Glyoxalase-I activity and cell cycle regulation in yeast

The status of glyoxalase-I was explored in exponentially growing and G₁ arrested temperature sensitive (ts) cell division cycle (cdc) mutants of Saccharomyces cerevisiae. It was observed that the specific activity of this enzyme was correlated with overall growth status. The activity was high in actively growing cells and was low in G₁ arrested cells. Specific activities of glyoxalase-I were also low in G₁ arrested prolonged stationary phase (PSP) cells of S. cerevisiae and Candida albicans. The activity of glyoxalase-I recovered when G₁ arrested S. cerevisiae (ts) cells were allowed to regrow under permissive conditions. Results demonstrate that although glyoxalase-I activity is a good indicator of cell growth status, it is not involved in cell cycle regulation of this eukaryotic organism.     

Voxel-Based Texture Analysis of the Brain

This paper presents a novel voxel-based method for texture analysis of brain images. Texture analysis is a powerful quantitative approach for analyzing voxel intensities and their interrelationships, but has been thus far limited to analyzing regions of interest. The proposed method provides a 3D statistical map comparing texture features on a voxel-by-voxel basis. The validity of the method was examined on artificially generated effects as well as on real MRI data in Alzheimer''s Disease (AD). The artificially generated effects included hyperintense and hypointense signals added to T1-weighted brain MRIs from 30 healthy subjects. The AD dataset included 30 patients with AD and 30 age/sex matched healthy control subjects. The proposed method detected artificial effects with high accuracy and revealed statistically significant differences between the AD and control groups. This paper extends the usage of texture analysis beyond the current region of interest analysis to voxel-by-voxel 3D statistical mapping and provides a hypothesis-free analysis tool to study cerebral pathology in neurological diseases.     

Lead-210 content of food samples in India

Summary The paper presents results of measurements made on cereals and composite meal samples collected from Bombay market for their lead-210 content. The details of sampling and analytical chemistry procedures are also given. The Pb-210 contents of most of these samples were in the range of 1–5 pCi/kg of cereals samples. The concentrations in composite meal samples were mostly in the range of 1–3.5 pCi of²¹⁰Pb per composite meal. The assessment of daily intake of this isotope through food-stuffs has been made.     

The presence of the glycerol phosphate shuttle and energy dependent transhydrogenase in aortic mitochondria

A procedure has been devised for isolating tightly coupled mitochondria from the intima-media strip of pig aorta. The mitochondria exhibit respiratory control with succinate, pyruvate-malate, β-hydroxybutyrate or α-glycerol phosphate as substrate. Furthermore, submitochondrial particles from these preparations exhibit an energy-dependent reduction of NADP⁺ by NADH.     

Effect of phospholipase A on the structure and functions of membrane vesicles from Mycobacterium phlei.

The phospholipid composition of the electron transport particles and coupling factor-depleted electron transport particles of Mycobacterium phlei are the same, but they differ in contents. The accessibility of partially purified phospholipase A to these membrane phospholipids was found to be different. Treatment of membranes of Mycobacterium phlei with phospholipase A impairs the rate of oxidation as well as phosphorylation. The inhibition of phosphorylation can be reversed by washing the membranes with defatted bovine serum albumin. The reconstitution of membrane-bound coupling factor-latent ATPase activity to phospholipase A-treated depleted electron transport particles and their capacity to couple phosphorylation to oxidation of substrates remained unaffected after phospholipase A treatment. However, the pH gradient as measured by bromthymol blue was not restored after reconstitution of phospholipase A-treated depleted electron transport particles with membrane-bound coupling factor-latent ATPase. These findings show that the phosphorylation coupled to the oxidation of substrates can take place without a pronounced pH gradient in these membrane vesicles. The dye 1-anilino-8-naphthalene sulfonic acid (ANS) exhibited low levels of energized and nonenergized fluorescence in phospholipase A-treated membranes. This decrease in the level of ANS fluorescence in phospholipase A-treated membranes was found to be directly related to the amount of phospholipids cleaved. The decrease in the energy-dependent ANS response in phospholipase A-treated electron transport particles, as compared with untreated electron transport particles, was shown to be a result of a change in the apparent K-d of the dye-membrane complex, and of a decrease in the number of irreversible or slowly reversible binding sites, with no change in the relative quantum efficiency of the dye. The decrease in ANS fluorescence in phospholipase A-treated particles appears to be due to a decrease in the hydrophobicity of the membranes.     

Binding of nucleotides to purified coupling factor-latent ATPase from Mycobacterium phlei.

Binding studies of various nucleotides to the purified coupling factor-latent ATPase from Mycobacterium phlei have been carried out using gel filtration, equilibrium dialysis, and ultrafiltration methods. The purified latent ATPase binds 3 mol of ADP per mol of the enzyme with an apparent dissociation constant of 68 muM. Binding of nucleotides occurred in the decreasing order: ADP, epsilon-ATP, epsilon-ADP, UDP, adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), IDP, and adenosine 5'-(alpha,beta-methylene)diphosphate (AdoP(CH2)P). AMP-P(NH)P inhibits both soluble (Ki = 77 muM) and membrane-bound latent ATPase activity. However, AMP-P(NH)P does not affect oxidative phosphorylation in membrane vesicles of M. phlei. AMP-P(NH)P exhibits one binding site per molecule of the enzyme with a dissociation constant of 71 muM. After trypsin treatment of the enzyme, the binding of ADP decreases 35%, while AMP-P(NH)P binding remains unchanged. Moreover, AMP-P(NH)P binding was not displaced by ADP. Studies with sulfhydryl agents showed that, in contrast to AMP-P(NH)P, binding of at least 1 mol of ADP requires the participation of sulfhydryl groups. The results indicate that AMP-P(NH)P and ADP do not share a common binding site and that the latent ATPase enzyme has separate sites for ATP hydrolysis and ATP synthesis.     

Application of HPLC to study the kinetics of a branched bi-enzyme system consisting of hypoxanthine-guanine phosphoribosyltransferase and xanthine oxidase--an important biochemical system to evaluate the efficiency of the anticancer drug 6-mercaptopurine in ALL cell line

The thiopurine antimetabolite 6-mercaptopurine (6MP) is an important chemotherapeutic drug in the conventional treatment of childhood acute lymphoblastic leukemia (ALL). 6MP is mainly catabolized by both hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine oxidase (XOD) to form thioinosinic monophosphate (TIMP) (therapeutically active metabolite) and 6-thiouric acid (6TUA) (inactive metabolite), respectively. The activity of both the enzymes varies among ALL patients governing the active and the inactive metabolite profile within the immature lymphocytes. Therefore, an attempt was made to study the kinetic nature of the branched bi-enzyme system acting on 6MP and to quantitate TIMP and 6TUA formed when the two enzymes are present in equal and variable ratios. The quantification of the branched kinetics using spectrophotometric method presents problem due to the closely apposed lambda(max) of the substrates and products. Hence, employing an HPLC method, the quantification of the products was done with the progress of time. The limit of quantification (LOQ) of substrate was found to be 10nM and for products as 50 nM. The limit of detection (LOD) was found to be 1 nM for the substrate and the products. The method exhibited linearity in the range of 0.01-100 microM for 6MP and 0.05-100 microM for both 6TUA and TIMP. The amount of TIMP formed was higher than that of 6TUA in the bi-enzyme system when both the enzymes were present in equivalent enzymatic ratio. It was further found that enzymatic ratios play an important role in determining the amounts of TIMP and 6TUA. This method was further validated using actively growing T-ALL cell line (Jurkat) to study the branched kinetics, wherein it was observed that treatment of 50 microM 6MP led to the generation of 12 microM TIMP and 0.8 microM 6TUA in 6 h at 37 degrees C.