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.     

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.     

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.     

Macromolecular synthesis in mycobacteriophage I3 infected cells

DNA-, RNA- and protein synthesis have been studied inMycobacterium smegmatis cells infected with phage 13. The macromolecular synthesis continued until the end of latent period. Early RNA and protein synthesis were necessary prior to the commencement of DNA replication. The infecting phage DNA sedimented as larger than unit length of genome, after initiation of DNA synthesis. Although the host DNA was not degraded, 90 percent of the RNA synthesized after phage infection hybridized to phage DNA.     

Presence of nucleoside triphosphates and calcium associated with mycobacteriophage I3

The association of nucleoside triphosphate molecules and calcium ions with purified particles of mycobacteriophage I3 has been documented. The content of nucleoside triphosphate has been determined to be 118 molecules per phage particle by equilibrium dialysis against labelled ATP or 148 molecules per phage particle by the direct determination of labelled nucleoside triphosphate. The concentration of bound Ca²⁺ exhibited a high degree of variation between different batches, which may be due to the nonspecific binding of Ca²⁺ by the virus particles. However, the tightly bound Ca²⁺ not removable by dialysis against calciumspecific chelating agent, showed a constant value of 2985 atoms/phage particle.Abbreviations EGTA Ethylene glycol-bis (-aminoethylether)-N,N¹ tetraacetic acid - PFU plaque forming unit - NTP nucleoside triphosphate     

Design of High-Specificity Nanocarriers by Exploiting Non-Equilibrium Effects in Cancer Cell Targeting

Although targeting of cancer cells using drug-delivering nanocarriers holds promise for improving therapeutic agent specificity, the strategy of maximizing ligand affinity for receptors overexpressed on cancer cells is suboptimal. To determine design principles that maximize nanocarrier specificity for cancer cells, we studied a generalized kinetics-based theoretical model of nanocarriers with one or more ligands that specifically bind these overexpressed receptors. We show that kinetics inherent to the system play an important role in determining specificity and can in fact be exploited to attain orders of magnitude improvement in specificity. In contrast to the current trend of therapeutic design, we show that these specificity increases can generally be achieved by a combination of low rates of endocytosis and nanocarriers with multiple low-affinity ligands. These results are broadly robust across endocytosis mechanisms and drug-delivery protocols, suggesting the need for a paradigm shift in receptor-targeted drug-delivery design.