Biochemical Diagnosis of Common Gene Mutations in Galactosemia

Objective: Galactosemia is an inborn error of galactose metabolism that is inherited in an autosomal recessive trait. Classical galactosemia is caused by deficient activity of the galactose-1-phosphate uridyltransferase (GALT) enzyme that can result in galactosemia complications. Materials & Methods: 135 unrelated families, clinically suspected to galactosemia, were screened by qualitative measurement of galactose-1-phosphate uridyl transferase (GALT) activity in blood RBCs by using Beutler method. Results: Deficient enzyme activity (classical galactosemia) were confirmed in 16 families. All of these 16 families were submitted to the diagnosis of six common mutations in GALT gene including Q188R, K285N, S135L, L195P, X380R and Q169K by using PCR-RFLP method which resulted in detection of 68% of the mutated alleles. Eight patients were homozygote for Q188R mutation, while one patient homozygote for S135L mutation and one heterozygote for K285N mutation. Conclusion: Biochemnical diagnosis of Galactosemia in Grand infant hospital is very important and necessary

Nuclear factor erythroid-2 related factor 2 overexpressed mesenchymal stem cells transplantation, improves renal function, decreases injuries markers and increases repair markers in glycerol-induced Acute kidney injury rats

Objective(s):Recently cell therapy is a promising therapeutic modality for many types of disease including acute kidney injury (AKI). Due to the unique biological properties, mesenchymal stem cells (MSCs) are attractive cells in this regard. This study aims to transplant MSCs equipped with nuclear factor E2-related factor 2 (Nrf2) in rat experimental models of acute kidney and evaluate regeneration potential of injured kidney especially expression of injury and repaired biomarkers. Materials and methods:Nrf2 was overexpressed in bone marrow-derived MSCs by pcDNA.3.1 plasmid. AKI was induced using glycerol in rat models. The regenerative potential of Nrf2-overexpressed MSCs was evaluated in AKI-Induced animal models using biochemical and histological methods after transplantation. Expression of repaired genes, AQP1 and CK-18, as well as injury markers, Kim-1 and Cystatin C, was also assayed in engrafted kidney sections. Results:Our results revealed that transplantation of Nrf2-overexpressed MSCs into AKI-induced rats decreased blood urea nitrogen and creatinine and ameliorated kidney regeneration throughout 14 days. Upregulation of repaired markers and downregulation of injury markers were considerable 14 days after transplantation.  Conclusions: Overexpression of Nrf2 in MSCs suggests a new strategy to increase efficiency of MSC-based cell therapy in AKI

Expression and purification of recombinant human coagulation factor VII fused to a histidine tag using Gateway technology

Background. Factor VII is a plasma glycoprotein that participates in the coagulation process leading to generation of fibrin. Construction, expression and purification of recombinant FVII fused to poly histidin tag through gateway technology were aimed in this study. Methods. To construct entry clone, blunt-end FVII cDNA and subsequent PCR product isolated from HepG2 cell line was TOPO cloned into pENTR TOPO vector. To construct expression clone, LR recombination reaction was carried out between entry clone and destination vector, pDEST26. CHO c ells were transfected with 1 g of DNA of PDEST26 FVII using the FuGENE HD transfection reagent. Two cell lines that permanently expressed recombinant factor VII were established. The expression of recombinant FVII was confirmed by RT-PCR and ELISA. Culture medium containing his-FVII was added to the nickel-nitrilotriacetic acid resin c olumn and bound protein was eluted. The purified protein was detected by SDS-PAGE and western blot analysis. Biological activity of the recombinant factor VII was determined by prothrombin time assay using factor FVII-depleted plasma. Results. The results showed that human recombinant FVII successfully was cloned and accuracy of the nucleotide sequence of the gene and its frame in the vector were confirmed by DNA sequencing. Stable clones transfected with the construct expressed FVII mRNA and related protein but any expression was not detected in the CHO cells containing empty vector. A protein of about 52KDa was detected in SDS-PAGE and was further confirmed by western blot analysis. A three-fold decrease in clotting time was observed by using this rFVII. Conclusion. As we are a ware, this is the first report of expression of recombinant FVII fused with his-tag through gateway technology. The next steps including large scale expression, purification, activation and stabilization are underwa y