[Site directed mutagenesis in ifnbeta gene in order to increasing its mRNA stability and translation level using megaprimer PCR method]

Background and Objectives: Beta interferon [IFNbeta] protein is produced as a recombinant drug and used in treatment of some diseases like Multiple Sclerosis. In eukaryotic cells, IFNbeta mRNA is rapidly degraded and its halflife is too short. One of the contributing factors to this short half-life is presence of the AU rich element [ARE] in 3'UTR of this mRNA. This region has an inhibitory effect on translation too. Our aim in this research was to delete ARE from IFNbeta gene in order to increase its mRNA stability and translational level

Materials and Methods: In order to delete an 18 nucleotide sequence from ARE, the Megaprimer PCR technique was used. The PCR product was digested with EcoRI and BglII enzymes. The vector was partially digested with the same enzymes. The digested PCR product was purified and cloned into the vector. Then, the recombinant vectors were transfected into CHO cell line

Results: The first PCR reaction product contained a deletion mutation and was used as megaprimer in the second reaction. Partial digestion of the vector yielded a variety of fragments with different weights. The sufficient fragment was purified from the gel and used as a cloning vector. Final product of PCR was cloned into the vector. The accuracy of the cloning reaction was confirmed and the recombinant vector was transfected into CHO cell line

Conclusion: An 18 nucleotide region of IFNbeta mRNA was deleted. The influence of this microdeletion on mRNA stability and translational efficiency needs to be surveyed in future

Antimicrobial effects of Kelussia odoratissima extracts against food borne and food spoilage bacteria "in vitro"

The aim of this paper was to investigate the antibacterial potential of Kelussia odoratissima Mozff extract against Gram-negative and Gram-positive bacteria. Karafs-eKoohi with the scientific name of Kelussia odoratissima is an Iranian endemic edible plant in the middle region of Iran with enormous use as food, spice and medicinal herb. The antibacterial effect of the extracts was investigated using pour plate and disk diffusion methods. Minimum Inhibitory Concentration [MIC] and Minimum Bactericidal Concentration [MBC] were also studied using the dilution method. Repeated measure ANOVA was used for data analysis. The results showed that in disk diffusion method all concentrations of ethanolic extract had inhibitory effect against Bacillus subtilis and Staphylococcus aureus. Minimum Inhibitory Concentration [MIC] of Kelussia odoratissima leaves of aqueous and ethanolic extracts for Bacillus subtilis and Staphylococcus aureus were 16 and 8 mg/ml, and for Enterobacter aerogenes were 32 and 16 mg/ml, respectively. Minimum Bactericidal Concentration [MBC] of Kelussia odoratissima leaves of aqueous and ethanolic extracts for Bacillus subtilis and Staphylococcus aureus were 32 and 16 mg/ml, and for Enterobacter aerogenes were 64 and 32mg/ml, respectively. The results showed that the extract of Kelussia odoratissima had a satisfactory antimicrobial activity and the ethanolic extract of Kelussia odoratissima leaves had greater inhibitory effects on the strains studied compared to aqueous extract in vitro. A significant correlation was also observed between zone of inhibition and concentration of extracts

Down-regulation of CD14 transcripts in human glioblastoma cell line U87 MG

Pattern recognition receptors [PRRs] are the main sensors of pathogen and danger signals in innate immunity of which Toll Like Receptors [TLRs] are the most studied ones. The contribution of PRRs in cerebral inflammation induced by microbial infection, tissue damage and cancer has not extensively been addressed so far. Glioma is the most common tumor of the central nervous system and glioblastomas are the most common and most malignant primary brain tumors. The objectives of the present study were to investigate the expression of several PRRs including TLR2, TLR4, MyD88 and CD14 transcripts in human glioblastoma cell line U87 MG and compare their expression level with peripheral blood mononuclear cells [PBMC] obtained from healthy individuals. Touchdown PCR [TD-PCR] and Real-time quantitative PCR [qPCR] were applied to detect and quantify the expression level of TLR2, TLR4, MyD88 and CD14 transcript in U87 MG cell line and [PBMC] of healthy individuals. According to our results, human glioblastoma cell line U87 MG expresses TLR2, TLR4, MyD88 and CD14 transcripts in TDPCR. Moreover, the quantification of the expression of these genes revealed a highly significant down-regulation of CD14 and a slight up-regulation of TLR2 transcripts as compared to PBMC of healthy individuals. The lower expression level of CD14 in human glioblastoma cell line, might have a potential implication for CD14 mediated cerebral pathology

Association of genetic variants of beta-lactoglobulin gene with milk production in a herd and a superior family of Holstein cattle

Polymorphism of the beta-lactoglobulin [beta-LG] gene in 101 cows belonging to the Holstein herd and a superior cow, producing more than 150 Kg milk/day, together with four offsprings was investigated by the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism [PCR-RFLP] method. In the Holstein herd, the alleles A and B of the beta-LG gene had frequencies of 0.53 and 0.47, respectively. The genotypes AA, AB and BB of the beta-LG gene were estimated to have frequencies of 0.257, 0.544 and 0.198, respectively. Genotypes were distributed according to the Hardy-Weinberg equilibrium. Results indicated that the beta-LG genotypes significantly affected [P< 0.01] milk yield [genotype AA being more effective than genotype BB]. The superior cow and her progenies were all heterozygotes [AB]