Role of Arg-401 of cytosolic serine hydroxymethyltransferase in subunit assembly and interaction with the substrate carboxy group.

In an attempt to identify the arginine residue involved in binding of the carboxylate group of serine to mammalian serine hydroxymethyltransferase, a highly conserved Arg-401 was mutated to Ala by site-directed mutagenesis. The mutant enzyme had a characteristic visible absorbance at 425 nm indicative of the presence of bound pyridoxal 5'-phosphate as an internal aldimine with a lysine residue. However, it had only 0.003% of the catalytic activity of the wild-type enzyme. It was also unable to perform reactions with glycine, beta-phenylserine or d-alanine, suggesting that the binding of these substrates to the mutant enzyme was affected. This was also evident from the interaction of amino-oxyacetic acid, which was very slow (8.4x10(-4) s-1 at 50 microM) for the R401A mutant enzyme compared with the wild-type enzyme (44.6 s-1 at 50 microM). In contrast, methoxyamine (which lacks the carboxy group) reacted with the mutant enzyme (1.72 s-1 at 250 microM) more rapidly than the wild-type enzyme (0.2 s-1 at 250 microM). Further, both wild-type and the mutant enzymes were capable of forming unique quinonoid intermediates absorbing at 440 and 464 nm on interaction with thiosemicarbazide, which also does not have a carboxy group. These results implicate Arg-401 in the binding of the substrate carboxy group. In addition, gel-filtration profiles of the apoenzyme and the reconstituted holoenzyme of R401A and the wild-type enzyme showed that the mutant enzyme remained in a tetrameric form even when the cofactor had been removed. However, the wild-type enzyme underwent partial dissociation to a dimer, suggesting that the oligomeric structure was rendered more stable by the mutation of Arg-401. The increased stability of the mutant enzyme was also reflected in the higher apparent melting temperature (Tm) (61 degrees C) than that of the wild-type enzyme (56 degrees C). The addition of serine or serinamide did not change the apparent Tm of R401A mutant enzyme. These results suggest that the mutant enzyme might be in a permanently 'open' form and the increased apparent Tm could be due to enhanced subunit interactions.

A Haplotype at the UCP1 Gene Locus Contributes to Genetic Risk for Type 2 Diabetes in Asian Indians (CURES-72)

Background: The gene encoding for uncoupling protein-1 (UCP1) is considered to be a candidate gene for type 2 diabetes because of its role in thermogenesis and energy expenditure. The objective of the study was to examine whether genetic variations in the UCP1 gene are associated with type 2 diabetes and its related traits in Asian Indians. Methods: The study subjects, 810 type 2 diabetic subjects and 990 normal glucose tolerant (NGT) subjects, were chosen from the Chennai Urban Rural Epidemiological Study (CURES), an ongoing population-based study in southern India. The polymorphisms were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Linkage disequilibrium (LD) was estimated from the estimates of haplotypic frequencies. Results: The three polymorphisms, namely -3826A -> G, an A -> C transition in the 5'-untranslated region (UTR) and Met229Leu, were not associated with type 2 diabetes. However, the frequency of the A-C-Met (-3826A -> G-5'UTR A -> C-Met229Leu) haplotype was significantly higher among the type 2 diabetic subjects (2.67%) compared with the NGT subjects (1.45%, P < 0.01). The odds ratio for type 2 diabetes for the individuals carrying the haplotype A-C-Met was 1.82 (95% confidence interval, 1.29-2.78, P = 0.009). Conclusions: The haplotype, A-C-Met, in the UCP1 gene is significantly associated with the increased genetic risk for developing type 2 diabetes in Asian Indians.

Characterization and immune response of a protein produced by a cDNA clone of foot and mouth disease virus, type Asia 1 63/72

A 0.9 kb double stranded cDNA of foot and mouth disease virus (FMDV) Type Asia 1, 63/72 was cloned in an expression vector, pUR222. A protein of 38 kd was produced by the clone which reacted with the antibodies raised against the virus. A 20 kd protein which may be derived from the 38 kd protein contained the antigenic epitopes of the protein VP1 of the virus. Injection of 10-20 micrograms of the partially purified 38 and 20 kd proteins or a lysate of cells containing 240 micrograms of the proteins elicited high titers of FMDV specific antibodies in guinea pigs and cattle respectively. Also, at these concentrations, the proteins protected 5 of 8 guinea pigs and 3 of 8 cattle when challenged with a virulent virus.

The fatigue crack growth rate in L-72 Al-alloy plate specimens with cold worked holes

A fatigue crack growth rate study has been carried out on L-72 aluminium alloy plate specimens with and without cold worked holes. The cold worked specimens showed significantly increased fatigue life compared to unworked specimens. Computer software is developed to evaluate the stress intensity factor for non-uniform stress distributions using Green's function approach. The exponents for the Paris equation in the stable crack growth region for cold worked and unworked specimens are 1.26 and 3.15 respectively. The reduction in exponent value indicates the retardation in crack growth rate. An SEM study indicates more plastic deformation at the edge of the hole for unworked samples as compared to the worked samples during the crack initiation period.

Correlation of Calcification on Excised Aortic Valves by Micro-Computed Tomography with Severity of Aortic Stenosis

Background and aim of the study: The quantification of incidentally found aortic valve calcification on computed tomography (CT) is not performed routinely, as data relating to the accuracy of aortic valve calcium for estimating the severity of aortic stenosis (AS) is neither consistent nor validated. As aortic valve calcium quantification by CT is confounded by wall and coronary ostial calcification, as well as motion artifact, the ex-vivo micro-computed tomography (micro-CT) of stenotic aortic valves allows a precise measurement of the amounts of calcium present. The study aim, using excised aortic valves from patients with confirmed AS, was to determine if the amount of calcium on micro-CT correlated with the severity of AS. Methods: Each of 35 aortic valves that had been excised from patients during surgical valve replacement were examined using micro-CT imaging. The amount of calcium present was determined by absolute and proportional values of calcium volume in the specimen. Subsequently, the correlation between calcium volume and preoperative mean aortic valve gradient (MAVG), peak transaortic velocity (V-max), and aortic valve area (AVA) on echocardiography, was evaluated. Results: The mean calcium volume across all valves was 603.2 +/- 398.5 mm(3), and the mean ratio of calcium volume to total valve volume was 0.36 +/- 0.16. The mean aortic valve gradient correlated positively with both calcium volume and ratio (r = 0.72, p <0.001). V-max also correlated positively with the calcium volume and ratio (r = 0.69 and 0.76 respectively; p <0.001). A logarithmic curvilinear model proved to be the best fit to the correlation. A calcium volume of 480 mm(3) showed sensitivity and specificity of 0.76 and 0.83, respectively, for a diagnosis of severe AS, while a calcium ratio of 0.37 yielded sensitivity and specificity of 0.82 and 0.94, respectively. Conclusion: A radiological estimation of calcium amount by volume, and its proportion to the total valve volume, were shown to serve as good predictive parameters for severe AS. An estimation of the calcium volume may serve as a complementary measure for determining the severity of AS when aortic valve calcification is identified on CT imaging. The Journal of Heart Valve Disease 2012;21:320-327

Growth kinetics and immune response of chimeric foot-and-mouth disease virus serotype `O' produced through replication competent mini genome of serotype Asia 1, 63/72, in BHK cell lines

Regular vaccinations with potent vaccine, in endemic countries and vaccination to live in non-endemic countries are the methods available to control foot-and-mouth disease. Selection of candidate vaccine strain is not only cumbersome but the candidate should grow well for high potency vaccine preparation. Alternative strategy is to generate an infectious cDNA of a cell culture-adapted virus and use the replicon for development of tailor-made vaccines. We produced a chimeric `O' virus in the backbone of Asia 1 and studied its characteristics. The chimeric virus showed high infectivity titre (>10(10)) in BHK 21 cell lines, revealed small plague morphology and there was no cross reactivity with antiserum against Asia I. The virus multiplies rapidly and reaches peak at 12 h post infection. The vaccine prepared with this virus elicited high antibody titres.