The isolation and characterization of bacteriophages infecting obligately thermophilic strains of Bacillus

Twenty-four thermophilic bacteriophages have been isolated from diverse sources such as compost, soil, silage and rotting straw. Although considerable individual host specificity was observed, the phages were able to infect most of the major taxonomic groups of Bacillus thermophiles. The phages varied considerably in morphology and size; the phage heads were either cylindrical or polyhedral with tails varying in length between 15 and 500 nm. Most of the phages were stable at 50 degrees C for 4-5 h but at 70 degrees C the plaque-forming units decreased by between 10(2)- and 10(7)-fold in 2 h. The DNA of morphologically similar phages was examined by restriction enzyme analysis, and some differences in the DNA fragment patterns were found. Efficiency of plating data indicated that 'B. caldotenax' has a restriction and modification system. These phages may be valuable for the study of the genetics of thermophilic bacilli: transduction of 'B. caldotenax' and 'B. caldovelox' by phage JS017 has been observed.     

Inducible DNA polymerase I synthesis in a UV hyper-resistant mutant of Escherichia coli

A mutant of Escherichia coli which is more resistant to shortwave UV light than its wild-type parent strain and which can synthesise DNA polymerase I constitutively has been further analysed. It carries two mutational alleles which are located about 1.5 min apart and cotransducible by P1 with the argH locus. The two mutational alleles have been segregated and their analysis shows that one of them is responsible for UV hyper-resistance whereas the other mutation confers UV sensitivity. Recombinant plasmids carrying various sections of the polA regulatory region, linked to a galK gene, were introduced into the mutant strains. Analysis of galactokinase shows that the enzyme activity in the UV hyper-resistant mutant is increased. The results suggest that the synthesis of DNA polymerase I in E. coli is inducible.     

The use of cellulose xanthate for the immobilisation of biological molecules

The mercapto groups of cellulose xanthate can reversibly form disulphide bridges with L-cysteine. This property has been utilised for the immobilisation of a protein and an enzyme. These macromolecules, as polythiol derivatives, formed disulphide linkages with the matrix without serious disturbance of their active sites, became firmly bound to the xanthate, and were not eluted by normal washing conditions. Cellulose xanthate is a cheap, easily prepared matrix which permits a simple coupling reaction. The immobilisation process is selectively reversible.     

Glutamine Synthetase Isoenzymes in the Green Soil Alga Stichococcus bacillaris Naeg

Two forms of glutamine synthetase (GS₁ and GS₂) have been separated from cells of Stichococcus bacillaris by fast protein liquid chromatography. The activities of the two isoenzymes were influenced by the composition of the media employed; thiol reagents were essential for stabilizing GS₂ but they suppressed GS₁ activity. The activity of each isoenzyme was, therefore, determined following separate purification procedures. Growth conditions influenced both isoenzymes; GS₂ showed maximum activity under photoautotrophic conditions, whereas GS₁ showed maximum activity under heterotrophic conditions.     

Comparison of intestine and bone marrow radiosensitivity of the BALB/c and the C57BL/6 mouse strains and their B6CF1 offspring

The radiosensitivity as measured by LD50/6 or LD50/30 of the F1 hybrid B6CF1 (C57BL/6 X BALB/c) is similar to that of C57BL/6 mice but markedly different from BALB/c. The LD50/6 for BALB/c mice was about 8.8 Gy compared to 16.4 Gy for the B6CF1. The difference in LD50/6 between the parent strains or between BALB/c and the F1 hybrid could not be explained by any differences in crypt cell number, cell cycle time, or transit time. Likewise, the observed differences in the LD50/6 do not appear to result from marked differences in the radiosensitivity of marrow stem cells (CFU-S) since the D0's for the three genotypes of mice were similar. Also, there were no apparent differences in the red blood cell contents of several enzymes associated with antioxidant defenses. The microcolony assay was used to determine the D0 for the crypt clonogenic cells and the D0 values for 60Co gamma rays were about 0.8 Gy for BALB/c mice and 1.4 Gy for B6CF1 mice. However, the D0 values for JANUS fission neutrons were similar; 0.6 Gy for the BALB/c mice and 0.5 for the B6CF1 mice. A comparison of clonogenic cell kinetics, using prolonged colcemid block to distinguish between slowly and rapidly cycling cells suggest that, normally, the stem cells are slowly cycling in both the BALB/c and the B6CF1 hybrid. However, the stem cells of the B6CF1 appear to go into rapid cell cycle more rapidly than those of the BALB/c following irradiation or prolonged colcemid treatment. The more rapid recovery in intestinal epihelial cell production in the B6CF1 hybrid after irradiation may provide an increased mucosal barrier and may, in part, explain the difference in the response to radiation compared to that in the BALB/c.     

Elevation of glutathione in phenylalanine mustard-resistant murine L1210 leukemia cells

Murine L1210 leukemia cells resistant to the antineoplastic agent L-phenylalanine mustard have a 1.5-2.0-fold elevation in their cellular GSH and GSSG content as compared to drug-sensitive cells. Cellular uptake of L-[U-14C]cystine and its incorporation into GSH of the resistant tumor are correspondingly elevated. Synthesis of gamma-glutamylcysteine, GSH, and GSSG is elevated 1.5-2.0-fold in cell-free preparations of the resistant tumor. This increased synthesis of GSH is attributed to increased cellular content (1.6-fold) of gamma-glutamylcysteine synthetase. GSH synthetase activity is equivalent in both drug-sensitive and -resistant cells. Investigation into the hydrolysis of selected peptides by cell-free preparations of both sensitive and resistant tumors suggest that aminopeptidase M participates in the formation of L-cysteine from L-Cys-Gly. This is supported by the observation that these preparations readily degrade L-Leu-p-nitroanilide and L-Ala-L-Ala-L-Ala, known substrates for aminopeptidase M, but not dipeptidase. The failure of the tumors to degrade Gly-D-Ala, a dipeptidase substrate, and the marked inhibition of L-Ala-Gly, L-Cys-Gly, and L-Ala-L-Ala-L-Ala hydrolysis by Bestatin further support a role for aminopeptidase M in the generation of L-cysteine from L-Cys-Gly. These results suggest that the drug-resistant tumor cell has developed an efficient mechanism for maintenance of elevated GSH which involves both gamma-glutamyl transpeptidase-initiated catabolism of GSH to cysteine and its reutilization by gamma-glutamylcysteine synthetase.     

Evidence for a sex pheromone metabolizing cytochrome P-450 mono-oxygenase in the housefly

Direct evidence is presented for the role of a cytochrome P-450 monooxygenase (called mixed-function oxidase, or polysubstrate mono-oxygenase, PSMO) in the metabolism of the sex pheromone (Z)-9-tricosene to its corresponding epoxide and ketone in the housefly. A secondary alcohol, most likely an intermediate in the conversion of the alkene to the ketone, was also tentatively identified. The results of in vivo and in vitro experiments showed that the PSMO inhibitors, piperonyl butoxide (PB) and carbon monoxide, markedly inhibited the formation of epoxide and ketone from (9,10-³H) (Z)-9-tricosene. An examination of the relative rates of (Z)-9-tricosene metabolism showed that males exhibited a higher rate of metabolism than females with the antennae of males showing the highest activity of any tissue/organ examined. The major product from all tissues/organs was the epoxide. Data from experiments with subcellular fractions showed that the microsomal fraction had the majority of enzyme activity, which was strongly inhibited by PB and CO and required NADPH and O₂ for activity. A carbon monoxide difference spectrum with reduced cytochrome showed maximal absorbance at 450 nm and allowed quantification of the cytochrome P-450 in the microsomal fraction of 0.410-nmol cytochrome P-450 mg^?1 protein. Interaction of (Z)-9-tricosene with the cytochrome P-450 resulted in a type I spectrum, indicating that the pheromone binds to a hydrophobic site adjacent to the heme moiety of the oxidized cytochrome P-450.     

Neurotensin receptors in canine intestinal smooth muscle: preparation of plasma membranes and characterization of (Tyr3-125I)-labelled neurotensin binding

To study the binding of (Tyr3-125I)-labelled neurotensin to intestinal muscle, plasma membranes have been purified from dog intestinal circular smooth muscle. Purification was done by differential centrifugation followed by separation on a sucrose gradient. Electron microscopic study revealed that the dissected circular muscles used as the source of membranes were free of myenteric plexus and that the plasma membrane fraction obtained was free of any mitochondria or synaptosomes. The fraction used was obtained at the interface of 14%-33% sucrose density on the gradient and was 25-times enriched in the plasma membrane marker enzyme 5'-nucleotidase activity as compared to post-nuclear supernatant. This fraction contained negligible activity of mitochondrial membrane marker enzyme cytochrome c oxidase and low activity of a putative endoplasmic reticulum marker enzyme NADPH-cytochrome-c reductase. This membrane fraction contained a high density of neurotensin binding sites. This binding was studied by kinetic and by saturation approaches. Analysis of data from saturation binding studies by the computer programs (EBDA and LIGAND) suggested the presence of a two-site model (Kd1 = 0.118 nM, Kd2 = 3.18 nM, Bmax1 = 9.73 fmol/mg and Bmax2 = 129.8 fmol/mg). A part of specifically bound neurotensin was rapidly dissociated. No cooperativity between the two receptor types could be detected. A kinetic analysis of binding gave the Kd value equal to 0.107 nM. Carboxy terminal amino acid residues 8-13 were found to be essential for the binding activity and replacement of Tyr11 by tryptophan reduced the affinity of the peptide by 10 times in displacement studies. Binding was modulated by sodium ions and a guanine nucleotide Gpp[NH]p. MgCl2, CaCl2 and KCl were also found to reduce the specific binding. Evidence was found of a high specific binding to another membrane fraction poor in plasma membranes and rich in synaptosomes. We concluded that plasma membrane of canine intestinal circular muscle contains neurotensin receptors with recognition properties distinct from those obtained in previous studies of neurotensin binding sites in murine tissues. Another neurotensin binding site may be present on neuronal membranes.     

Isolation and preliminary characterization of a 2-chlorobenzoate degrading Pseudomonas

Pseudomonas sp. strain B-300, which is able to utilize 2-chlorobenzoic acid, was isolated from a soil sample by enrichment culture. This strain was shown to grow on 2-chlorobenzoic acid and to completely degrade the substrate with concomitant chlorine ion release. Concentrations of 2-chlorobenzoic acid higher than 0.5% (w/v) were toxic to the cells. Our study also suggested that in the presence of glucose, 2-chlorobenzoic acid is converted to catechol or chlorocatechol; these are in turn transformed to muconic and chloromuconic acid, respectively, suggesting a repression by glucose of some of the degradation pathway enzymes. A similar scheme was already described for 3-chlorobenzoate degradation by pAC25 plasmid.