Reduction of methylglyoxal in Escherichia coli K12 by an aldehyde reductase and alcohol dehydrogenase

Two enzymes, one NADPH-dependent and another NADH-dependent which catalyze the reduction of methylglyoxal to acetol have been isolated and substantially purified from crude extracts of Escherichia coli K₁₂ cells. Substrate specificity and formation of acetol as the reaction product by both the enzymes, reversibility of NADH-dependent enzyme with alcohols as substrates and inhibitor study with NADPH-dependent enzyme indicate that NADPH-dependent and NADH-dependent enzymes are identical with an aldehyde reductase (EC 1.1.1.2) and alcohol dehydrogenase (EC 1.1.1.1) respectively. The K_m for methylglyoxal have been determined to be 0.77 mM for NADPH-dependent and 3.8 mM for NADH-dependent enzyme. Stoichiometrically equimolar amount of acetol is formed from methylglyoxal by both NADPH- and NADH-dependent enzymes. In phosphate buffer, both the enzymes are active in the pH range of 5.8–6.6 with no sharp pH optimum. Molecular weight of both the enzymes were found to be 100,000 ± 3,000 by gel filtration on a Sephacryl S-200 column. Both NADPH- and NADH-dependent enzymes are sensitive to sulfhydryl group reagents.     

Impairment of raw 264.7 macrophage function by antiarrhythmic drugs

The effect of the antiarrhythmic drugs lidocaine, quinidine and procainamide on macrophage function was investigated in RAW 264.7 mouse monocytic macrophage cell. Cells stimulated by either zymosan or phorbol ester were found to generate both superoxide (O ₂ ^– ) and H₂O₂. The production of O₂ was detected as superoxide dismutase inhibitable ferricytochrome c reduction. H₂O₂ production was monitored in both chemical and flow cytometric fluorescent assays. Although all three drugs inhibited both O₂ and H₂O₂ release in a dose dependent manner, only quinidine was found to have significant inhibitory effects. The amounts of quinidine required to cause a 50% inhibition in O₂ production in zymosan and phorbol ester stimulated cells were found to be 250 M and 300 M, respectively and the amounts required to cause one-half optimum levels of H₂O₂ production in these cells were found to be 50 M and 100 M, respectively. The effect of these drugs on O₂ producing NADPH oxidase was investigated and only procainamide was found to have a significant effect (p<0.001) in inhibiting the oxidase activity. Lidocaine and quinidine had no significant effect on the activation of the respiratory burst oxidase. A sensitive and convenient differential phagocytosis assay was devised on the basis of number of particles engulfed by individual phagocytes using flow cytometric techniques. It appears to be remarkably free of interference and was applied to investigate the role of antiarrhythmic drugs on the phagocytosis of fluorescent latex beads. All three antiarrhythmic drugs inhibited phagocytosis of latex beads in a dose dependent manner irrespective of the number of particles phagocitized by the cells. The results of these studies do not conclusively establish a mechanism of action of these drugs on the generation of O₂ and H₂O₂ by stimulated macrophages; nevertheless, it is interesting that all three drugs inhibited the phagocytic activity.     

Proteolytic cleavage of bovine herpesvirus 1 (BHV-1) glycoprotein gB is not necessary for its function in BHV-1 or pseudorabies virus.

Glycoprotein B homologs represent the most highly conserved group of herpesvirus glycoproteins. They exist in oligomeric forms based on a dimeric structure. Despite the high degree of sequence and structural conservation, differences in posttranslational processing are observed. Whereas gB of herpes simplex virus is not proteolytically processed after oligomerization, most other gB homologs are cleaved by a cellular protease into subunits that remain linked via disulfide bonds. Proteolytic cleavage is common for activation of viral fusion proteins, and it has been shown that herpesvirus gB homologs are essential for membrane fusion events during infection, e.g., virus penetration and direct viral cell-to-cell spread. To analyze the importance of proteolytic cleavage for the function of gB homologs, we isolated a mutant bovine herpesvirus 1 (BHV-1) expressing a BHV-1 gB that is no longer proteolytically processed because of a deletion of the proteolytic cleavage site and analyzed its phenotype in cell culture. We showed previously that BHV-1 gB can functionally substitute for the homologous glycoprotein in pseudorabies virus (PrV), based on the isolation of a PrV gB-negative PrV recombinant that expresses BHV-1 gB (A. Kopp and T. C. Mettenleiter, J. Virol, 66:2754-2762, 1992). Therefore, we also isolated a mutant PrV lacking PrV gB but expressing a noncleavable BHV-1 gB. Our results show that cleavage of BHV-1 gB is not essential for its function in either a BHV-1 or a PrV background. Compared with the PrV recombinant expressing cleavable BHV-1 gB, deletion of the cleavage site in the recombinant PrV did not detectably alter the viral phenotype, as analyzed by plaque assays, one-step growth kinetics, and penetration kinetics. In the BHV-1 mutant, the uncleaved BHV-1 gB was functionally equivalent to the wild-type protein with regard to penetration and showed only slightly delayed one-step growth kinetics compared with parental wild-type BHV-1. However, the resulting plaques were significantly smaller, indicating a role for proteolytic cleavage of BHV-1 gB in cell-to-cell spread of BHV-1.     

Synergistic interaction between two bacterial isolates in the degradation of carbofuran

The dominant bacteriaPseudomonas sp. andArthrobacter sp. were isolated from the standing water of carbofuran-retreatedAzolla plot.Arthrobacter sp. hydrolysed carbofuran added to the mineral salts medium as a sole source of carbon and nitrogen while no degradation occurred withPseudomonas sp. Interestingly, when the medium containing carbofuran was inoculated with bothArthrobacter sp. andPseudomonas sp., a synergistic increase in its hydrolysis and subsequent release of CO₂ from the side chain was noticed. This synergistic interaction was better expressed at 25° C than at 35° C. Likewise, related carbamates, carbaryl, bendiocarb and carbosulfan were more rapidly degraded in the combined presence of both bacterial isolates.     

Isozymic pattern of lactate dehydrogenase in cases of bancroftian filariasis.

Isozymic patterns of lactate dehydrogenase (E.C. 1.1.1.27) by polyacrylamide gel electrophoresis (PAGE) were observed in various categories of filariasis and controls, i.e. asymptomatic microfilaraemia and symptomatic amicrofilaraemia, endemic normal and non-endemic normal. Lactate dehydrogenase (LDH) activity was also observed amongst the above categories of patients. An increase in enzyme activity and change in the isozymic pattern was observed in the above categories of filaria infected serum. LDH activity doubled in asymptomatic microfilaraemia whereas in symptomatic amicrofilaraemia the increase in LDH activity was thirtyfold. The isozymic pattern of microfilaraemic cases showed the presence of three bands B4, A1B3, A2B2, which are quite thick as compared to normal healthy subjects, whereas the patients with symptomatic amicrofilaraemia showed marked elevation of serum LDH-4 or A3B1. The LDH was partially purified by combined treatment of (NH4)2SO4 fractionation and gel filtration. The isozymic pattern of purified LDH showed a similar pattern.     

Variability of milk fat globule membrane protein gene between cattle and riverine buffalo.

A study on butyrophilin (BTN) gene was conducted to detect variability at nucleotide level between cattle and buffalo. Hae III PCR-RFLP was carried out in crossbred cattle and it revealed polymorphism at this locus. Three genotypes namely, AA, BB and AB and two alleles were observed with frequencies 0.78, 0.17, 0.04 and 0.87, 0.13, respectively. The sequences of different cattle, buffalo and sheep breeds have been reported in the EMBL gene bank with accession numbers: AY491468 to AY491475. The nucleotides, which have been substituted from allele A to B, were found to be C to G (71st nucleotide), C to T (86th nucleotide), A to T (217th nucleotide), G to A (258th nucleotide), A to C (371st nucleotide) and C to T (377th nucleotide). The nucleotide substitution at 71st, 86th and 377th position of the fragment were expected to be a silent mutation where as nucleotide changes at 217th, 258th and 371st positions were expected to be substituted by lysine with arginine, valine with isoleucine and leucine with proline in allele B. The differences of nucleotides and amino acids between cattle, buffalo and sheep breeds have been revealed and on the basis of nucleotide as well as protein variability the phylogenetic diagram have been developed indicating closeness between cattle and buffalo.     

Growth,photosynthetic pigment content and oil yield ofPogostemon cablin grown under sun and shade conditions

Patchouli (Pogostemon cablin Benth.) plants grown under shade (LI) showed an increased height due to internodal elongation, leaf area, leaf area index, and chlorophyll (Chl)b, Chl (a+b) and carotenoid (Car) contents compared to the plants grown in sunlight (HI). The number of branches and green leaves decreased under LI treatment, with a marginal variation in the patchouli oil yield. A comparison between the relative contents of photosynthetic pigments indicated that Chlb and Car accumuled preferentially over Chla in the LI grown plants.