Genetic evidence for distinct catalytic and regulatory subunits in yeast phosphofructokinase

Rat liver mitochondria contain an ATP-dependent proteolytic system which is localized on the outside of the inner membrane. It is capable of utilizating both the ATP produced within the mitochondria as well as that supplied externally. The system is dependent on Ca²⁺. Its physiological function is seen in the normal breakdown of mitochondria during their turnover. The system may be selective for the breakdown of the inner membranes.     

Genotype-phenotype relationship of <Emphasis Type="Italic">F7</Emphasis> R353Q polymorphism and plasma factor VII coagulant activity in Asian Indian families predisposed to coronary artery disease

Elevated factor VII (FVII) level is a risk factor for coronary artery disease (CAD). We investigated the role of R353Q polymorphism in the F7 gene in 139 Indian families with CAD, comprising of 222 affected subjects, 105 unaffected subjects and 126 affected sibling pairs. Plasma per cent FVIIc activity (FVII.c activity) differed significantly across R353Q genotype (P < 0.0001). Frequency of subjects with RR and QQ genotypes were higher in 4th quartile and 1st quartile of FVII.c activity, respectively (P < 0.0001). F7 R353Q SNP was able to explain up to 7% of variation in FVII.c activity by regression analysis and an additive genetic component of variance of 28.04% by heritability analysis. Quantitative trait loci analysis showed suggestive linkage evidence of F7 SNP with per cent FVII.c activity (LOD score −1.82; P = 0.002). Individuals with RR and RQ genotypes carried an OR of 2.071 (95% c.i. = 1.506−2.850) and 2.472 (95% c.i. = 1.679−3.641), respectively, towards CAD risk. There was significant correlation of FVII.c activity with lipid markers, particularly among those with RR and RQ genotype after covariate adjustment. In conclusion, the F7 R353Q SNP appears to moderately influence plasma FVII.c activity and risk of CAD in Indians.     

A novel 9-bp insertion detected in steroid 21-hydroxylase gene (CYP21A2): prediction of its structural and functional implications by computational methods

Background

Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia (CAH). Detection of underlying mutations in CYP21A2 gene encoding steroid 21-hydroxylase enzyme is helpful both for confirmation of diagnosis and management of CAH patients. Here we report a novel 9-bp insertion in CYP21A2 gene and its structural and functional consequences on P450c21 protein by molecular modeling and molecular dynamics simulations methods.

Methods

A 30-day-old child was referred to our laboratory for molecular diagnosis of CAH. Sequencing of the entire CYP21A2 gene revealed a novel insertion (duplication) of 9-bp in exon 2 of one allele and a well-known mutation I172N in exon 4 of other allele. Molecular modeling and simulation studies were carried out to understand the plausible structural and functional implications caused by the novel mutation.

Results

Insertion of the nine bases in exon 2 resulted in addition of three valine residues at codon 71 of the P450c21 protein. Molecular dynamics simulations revealed that the mutant exhibits a faster unfolding kinetics and an overall destabilization of the structure due to the triple valine insertion was also observed.

Conclusion

The novel 9-bp insertion in exon 2 of CYP21A2 genesignificantly lowers the structural stability of P450c21 thereby leading to the probable loss of its function.     

Co-polymeric hydrophilic nanospheres for drug delivery: release kinetics, and cellular uptake

Nanobiotechnology focuses on the biological effects and applications of nanoparticles that include nano-safety, drug encapsulation and nanotherapeutics. The present study focuses on hydrophilic nanospheres of copolymers N-isopropylacrylamide [NIPAAM] and vinyl pyrrolidone [VP], encapsulating a bioactive derivative of 5-fluorouracil-hexyl-carbamoyl fluorouracil (HCFU). The size of the nanospheres was approximately 58 nm and the surface charge measured was -15.4 mV. Under optimal conditions, the yield was >80%, and the drug loading was 2%. The entrapment efficiency was approximately 75%. Wide-angle X-ray diffraction analysis showed that the entrapped HCFU was present in an amorphous state, which has higher water solubility compared with the crystalline state. Slow drug release from nanospheres was observed in PBS and serum, with approximately 80% released at 37 degrees C after 72 h. The HCFU loaded polymeric nanospheres have been found to be stable in whole blood having negligible RBC toxicity. Cytotoxicity in Mia-Paca 3, pancreatic cancer cell line was done in a 24-72 h assay. Dose dependant cytotoxicity was observed when incubated with various concentrations of HCFU loaded polymeric nanospheres while HCFU per se (<1 mg) showed 90% toxicity within 24 h.     

Polymorphisms in the pituitary growth hormone gene and its receptor associated with coronary artery disease in a predisposed cohort from India

We investigated the promoter polymorphisms of the pituitary growth hormone gene (GH1) and exon 3 deletion polymorphism (GHRd3) in its receptor gene (GHR) in 299 angiographically proven patients with coronary artery disease (CAD) and 231 asymptomatic controls enrolled in the ongoing Indian Atherosclerosis Research Study. Real time PCR based analysis of the GHR variant showed significant association of the GHRd3 deletion allele with CAD (OR 0.48, 95% CI: 0.30–0.76, P = 0.0014) and a dominant model of inheritance (Akaike information criterion = 482). The deletion allele showed significant association with high plasma HDL-c levels (P = 0.001). Sequencing of the proximal promoter region of GH1 revealed 12 novel polymorphisms and a TAGA haplotype constituted by the functional SNPs rs2005171, rs11568828, rs2005172 and rs6171, that showed significant association with CAD alone (adjusted OR of 3.31 (95% CI = 1.33–8.29, P = 0.011) and in CAD patients with diabetes (P = 0.019). Mean standardized height was associated with three of the four haplotype-tagging SNPs in the cohort (P ≤ 0.03). Eleven of the 12 polymorphic promoter SNPs contributed to 14.7% of variation in height in females in the whole dataset (P = 0.029). CAD patients with history of stroke exhibited marginally significantly lower mean height as compared to rest of the cohort (P < 0.006). In conclusion, genetic polymorphisms in the GHR gene and its ligand, GH1, may modulate the risk of CAD in the Asian Indian population.     

A Slantlet transform based intelligent system for magnetic resonance brain image classification

The present paper proposes the development of a new approach for automated diagnosis, based on classification of magnetic resonance (MR) human brain images. Wavelet transform based methods are a well-known tool for extracting frequency space information from non-stationary signals. In this paper, the proposed method employs an improved version of orthogonal discrete wavelet transform (DWT) for feature extraction, called Slantlet transform, which can especially be useful to provide improved time localization with simultaneous achievement of shorter supports for the filters. For each two-dimensional MR image, we have computed its intensity histogram and Slantlet transform has been applied on this histogram signal. Then a feature vector, for each image, is created by considering the magnitudes of Slantlet transform outputs corresponding to six spatial positions, chosen according to a specific logic. The features hence derived are used to train a neural network based binary classifier, which can automatically infer whether the image is that of a normal brain or a pathological brain, suffering from Alzheimer's disease. An excellent classification ratio of 100% could be achieved for a set of benchmark MR brain images, which was significantly better than the results reported in a very recent research work employing wavelet transform, neural networks and support vector machines.     

A novel salt-tolerant L-myo-inositol-1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka, a halophytic wild rice: molecular cloning, bacterial overexpression, characterization, and functional introgression into tobacco-conferring salt tolerance phenotype

l-myo-Inositol-1-phosphate synthase (EC 5.5.1.4, MIPS), an evolutionarily conserved enzyme protein, catalyzes the synthesis of inositol, which is implicated in a number of metabolic reactions in the biological kingdom. Here we report on the isolation of the gene (PINO1) for a novel salt-tolerant MIPS from the wild halophytic rice, Porteresia coarctata (Roxb.) Tateoka. Identity of the PINO1 gene was confirmed by functional complementation in a yeast inositol auxotrophic strain. Comparison of the nucleotide and deduced amino acid sequences of PINO1 with that of the homologous gene from Oryza sativa L. (RINO1) revealed distinct differences in a stretch of 37 amino acids, between amino acids 174 and 210. Purified bacterially expressed PINO1 protein demonstrated a salt-tolerant character in vitro compared with the salt-sensitive RINO1 protein as with those purified from the native source or an expressed salt-sensitive mutant PINO1 protein wherein amino acids 174-210 have been deleted. Analysis of the salt effect on oligomerization and tryptophan fluorescence of the RINO1 and PINO1 proteins revealed that the structure of PINO1 protein is stable toward salt environment. Furthermore, introgression of PINO1 rendered transgenic tobacco plants capable of growth in 200-300 mm NaCl with retention of approximately 40-80% of the photosynthetic competence with concomitant increased inositol production compared with unstressed control. MIPS protein isolated from PINO1 transgenics showed salt-tolerant property in vitro confirming functional expression in planta of the PINO1 gene. To our knowledge, this is the first report of a salt-tolerant MIPS from any source.     

Phosphorylation of mammalian translation initiation factor 5 (eIF5) in vitro and in vivo

Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S initiation complex (40S•eIF3•AUG•Met-tRNA_f•eIF2•GTP) and, acting as a GTPase activating protein, promotes the hydrolysis of bound GTP. We isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate purified bacterially expressed recombinant rat eIF5. Physical, biochemical and antigenic properties of this kinase identify it as casein kinase II (CK II). Mass spectrometric analysis of maximally in vitro phosphorylated eIF5 localized the major phosphorylation sites at Ser-387 and Ser-388 near the C-terminus of eIF5. These serine residues are embedded within a cluster of acidic amino acid residues and account for nearly 90% of the total in vitro eIF5 phosphorylation. A minor phosphorylation site at Ser-174 was also observed. Alanine substitution mutagenesis at Ser-387 and Ser-388 of eIF5 abolishes phosphorylation by the purified kinase as well as by crude reticulocyte lysates. The same mutations also abolish phosphorylation of eIF5 when transfected into mammalian cells suggesting that CK II phosphorylates eIF5 at these two serine residues in vivo as well.