Characterization of beta-lactamase from Mycobacterium butyricum ATCC 19979

beta-lactamase has been purified to a homogeneous state from Mycobacterium butyricum ATCC 19979. The molecular weight (Mr = 29,000) and the isoelectric point (4,0) of the enzyme have been determined. The enzyme showed both penicillinase and cephalosporinase activity, but had relatively more of the former. With respect to substrate-profile the enzyme resembled the plasmid specified TEM-type beta-lactamases commonly encountered in Gram-negative bacteria. The enzyme was insensitive to p-chloromercuribenzoate, sodium chloride, or iodine inhibition.     

Purification of larvicidal protein from Bacillus sphaericus 1593

Coat proteins from the spores of Bacillus sphaericus 1593 were separated by preparative polyacrylamide gel electrophoresis. Neutralising antibodies were raised against a single protein band exhibiting toxicity to mosquito larvae. IgG was purified and coupled to CNBr-activated Sepharose 4B to be used as an immunoaffinity matrix. The larvicidal protein was purified to electrophoretic homogeneity using this immunoaffinity column. The single protein species resolved into four peptides of molecular weights 42.6, 44.1, 50.7 and 51.3 KDa on polyacrylamide gel electrophoresis under denaturing conditions. This protein contained 12% carbohydrates. The purified protein exhibited an LC50 value of 8.3 +/- 1.6 ng/ml when tested against early third instar larvae of the mosquito Culex pipiens var quinquefasciatus.     

Characterization of genomic DNA of mycobacteriophage I3

The GC content of mycobacteriophage I3 DNA is 67% as determined from thermal melting analysis, buoyant density in CsCl gradient, and 5-deoxymononucleotide analysis. High-resolution melting of I3 DNA revealed that the base distribution is random. Studies with methylation-specific restriction enzymes and high voltage electrophoretic analysis of 5-deoxymononucleotides did not indicate the presence of any unusual or methylated bases in I3 DNA. The molecular size of I3 DNA is estimated to be about 135 Kbp, based on the restriction fragment size distributions and sedimentation in sucrose gradients. The restriction cleavage pattern by a variety of restriction endonucleases has been determined. The circularly permuted nature of I3 DNA, indicated from the restriction patterns, has been confirmed by Southern blot hybridizations and 5 end group analysis.     

Analysis of nuclear proteins from silk glands ofBombyx mori

A gentle method for the isolation of nuclei from developing silk glands ofBombyx mori has been standardized. The nuclei, whether isolated or directly visualizedin situ within the silk glands, exhibit complex morphology. The nuclei occupy almost the entire volume of the gigantic silk gland cells. Although the isolated nuclei still retain their ramified morphology, being polyploid they are fragile and often become fragmented. The histone and low-salt-extractable proteins from nuclei isolated from the middle and posterior silk glands on different days of the fourth and fifth instars of larval development have been analysed. The histones did not show any stage- or tissue-specific variations whereas the low-salt-extractable proteins showed some developmental stage specific variation. Using the antibody raised against one such protein, its absence in the early stage of development has been confirmed by Western blotting techniques. This developmental stage specific protein may be functionally linked to some activities responsible for boosting up the production of silk or silk-related proteins during the fifth instar of larval development.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Presence of mycobacteriophage I3-like DNA sequences in the genome of its host Mycobacterium smegmatis

The genomes of phage I3 and its host Mycobacterium smegmatis have been compared. From thermal melting studies the GC contents of DNA from mycobacteriophage I3 and its host M. smegmatis were found to be 66%. A new method, based only on the initial rates of reassociation, has been developed for calculating the DNA homology. Analysis of DNA reassociation kinetics suggested the presence of one equivalent of the phage I3 genome within the M. smegmatis genome. Southern analysis revealed the presence of almost all of the phage I3 specific sequences within the host genome.     

An oxygenase from guinea-pig liver which catalyses sulphoxidation

A mono-oxygenase catalysing the conversion of 2-ethyl-4-thioisonicotinamide (ethionamide) into its sulphoxide was purified from guinea-pig liver homogenates. The enzyme required stoicheiometric amounts of oxygen and NADPH for the sulphoxidation reaction. The purified protein is homogeneous by electrophoretic, antigenic and chromatographic criteria. The enzyme has mol.wt. 85000 and it contains 1g-atom of iron and 1mol of FAD per mol, but not cytochrome P-450. The enzyme shows maximal activity at pH7.4 in a number of different buffer systems and the K(m) values calculated for the substrate and NADPH are 6.5x10(-5)m and 2.8x10(-5)m respectively. The activation energy of the reaction was calculated to be 36kJ/mol. Under optimal conditions, the molecular activity of the enzyme (mol of substrate oxidized/min per mol of enzyme) is calculated to be 2.1. The oxygenase belongs to the class of general drug-metabolizing enzymes and it may act on different compounds which can undergo sulphoxidation. The mechanism of sulphoxidation was shown to be mediated by superoxide anions.     

Involvement of the superoxide anion in sulphoxidation (Short Communication)

Sulphoxidation of compounds capable of undergoing biological sulphoxidation has been demonstrated in a model system (NADH-phenazine methosulphate-O(2)), known to generate superoxide anions (O(2) (-)). Addition of superoxide dismutase to this system results in complete inhibition, suggesting the involvement of O(2) (-) in sulphoxidation.     

Nicotinamide-adenine dinucleotide-glycohydrolase activity in experimental tuberculosis

1. The specific NAD-glycohydrolase activity is increased 70 and 50% over the normal in lung and liver tissues respectively of tuberculous mice. 2. Concomitant with the increase in the NAD-glycohydrolase activity, the NAD–isonicotinic acid hydrazide-exchange activity also is increased in infection. The isonicotinic acid hydrazide analogue of NAD formed by the lung enzyme from tuberculous mice has been isolated and identified. 3. The increased NAD-glycohydrolase activity in infection has been shown to be of host-tissue origin and not due to the activation of the bacterial enzyme on growth of the organism in vivo. 4. In addition to NAD, NMN and NADP also participate in the exchange reaction with isonicotinic acid hydrazide catalysed by NAD glycohydrolase. The interference of the drug at the nucleotide level of metabolism is therefore suggested.     

DETERMINATION OF GUSTATORY SUCROSE THRESHOLD IN WATER BY USE OF A LINEAR SUCROSE GRADIENT

A novel experimental method was developed which allows the determination of the threshold concentration of sucrose by use of a linear sucrose gradient in water. With this method a continuous tasting of the test-liquid is possible. A panel of 15 persons experienced in taste-testing was used. Three gradients of different steepness were applied: 0 to 1.5% (w/w) sucrose in 2 min (I), 3 min (II) and 4 min (III). The results of the new method were compared with those of the standard method (DIN). With gradients I and II we found values which were significantly higher than those of the standard method (I: 0.49% (w/w); II: 0.46% (w/w); DIN: 0.31% (w/w)), whereas with gradient III the same threshold value was found as with the DIN-Method (III: 0.32% (w/w)).