Single nucleotide polymorphisms in PON1 gene and their relation with PON1 activity in Vokkaliga population of Mysore, South India

The cardio protective functions of HDL are attributed to the HDL-associated proteins like Paraoxonase 1 (PON1). PON1 is associated with HDL through a N-terminal signal peptide. HDL-associated PON1 alone is cardio protective. Indians have normal HDL levels and yet a high risk of cardiovascular diseases. Hence, the presence of polymorphisms in the promoter region and first exon of PON1 were investigated in 256 individuals to find out whether the polymorphisms could be the cause of the high risk. Out of these 256 individuals, 195 were from Vokkaliga community and the rest were from other communities. Whole blood was collected, plasma and cells were separated. Plasma was used for determination of PON1 activity and for lipid analysis. Cells were used for DNA extraction.DNA was sequenced from -1246 to +203 of the PON1 gene. Nine single nucleotide polymorphisms(SNPs) were identified of which three were new SNPs, two of which were in the 5’ flanking region of the putative mRNA for the protein but none in the first exon. Our results show that the low active PON1 phenotype was predominant in the Vokkaliga population

Purification of human serum paraoxonase: a simple and rapid method

Paraoxonase/arylesterases (EC.3.1.8.2) is an enzyme found tightly associated with high density lipoprotein particle in serum. Because of its unique enzyme activity, antioxidant property and its role as an anti atherosclerotic molecule, various methods are used for its purification from human serum. Methods involved in its purification are elaborate and complicated. Also the yield and final activity are highly variable. Here, we report a 2 step method of purification involving affinity chromatography on cibacron blue sepharose followed by gel filtration on sephadex G50.The final preparation was 27.7 fold purified compared with the serum and gave a single band in SDS-PAGE by silver staining

Anticoagulant effect of Naja naja venom 5′nucleotidase: Demonstration through the use of novel specific inhibitor, vanillic acid

The snake venom proteins affect hemostasis by either advancing/delaying blood coagulation. Apart from proteases and phospholipase A2s (PLA2s), 5′nucleotidase is known to affect hemostasis by inhibiting platelet aggregation. In this study, the possible involvement of Naja naja venom 5′nucleotidase in mediating anticoagulant affect is evaluated. Vanillic acid selectively and specifically inhibited 5′nucleotidase activity among other enzymes present in N. naja venom. It is a competitive inhibitor as evident of inhibition relieving upon increased substrate concentration. Vanillic acid dose dependently inhibited the anticoagulant effect of N. naja venom up to 40. This partial involvement of 5′nucleotidase in mediating anticoagulant effect is substantiated by concanavalin-A (Con-A) inhibition studies. Con-A, competitively inhibited in vitro protease and 5′nucleotidase activity up to 100. However, it did not exhibit inhibitory activity on PLA2. The complete inhibition of anticoagulant effect by Con-A upon recalcification time suggests the participation of both 5′nucleotidase and protease in mediating anticoagulant effect of N. naja venom. Vanillic acid and Con-A inhibition studies together suggest that probably 5′nucleotidase interacts with one or more factors of intrinsic pathway of blood coagulation to bring about anticoagulant effect. Thus, this study for the first time demonstrates the involvement of 5′nucleotidase in mediating N. naja venom anticoagulant effect

rAAV-compatible MiniPromoters for restricted expression in the brain and eye

Background: Small promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters–however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo. Methods: For much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were “cut down” to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP. Results: The data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia. Conclusions: Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.Medicine, Faculty ofOther UBCNon UBCMedical Genetics, Department ofPsychiatry, Department ofReviewedFacult