involved in cell proliferation. In the present study, the RNAi system was utilized in order to reduce the expression of specific transcript of OCC-1 gene

Methods: the silencer structure was designed and cloned in the expression vector pRNA-H1.l / Neo which has a promoter for RNApolIII. Plasmid was transferred into the colorectal cancer cell line, and the expression of transcript was compared to the control samples at 24 and 72 h post transfection by Real-Time PCR

Results: the expression of transcript was reduced about 25 % at 72 h post transfection following shRNA transfection, compared to the control sample

Conclusion: the designed shRNA structure successfully reduced the expression of target gene; accordingly, it can be used in future studies in order to investigate the effect of these transcripts on development and progression of colorectal cancer

Association of ANRIL expression with coronary artery disease in type 2 diabetic patients

Objective: ANRIL is an important antisense noncoding RNA gene in the INK4 locus [9p21.3], a hot spot region associated with multiple disorders including coronary artery disease [CAD], type 2 diabetes mellitus [T2DM] and many different types of cancer. It has been shown that its expression is dysregulated in a variety of immune-mediated diseases. CAD is a major problem in T2DM patients and the cause of almost 60% of deaths in these patients worldwide. The aim of the present study was to compare the expression level of ANRIL between T2DM patients with and without CAD

Materials and Methods: In this case-control study, we examined ANRIL expression in peripheral blood mononuclear cell samples by quantitative reverse transcriptionpolymerase chain reaction [RT-qPCR] in 64 T2DM patients with and without CAD [33 CAD+ and 31 CADpatients respectively, established by coronary angiography]

Results: Expression analysis revealed that ANRIL was up regulated [2.34-Fold, P=0.012] in CAD+ versus CAD diabetic patients. Data from receiver operating characteristic [ROC] curve analysis has shown that ANRIL could act as a potential biomarker for detecting CAD in diabetic patients

Conclusion: The expression level of ANRIL is associated with presence of CAD in diabetic patients and could be considered as a potential peripheral biomarker

rs1127354 polymorphism in ITPA is associated with susceptibility to infertility

Objective: Infertility is a common human disorder which is defined as the failure to conceive for a period of 12 months without contraception. Many studies have shown that the outcome of fertility could be affected by DNA damage. We attempted to examine the association of two SNPs [rs1127354 and rs7270101] in ITPA, a gene encoding a key factor in the repair system, with susceptibility to infertility

Materials and Methods: This was a case-control study of individuals with established infertility. Blood samples were obtained from 164 infertile patients and 180 ethnically matched fertile controls. Total genomic DNA were extracted from whole blood using the standard salting out method, and stored at -20 C. Genotyping were based on mismatch polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP] method in which PCR products were digested with the XmnI restriction enzyme and run on a 12% polyacrylamide gel

Results: All genotype frequencies in the control group were in Hardy-Weinberg equilibrium. A significant association [in allelic, recessive and dominant genotypic models] was observed between infertile patients and healthy controls based on rs1127354 [P=0.0001], however, no significant association was detected for rs7270101. Also, gender stratification and analysis of different genotype models did not lead to a significant association for this single- nucleotide polymorphis [SNP]

Conclusion: ITPA is likely to be a genetic determinant for decreased fertility. To the best of our knowledge, this is the first report demonstrating this association, however, given the small sample size and other limitations, genotyping of this SNP is recommended to be carried out in different populations with more samples

Functional surface display of laccase in a phenol-inducible bacterial circuit for bioremediation purposes

Background: Phenolic compounds, which are produced routinely by industrial and urban activities, possess dangers to live organisms and environment. Laccases are oxidoreductase enzymes with the ability of remediating a wide variety of phenolic compounds to more benign molecules. The purpose of the present research is surface display of a laccase enzyme with adhesin involved in diffuse adhesion [AIDA-I] autotransporter system on the surface of Escherichia coli cells for bioremediation of phenolic compounds

Methods: The expression of laccase was regulated by a phenol-responsive promoter [a 54 promoter]. The constitutively-expressed CapR transcription activator was able to induce laccase expression in the presence of phenolic compounds

Results: Western blot analysis showed the expression and correct transfer of the enzyme to the outer membrane of E. coli cells in the presence of phenol. Activity assay confirmed the correct folding of the enzyme after translocation through the autotransporter system. HPLC analysis of residual phenol in culture medium showed a significant reduction of phenol concentration in the presence of cells displaying laccase on the surface

Conclusion: Our findings confirm that autodisplay enables functional surface display of laccase for direct substrate-enzyme availability by overcoming membrane hindrance

C1019T polymorphism in the connexin 37 gene and myocardial infarction risk in premature coronary artery disease

Background: the C1019T polymorphism of the connexin-37 [GJA4] gene is a single-nucleotide polymorphisms involved in atherosclerotic plaque rupture and atherosclerosis predisposition. We examined the association between the C1019T polymorphism of the GJA4 gene and the occurrence of myocardial infarction [MI] in patients with premature coronary artery disease [CAD]

Methods: our study recruited 1000 patients with the final diagnosis of premature CAD and classified them into 2 groups: with a history of MI [n = 461] and without it [n = 539]. The polymorphism variants were determined via the PCR-RFLP, and then genotyping was conducted through the high-resolution melting method. From a total of 1000 patients, 554 patients, who had been previously followed-up with a median follow-up time of 45.74 months vis-à-vis long-term major adverse cardiac events, were enrolled in this retrospective cohort phase

Results: the frequencies of the wild, heterozygous, and mutant genotypes of the C1019T polymorphism were 54.0%, 40.6%, and 5.4% in the MI group and 49.2%, 43.2%, and 7.6% in the non-MI group [p value = 0.187]. After adjustment for the baseline covariates, no difference was found between the MI and non-MI groups apropos the frequency of the heterozygous genotype [p value = 0.625] and the mutant genotype [p value = 0.452]. Regarding the level of human connexin-37, the serum level of this marker was not different between the MI and non-MI groups

Conclusion: the C1019T polymorphism of the GJA4 gene may not be useful for predicting the occurrence of MI in patients with premature CAD. The presence of this polymorphism in such patients may also have a low value for predicting long-term CAD complications

Association between the hepatic lipase promoter region polymorphism [-514 C/T] and the presence and severity of premature coronary artery disease

Background: hepatic lipase [HL] plays a crucial role in lipid metabolism, but there is debate about whether HL acts in a more pro- or more anti-atherogenic fashion. We aimed to examine the relationship between the -514 C/T polymorphism within the HL gene [LIPC] and the risk of angiographically determined premature coronary artery disease [CAD]

Methods: four hundred seventy-one patients with newly diagnosed angiographically documented [>/= 50% luminal stenosis of any coronary vessel] premature CAD were compared to 503 controls [subjects with no luminal stenosis in coronary arteries]. A real-time polymerase chain reaction and high-resolution melting analysis was used to distinguish between the genotypes

Results: there was no significant difference in the distribution of -514 C/T genotypes between the 2 groups in the whole population or in the men, but the examined polymorphism was found to be associated with the presence of CAD in the women [p value = 0.029]. After the application of a multiple logistic regression model, the minor T allele of the LIPC gene was not found to be independently associated with the presence of CAD either in the total population [adjusted OR = 0.97, 95% CI = 0.75-1.25; p value = 0.807] or in the women [adjusted OR = 0.91, 95% CI = 0.59-1.40; p value = 0.650] and in the men [adjusted OR = 1.15, 95% CI = 0.81-1.64; p value = 0.437] separately

Conclusion: our findings suggest that there is no relationship between the LIPC -514 C/T and the risk of premature CAD or its severity in patients undergoing coronary angiography

Up-regulation of FOXC2 and FOXQ1 is associated with the progression of gastric-type adenocarcinoma

Objective: Forkhead box [FOX] proteins are important regulators of the epithelial-to-mesenchymal transition [EMT], which is the main mechanism of cancer metastasis. Different studies have shown their potential involvement in progression of cancer in different tissues such as breast, ovary and colorectum. In this study, we aimed to analyze the expression of genes encoding two FOX proteins in gastric adenocarcinoma

Materials and Methods: In this experimental case-control study, the expression of FOXC2 and FOXQ1 was examined in 31 gastric adenocarcinoma tumors and 31 normal adjacent gastric tissues by reverse transcription polymerase chain reaction [PCR]

Results: The expression of both genes was significantly up-regulated in gastric adenocarcinoma tumors compared with the normal tissues [P<0.05]. The differential expression of these two genes was also correlated with the grade of tumors [P<0.01]

Conclusion: We show that up-regulation of FOXC2 and FOXQ1 are likely to be involved in the progression of gastric adenocarcinoma

convenience of single homology arm donor DNA and CRISPR/Cas9-nickase for targeted insertion of long DNA fragment

Objective: CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid

Materials and Methods: In this experimental study, single homology arm donor was applied along with a single guide RNA [sgRNA] specific to the homology region, and either Cas9 or its mutant nickase variant [Cas9n]. Using Pdx1 gene as the target locus the functionality of this system was evaluated in MIN6 cell line and murine embryonic stem cells [ESCs]

Results: Both wild type Cas9 and Cas9n could conduct the knock-in process with this system. We successfully applied this strategy with Cas9n for generation of Pdx1GFP knock-in mouse ESC lines. Altogether, our results demonstrated that a combination of a single homology arm donor, a single guide RNA and Cas9n is capable of precisely incorporating DNA fragments of multiple kilo base pairs into the targeted genomic locus

Conclusion: While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies

Production of specific IgY Helicobacter pylori recombinant OipA protein and assessment of its inhibitory effects towards attachment of H. pylori to AGS cell line

PURPOSE: The common triple therapy for Helicobacter pylori is challenged by the increasing cases of antibiotic resistant infections, raising the need to explore alternative therapies. Oral administration of egg yolk immunoglobulin Y (IgY) has been previously reported as a means of passive immunization therapy for H. pylori infections. In this work, we investigated the inhibitory effect of IgY on the attachment of H. pylori to AGS cell line. MATERIALS AND METHODS: Recombinant OipA was prepared. Hens were immunized with recombinant protein three times. IgY was purified from egg yolks of immunized hens using polyethylene glycol precipitation method. The inhibitory effect of the specific immunoglobulin was evaluated in AGS cell line infected with H. pylori. RESULTS: The presence of recombinant OipA (30 kD) was confirmed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunization of hens was confirmed using enzyme-linked immunosorbent assay. The purified IgY from egg yolks were assessed using SDS-PAGE and confirmed by western blot. CONCLUSION: The results showed that IgY-OipA had inhibitory effect on attachment of H. pylori to AGS cell line and may be utilized as a therapeutic or prophylaxis material.

[One-step synthesis of anionic PEG-citrate G2 dendrimer and evaluation of its cytotoxicity in two cell lines]

Objective: Dendrimers are three-dimensional nanostructures that have numerous applications in medicine, including drug delivery and imaging. Although anionic dendrimer polyethylene glycol-citrate has a high potential to increase solubility of waterinsoluble drugs and drug delivery, its multi-step synthesis procedure is time consuming. In addition, toxic substances such as dichloromethane are used in its synthesis procedure. In this study, we have developed a simple one-step synthesis method using green chemistry

Methods: We examined four different methods to improve the synthesis method of this dendrimer. Products were characterized by FTIR, LC-MS and DLS. Cytotoxicity was assessed by the XTT method

Results: We synthesized a G2 polyethylene glycol-citrate dendrimer in one-step without purifying G1. This process was chosen as a beneficial method for synthesis of the G2 dendrimer. When compared with previous methods, this procedure had higher efficiency and greatly reduced response. This procedure used nontoxic materials. XTT assay results showed that this dendrimer created by green chemistry had no cytotoxicity in Hela and Vero cells up to a concentration of 800 microM

Conclusion: One-step synthesis of anionic polyethylene glycol-citrate G2 dendrimer is a simple, beneficial production method. The dendrimer is biocompatible and can be used as a suitable carrier for drug delivery purposes

[Comparison of snRNA-U6 and microRNA-16 for identification of suitable endogenous control gene for microRNA gene expression analysis under dendrosomal curcumin treatment in hepatocellular carcinoma cell lines]

Objective: MicroRNAs [miRNAs] are single-stranded small RNAs 18-25 nucleotides in length that regulate gene expression through translational inhibition and mRNA cleavage. Aberrant expression of miRNAs contribute to several diseases. This has increased interest in profiling the expressions of these molecules. Real-time quantitative PCR [RQ-PCR] is a sensitive, quantitative technique for gene expression assessment. To correct for systematic variables such as the amount of starting template, RNA quality and enzymatic efficiencies, RQ-PCR data is commonly normalized to an endogenous control gene which is stably-expressed across the test sample set. To avoid occurring further error in the quantification of gene expression data, it is necessary that candidate endogenous controls be validated in the samples of interest. In this study the expression of miRNA-16 and small nuclear RNA [snRNA]-U6 in hepatocellular carcinoma [HCC] cell lines under dendrosomal curcumin treatment were evaluated to identify appropriate endogenous controls for dendrosomal curcumin-related miRNA expression assays

Methods: HCC cell lines were treated with dendrosomal curcumin. Dendrosomal curcumin entry into HepG2 and HuH-7 cells was assessed by fluorescent microscopy images. RNA was extracted and cDNA, after polyA polymerization, was synthesised. Then, we performed gene expression assays using RQ-PCR

Results: In this treatment condition, miRNA-16 for HepG2, snRNA-U6 and the combined miRNA-16 and snRNA-U6 for HuH-7 were suitable endogenous controls

Conclusion: These genes are appropriate endogenous controls for miRNA expression assays in HCC cell lines under treatment with dendrosomal curcumin. There are stable, non-significant expression changes of these genes

[ study of the effect of human prothrombin's signal peptide on the secretion efficiency of recombinant human FIX in the HEK293T cell line]

Objective: Eukaryotic proteins generally have signal peptides which are not only crucial for their secretion efficiencies but are important for their expression levels. Coagulation factor IX [FIX] is a glycoprotein that plays a fundamental role in the blood coagulation pathway. Reduced levels or dysfunctional FIX are associated with hemophilia B. This study investigates the function of the human prothrombin signal peptide in an attempt to improve the human FIX [hFIX] secretion efficiency in a heterologous expression system. With this aim, we have used the SignalP and PrediSi programs for in silico evaluation of the signal peptide efficiency prior to conducting this experiment

Methods: We used molecular techniques to amplify and join the coding region of the human prothrombin signal peptide to the cDNA of mature hFIX. The chimeric fragment was examined for transient expression in a mammalian cell line [HEK293T] in comparison with the native hFIX, under a CMVp regulation. Using the neural networkbased prediction programs, we evaluated the scores for cleavage position and secretion efficiency of the human prothrombin and hFIX signal peptides. The expression efficiencies of hFIX expressed by the recombinant cells were analyzed by RT-PCR and ELISA

Results: In silico analysis more efficiently predicted the human prothrombin signal peptide with a high score compared to the native hFIX signal peptide. This data was confirmed by the RT-PCR and ELISA results obtained from expression analyses at the RNA and protein levels, respectively

Conclusion: The present study showed that the signal peptide derived from the human prothrombin has the potential for efficient secretion of hFIX as evidenced by the results taken from a transient expression system. The results were consistent with in silico analysis. This replacement could be evaluated in a stable state condition

[Decrease of inosine triphosphatase gene expression in human gastric adenocarcinoma tumors]

Objective: A pure nucleotide pool is essential for correct DNA replication in addition to the prevention of mutagenesis and abnormalities in a living cell. Inosine triphosphatase [ITPase] is a critical enzyme for the removal of deaminated rough purine nucleotides such as inosine from the nucleotide pool. It has been shown that abnormal function and expression of the ITPA gene is followed by an increased substitution mutation rate in the genome. This study compares the ITPA gene expression level between human adenocarcinoma tumors and their normal marginal tissues

Method: We examined ITPA gene expression in 24 pairs of gastric adenocarcinoma tumors and their normal adjacent tissues by quantitative real-time PCR

Result: There was reduced ITPA gene expression in tumor tissues compared with the adjacent normal tissues. The decline in ITPA gene expression was more significant in the higher grade samples

Conclusion: ITPA is involved in omitting deaminated purines from the nucleotide pool. Therefore its abnormal function increases the frequency of mutations and causes higher genomic instability. Our data suggest that lower expression of ITPA can be considered a risk factor for the development and progression of gastric cancer

6-Methoxy Podophyllotoxin induces Apoptosis via inhibition of TUBB3 and TOPIIA Gene expressions in 5637 and K562 Cancer cell lines

Objective: Podophyllotoxin [PTOX], a natural compound in numerous plants, contains remarkable biological properties that include anti-tumor, anti-viral such as anti-human im-munodeficiency virus [HIV] activities. In order to avoid its adverse effects, various compounds have been derived from PTOX. 6-methoxy PTOX [MPTOX] is one of the natural PTOX derivatives with an extra methoxy group. MPTOX is mostly isolated from the Linum species. This study has sought to determine the biological effects of MPTOX on cancer cell lines, 5637 and K562

Materials and Methods: In this experimental study, we treated the 5637 and K562 cancer cell lines with MPTOX in a dose- and time-dependent manner. Apoptosis was examined by flow cytometry and viability rate was analyzed by the MTT assay. Expressions of the tubulin [TUBB3] and topoisomerase II [TOPIIA] genes were determined by real-time polymerase chain reaction [PCR]

Results: Treatment with MPTOX led to significant induction of apoptosis in cancer cells compared to control cells. Gene expression analysis showed reduced levels of TUBB3 and TOPIIA mRNA following MPTOX treatment

Conclusion: MPTOX inhibited TUBB3 and TOPIIA gene expression and subsequently induced cell death through apoptosis. These results suggested that MPTOX could be considered a potential anti-tumor agent

effect of sortilin silencing on ovarian carcinoma cells

Our preliminary data on the protein expression of SORT1 in ovarian carcinoma tissues showed that sortilin was overexpressed in ovarian carcinoma patients and cell lines, while non-malignant ovaries expressed comparably lower amount of this protein. In spite of diverse ligands and also different putative functions of sortilin [NTR3], the function of overexpressed sortilin in ovarian carcinoma cells is an intriguing subject of inquiry. The aim of this study was, therefore, to investigate the functional role of sortilin in survival of ovarian carcinoma cell line. Expression of sortilin was knocked down using RNAi technology in the ovarian carcinoma cell line, Caov-4. Silencing of SORT1 expression was assessed using real-time qPCR and Western blot analyses. Apoptosis induction was evaluated using flow cytometry by considering annexin-V FITC binding. [3H]-thymidine incorporation assay was also used to evaluate cell proliferation capacity. Real-time qPCR and Western blot analyses showed that expression of sortilin was reduced by nearly 70-80% in the siRNA transfected cells. Knocking down of sortilin expression resulted in increased apoptosis [27.5 +/- 0.48%] in siRNA-treated ovarian carcinoma cell line. Sortilin silencing led to significant inhibition of proliferation [40.1%] in siRNA-transfected Caov-4 cells as compared to mock control-transfected counterpart [p<0.05]. As it was suspected from overexpression of sortilin in ovarian tumor cells, a cell survival role for sortilin can be deduced from these results. In conclusion, the potency of apoptosis induction via silencing of sortilin expression in tumor cells may introduce sortilin as a potential candidate for developing a novel targeted therapy in patients with ovarian carcinoma

[Dendrosomal curcumin upregulates expression of the long noncoding RNA gene MEG3 in U87MG glioblastoma cells]

Glioblastoma is an invasive tumor of the central nervous system. Epigenetic therapy of cancer is potentially very useful in reversing some of cancer defects due to reversibility of epigenetic alterations. MEG3 is a tumor suppressor long non-coding RNA [lncRNA] that expresses in the majority of normal tissues. Methylation of the MEG3 promoter region elicits a decrease in its expression in glioblastoma cells. Bioactive nutrients including curcumin offer great potential in altering DNA methylation status. Herein, we aim to investigate the epigenetic-based role of dendrosomal-curcumin [DNC] in upregulation of MEG3 expression in glioblastoma cells. We evaluated DNC entrance to U87MG cells with the use of the fluorescent characteristics of curcumin. Next we performed the MTT assay to evaluate DNC and dendrosome effects on cell viability. The ability of DNC to boost expression of MEG3 in DNA methylation regulation was accomplished by a study of the relative expressions of MEG3 and DNA methylation regulator enzymes, DNA methyltransferases [DNMT1, DNMT3A and 3B] using semi-quantitative and quantitative PCR. We observed the entrance of DNC into U87MG cells. DNC significantly caused U87MG cell death in a time and dose-dependent manner. However dendrosome did not show any toxic effect on this cell line. Data acquired from gene expression assays determined that DNC upregulated MEG3 expression [P<0.05] and downregulated DNMT3B expression [P<0.05]. There was no significant effect on DNMT1, 3A expression in U87MG cells. The data showed that DNC could awaken epigenetically silenced tumor suppressor genes through an ambiguous route in glioblastoma cells. Notwithstanding, DNA hypomethylation has occurred by downregulation of DNMTs, inactive DNA demethylation and or active DNA demethylation, subsequently tumor suppressor genes such as MEG3 a cell growth regulator overexpressed. We concluded that DNC has useful characteristics in epigenetic therapy of glioblastoma

Biological activity of recombinant accessory cholerae enterotoxin [Ace] on rabbit ileal loops and antibacterial assay

Vibrio cholerae [V. cholerae] causes a potentially lethal disease named cholera. The cholera enterotoxin [CT] is a major virulence factor of V. cholerae. In addition to CT, V. cholerae produces other putative toxins, such as the zonula occludens toxin [Zot] and accessory cholera enterotoxin [Ace]. The ace gene is the third gene of the V. cholerae virulence cassette. The Ace toxin alters ion transport, causes fluid accumulation in ligated rabbit ileal loops, and is a cause of mild diarrhea. The aim of this study is the cloning and overexpression of the ace gene into Escherichia coli [E. coli] and determination of some characteristics of the recombinant Ace protein. In this experimental study, the ace gene was amplified from V. cholerae strain 62013, then cloned in a pET28a expression vector and transformed into an E. coli [DH5 alpha] host strain. Subsequently, the recombinant vector was retransformed into E. coli BL21 for expression, induced by isopropythio-beta-D-galctoside [IPTG] at a different concentration, and examined by SDS-PAGE and Western blot. A rabbit ileal loop experiment was conducted. Antibacterial activity of the Ace protein was assessed for E. coli, Stapylococcus aureus [S. aureus], and Pseudomonas aeruginosa [P. aeruginosa]. The recombinant Ace protein with a molecular weight of 18 kDa [dimeric form] was expressed in E. coli BL21. The Ace protein showed poor staining with Coomassie blue stain, but stained efficiently with silver stain. Western blot analysis showed that the recombinant Ace protein reacted with rabbit anti-V. cholerae polyclonal antibody. The Ace protein had antibacterial activity at a concentration of >/= 200 micro g/ml and caused significant fluid accumulation in the ligated rabbit ileal loop test. This study described an E. coli cloning and expression system [E. coli BL21- pET-28a-ace] for the Ace protein of V. cholerae. We confirmed the antibacterial properties and enterotoxin activity of the resultant recombinant Ace protein

Gene expression and activity of phenyl alanine amonia-lyase and essential oil composition of Ocimum basilicum L at different growth stages

Phenylalanine amonia-lyase [PAL] is one of the most important enzymes that plays a key role in regulation of phenylpropanoid production in plants. It catalyzes the first step of the phenylpropanoid pathway in which L-phenylalanine is deaminated to trans-cinnamic acid. This step is significant for metabolic engineering and hyper-expression of the major phenylpropanoid, methyl chavicol. We followed gene expression and activity of PAL in Ocimum basilicum L. at different stages of growth including seedling, beginning and middle of growth phase, budding stage and flowering, and their correlation with final concentration of phenylpropanoid compounds. The level of gene expression was monitored by semi quantitative RT-PCR and phenylpropanoid compounds were identified by gas chromatography/mass spectrometry [GC/MS]. PAL activity was assayed using spectrophotometer. The results indicated that the level of gene expression and activity of PAL enzyme are altered during the plant development, where the highest expression and activity [0.851 [micromol cinnamic acid/mg/min] was achieved at budding stage. In this experiment, changes of methylchavicol content were correlated to the transcription and activity of PAL enzyme

study of the association between SNP8NRG241930 in the 5' end of neuroglin 1 gene with schizophrenia in a group of Iranian patients

Neuregulin1 [NRG1] gene is among the most promising candidate genes for schizophrenia. This gene is located on 8p22-p12, a region with a reported linkage to schizophrenia. Several studies have reported an association between schizophrenia and the 5? end polymorphisms in this gene. However, some studies have failed to confirm the role of NRG1 gene in the pathogenesis of schizophrenia. In the current study, we attempt to examine the association of SNP8NRG241930 from the NRG1 gene with schizophrenia in an Iranian population. It is noteworthy that there has been no report on the NRG1 association with schizophrenia in a population from the Middle East region. Genomic DNA samples were obtained via isolation from the peripheral blood cells of 95 unrelated subjects with schizophrenia and 95 matched healthy controls from southwest Iran. SNP8NRG241930 was genotyped by PCRRFLP using ScaI as a restriction endonuclease enzyme. Association of the SNP with schizophrenia was examined using the chi-square test. The frequency difference of alleles and genotypes between the two groups were compared. P?0.05 was considered significant. Statistical analysis on the studied polymorphism showed that both case and control groups were in Hardy-Weinberg equilibrium. The frequency of high risk allele [G allele] was 72.6% in patients, while this number was 56.8% in controls. The genotype frequencies in the patient group were as follows: GG [54%], GT [38%] and TT [8%] vs. genotype frequencies in the control group of: GG [26%], GT [63%] and TT [11%]. Considering allele and genotype frequencies, a significant association was observed between schizophrenia and SNP8NRG241930.The current study adds weight to the idea that some functional polymorphisms could exist in the 5? end of the NRG1 gene which increase susceptibility to schizophrenia. This is the first time that supportive evidence shows an involvement of the NRG1 locus in schizophrenia in an Iranian sample population