Comparison of wild type and mutated [mHuIFN- beta 27-101] Interferon binding to the IFNRA receptor by Molecular Docking

Introduction: Interferon beta is one of the members of type I interferons. Creating R27T and V101F mutations is one of the important researches performed to improve function, decrease immunogenicity, increase expression and increase half-life of interferon beta. In this study, the effects of R27T and V101F mutations on interferon beta binding to interferon receptors were studied by molecular docking

Method: This study was performed through Bioinformatics methods. The required crystal structures were provided by the RCSB server. The simulations of R27T and V101F mutations were performed using online Rosetta Backrub software. Comparison of access to the solvent for the amino acids in the created structures was performed using asaview online server. Also, the effect of mutations on the structure and protein folding was investigated by the online Hope server and SPDBV software. The molecular docking between HuIFN- beta and the external region of IFNAR receptor was performed using the online ClusPro2 protein-protein docking server

Results: The comparison of the values of the negative binding energy [ DELTAGbind] obtained from protein-protein molecular docking between IFNAR receptor and HuIFN- beta, mHuIFN- beta-27, mHuIFN- beta-101 and mHuIFN- beta-27-101 ligands did not show a significant difference [P> 0.99]

Conclusion: Regarding these results, it can be concluded that the produced mutations do not have a negative effect on the forming of rHuIFN- beta/IFNAR complex and does not interfere with the binding of the interferon beta to the receptor and thus improves the quality of the produced rHuIFN- beta

Screening of two neighboring CFTR mutations in Iranian infertile men with non-obstructive azoospermia

The genetic association between cystic fibrosis transmembrane conductance regulator [CFTR] gene mutations and male infertility due to congenital bilateral absence of vas deferens [CBAVD] is well established. Mutant CFTR, however may also be involved in the etiology of male infertility in non-CBAVD cases. The present study was conducted to estimate the frequency of DELTA I507 and DELTA F508 CFTR gene mutations in Iranian infertile males. We undertook the first study of association between these CFTR mutations and non-obstructive azoospermia in Iran. In this case-control study, 100 fertile healthy fathers and 100 non-obstructive azoospermia's men were recruited from Isfahan Infertility Center [IIC] and Sari Saint Mary's Infertility Center, between 2008 and 2009. Screening of F508del and I507del mutations was carried out by the multiplex-ARMS-PCR. Significance of differences in mutation frequencies between the patient and control groups was assessed by Fisher's exact test. The DELTA F508 was detected in three patients. However there are no significant association was found between the presence of this mutated allele and infertility [OR=9.2 [allele-based] and 7.2 [individual-based], P=0.179]. None of the samples carried the DELTA I507 mutation. Altogether, we show that neither DELTA I507 nor DELTA F508 is involved in this population of Iranian infertile males with non-obstructive azoospermia

Association between polymorphisms of exon 12 and exon 24 of JHDM2A gene and male infertility

Background: Some dynamic changes occurs during spermatogenesis such as histone removal and its replacement with transition nuclear protein and protamine. These proteins are required for packing and condensation of sperm chromatin. JHDM2A is a histone demethylase that directly binds to promoter regions of Tnp1 and Prm1 genes and controls their expression by removing H3K9 at their promoters

Objective: The association between polymorphisms of exon 12 and exon 24 in JHDM2A gene and male infertility were evaluated for the first time

Materials and Methods: In this experimental study, 400 infertile men [oligospermia and azoospermia] and normal healthy fathers were evaluated [n=200]. Single Strand Conformation Polymorphism [SSCP-PCR] and polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP] methods were used for screening any polymorphisms that are exist in exon 12 and exon 24

Results: Exon 24 PCR products were analyzed by RFLP but no polymorphism was found in this exon at the restriction site of EcoRV enzyme. Our monitoring along the whole nucleotides of exon 12 and exon 24 were continued using SSCP method, but we found no change along these exons

Conclusion: Generally, this study evaluated the association between polymorphisms in exon 12 and exon 24 of JHDM2A gene and male infertility which suggests that polymorphisms of these exons may not be associated with the risk of male infertility

[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

New insights into VEGF-A alternative splicing: key regulatory switching in the pathological process

Vascular endothelial growth factor [VEGF-A] is one of the most important regulatory factors in pathological and physiological angiogenesis. Alternative splicing is a complicated molecular process in VEGF-A gene expression which adds complexity to VEGF-A biology. Among all VEGF-A exons, alternative splicing of exon 8 is the key determinant of isoform switching from pro-angiogenic VEGF-xxx to anti-angiogenic VEGF-xxxb. This is known as a key molecular switching in many pathological situations. In fact, the balance between VEGF-xxx and VEGF-xxxb isoforms is a critical controlling switch in both conditions of health and disease. Here, the properties of VEGF-xxx and VEGF-xxxb isoforms were discussed and their regulatory mechanism and their roles in certain pathological processes were evaluated. In summary, it was suggested that C-terminal VEGF-A alternative splicing can provide a new treatment opportunity in angiogenic diseases

Exon 10 CFTR gene mutation in male infertility

About 10% of infertilities with obstructive azoospermia are congenital and caused by CF gene mutations. M469I mutation was observed for the first time in Taiwanese patients. This mutation not only causes CF, but also may be the origin of infertility too. In this study, we aimed in designing a rapid, reliable RFLP-PCR procedure for detection of M469I mutation. The correlation and association between M469I mutation with infertility was investigated in this study. One hundred ten patients [90 non obstructive and 20 obstructive] and 60 normal individuals were considered in this study. M469I mutation was detected using RFLP-PCR. This technique was completely designed for M469I genotyping, for the first time in our study. Amplification of the region surrounding the mutation in exon 10 of CFTR gene was then performed. RFLP analysis was carried out using the NdeI restriction enzyme. All genomic DNA samples were genotyped successfully. M469I mutation was observed only in patients group. Therefore, genotype containing mutant allele [GT] has been detected only in the patients group. There was no significant correlation between GT and TT genotypes with infertility [p=0.437]. The M469I mutation has only been observed in Exon 10 CFTR gene of infertile patients, not in the control group. This mutation causes congenital bilateral absence of vaz deferens and finally infertility. This indicates a strong association between the M469I mutation and male infertility. Therefore, this is a CF-causing CFTR mutation that could be considered as a cause of infertility

Amplification of GC-rich putative mouse PeP promoter using betaine and DMSO in ammonium sulfate polymerase chain reaction buffer

Recently, we have shown that peroxisomal protein expression was induced upon retinoic acid treatment in mouse embryonic stem cells during the process of neurogenesis. Thus, characterization of the respective promoter could elucidate the molecular aspects of transcriptional regulation of this gene. Using the conventional software programs for promoter prediction, a putative promoter region was identified approximately 561 bp upstream of the peroxisomal protein coding sequence. In order to clone this region with a GC-content of 71.01%, a cocktail of ammonium sulfate buffer supplied with two additive components, betaine and dimethyl sulfoxide, and a high concentration of MgCl[2] was used. The modulated polymerase chain reaction composition significantly improved the amplification of GC-rich DNA target sequences. Improved amplification of this region was due to reduction in the formation of secondary structures by the GC-rich region. Therefore, this polymerase chain reaction composition could be generally used to facilitate the amplification of other GC-rich DNA sequences as verified by amplification of different GC rich regions

Molecular analysis of the clavulanic acid regulatory gene isolated from an Iranian strain of streptomyces clavuligerus, PTCC 1709

The clavulanic acid regulatory gene [claR] is in the clavulanic acid biosynthetic gene cluster that encodes ClaR. This protein is a putative regulator of the late steps of clavulanic acid biosynthesis. The aim of this research is the molecular cloning of claR, isolated from the Iranian strain of Streptomyces clavuligerus [S. clavuligerus]. In this experimental study, two different strains of S. clavuligerus were used [PTCC 1705 and DSM 738], of which there is no claR sequence record for strain PTCC 1705 in all three main gene banks. The specific designed primers were subjected to a few base modifications for introduction of the recognition sites of BamHI and ClaI. The claR gene was amplified by polymerase chain reaction [PCR] using DNA isolated from S. clavuligerus PTCC 1705. Nested-PCR, restriction fragment length polymorphism [PCR-RFLP], and sequencing were used for molecular analysis of the claR gene. The confirmed claR was subjected to double digestion with BamHI and ClaI. The cut claR was ligated into a pBluescript [pBs] vector and transformed into E. coli. The entire sequence of the isolated claR [Iranian strain] was identified. The presence of the recombinant vector in the transformed colonies was confirmed by the colony-PCR procedure. The correct structure of the recombinant vector, isolated from the transformed E. coli, was confirmed using gel electrophoresis, PCR, and double digestion with restriction enzymes. The constructed recombinant cassette, named pZSclaR, can be regarded as an appropriate tool for site directed mutagenesis and sub-cloning. At this time, claR has been cloned accompanied with its precisely selected promoter so it could be used in expression vectors. Hence the ClaR is known as a putative regulatory protein. The overproduced protein could also be used for other related investigations, such as a mobility shift assay

role of matrix metalloproteinase-3 functional 5A/6A promoter polymorphism in tumor cell progression and metastasis of breast cancer

In the human genome, chromosome 11 contains a cluster of matrix metalloproteinase [MMP] genes. Single nucleotide polymorphisms in the promoter region of MMP genes are important for MMP expression. A common adenine deletion polymorphism [5A] at position -1171 of the MMP-3 gene promoter [5-AAAAAACCAT-3 change to 5-AAAAACCAT-3] facilitates transcriptional factor binding and MMP-3 promoter activity. A case-control study was performed including 120 breast cancer patients [60 patients with metastatic activity and 60 patients without metastatic activity]; and 60 healthy controls. Whole blood samples were obtained from patients and healthy controls. Genomic DNA was extracted from samples and the MMP-3 5A/6A genotypes were determined using PCR-RFLP. MMP-3 genotype distributions between patients and controls were similar [OR= 0.89, 95%CI, 0.43-1.84, P= 0.047]. It was observed that the 5A allele was more frequent among patients with metastatic activity than controls [OR= 2.9, 95%CI, 0.94-8.9, P= 0.074]. Therefore, the 5A polymorphism in the MMP-3 promoter showed correlation with the metastasis group than patients without metastasis; both at the time of diagnosis. However our results do not show evidence for correlation between 5A/6A polymorphism and breast cancer susceptibility