Nucleotide sequence of the chromosomal gene coding for the aminoglycoside 6-adenylyltransferase from Bacillus subtilis Marburg 168

Gene aadK of Bacillus subtilis is 855 bp long and codes for aminoglycoside 6-adenylyltransferase.     

Nucleotide sequence of the glutamine synthetase gene (glnA) and its upstream region from Bacillus cereus

We have determined the complete nucleotide sequence of a 2.4 kb chromosomal EcoT22I-NspV fragment, containing the Bacillus cereus glnA gene (structural gene of glutamine synthetase). The deduced amino acid sequence indicates that the glutamine synthetase subunit consists of 444 amino acid residues (50,063 Da). Comparisons are made with reported amino acid sequences of glutamine synthetases from other bacteria. Upstrem of glnA we found an open reading frame of 129 codons (ORF129) preceded by the consensus sequence for a typical promoter. Maxicell experiments showed two polypeptide bands, with molecular weights in good agreement with that of glutamine synthetase and that of ORF129, in addition to vector-coded protein. It is possible that the product of this open reading frame upstream of glnA has a regulatory role in glutamine synthetase expression.     

Effects of temperature and pH on hemoglobin release from hydrostatic pressure-treated erythrocytes

The release of hemoglobin from human erythrocytes hemolyzed beforehand by hydrostatic pressure, osmotic pressure, and freeze-thaw methods was examined as a function of temperature (0-45 degrees C) and pH (5.5-8.8) at atmospheric pressure. Only in the case of high pressure (2,000 bar) did the release of hemoglobin increase significantly with decreasing temperature and pH. Maleimide spin label studies showed that the temperature and pH dependences of hemoglobin release were qualitatively explicable in terms of those of the conformational changes of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins showed the diminution of band intensities corresponding to spectrin, ankyrin, and actin in the erythrocytes hemolyzed by high pressure. Cross-linking of cytoskeletal proteins by diamide stabilized the membrane structure against high pressure and suppressed hemoglobin release. These results indicate that the disruption of cytoskeletal apparatus by high pressure makes the membrane more leaky.     

Isolation of a stable tetramer of the low molecular weight component of neurofilaments (NF-L)

When crude neurofilaments were dissolved in a solution containing 8 M urea and 1% beta-mercaptoethanol (beta-ME), the component proteins of the neurofilaments and other contaminating filaments were solubilized into monomeric forms. However, when reassembled filaments were solubilized again by the addition of urea to 8 M without beta-ME, several bands which seemed to be oligomeric forms of filament proteins were observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Among them, a band which appeared between microtubule-associated protein-1 (MAP-1) and fodrin was most remarkable. This band was also observed when a triplet mixture of the neurofilaments (NF-H, NF-M, NF-L) was reassembled. The molecular weight of this band was estimated to be 280 kDa. In addition, much of this component was easily isolated on DE-52 column chromatography of the reassembled crude neurofilament proteins with buffers containing 6 M urea, while the low molecular weight component of the neurofilaments (NF-L, 70 kDa) was hardly detected. Furthermore, the isolated 280 kDa component was reduced to NF-L on the addition of beta-ME to 1%. In contrast, the 280 kDa component was produced on dialysis of isolated NF-L against the assembly buffer. From these results, it is deduced that this component is the stable tetramer of NF-L which is produced through spontaneous interchain disulfide formation among protofilament tetramers.     

Direct evidence for nuclear and cytoplasmic colocalization of proteasomes (multiprotease complexes) in liver

Subcellular localization of the large multicatalytic protease complexes called proteasomes, which have been found in soluble fractions of various cells, was examined by biochemical, immunological, and immunohistological methods. Rat liver nuclei, purified by two different procedures, showed high activities for degrading [3H]methylcasein and various fluorogenic oligopeptides with neutral and weakly alkaline pH optima. On gel filtration, all of these peptidase activities were recovered in a single peak with the unusually large molecular weight of about 600,000. Properties of the proteolytic activity in crude extracts of the nucleus and the cytoplasm were very similar. Immunoelectrophoretic blot analysis showed the presence of appreciable concentrations of proteasomes with similar immunoreactivity in isolated nuclear and cytosolic fractions. Moreover, immunohistochemical staining of human liver showed that proteasomes were predominantly localized in the nuclear matrix but also were present diffusely in the cytoplasm of hepatocytes. These findings indicate the nuclear and cytoplasmic colocalization of proteasomes.     

Enhanced secretion of Escherichia coli beta-lactamase by a spontaneous erythromycin-resistant mutant of Bacillus subtilis

An extracellular-protease-deficient mutant, ME142, was isolated from Bacillus subtilis as a spontaneous erythromycin-resistant (Eryr) clone. This mutant showed conditional sporulation and only sporulated normally in the absence of erythromycin. In the presence of the antibiotic, sporulation was greatly reduced. Production of extracellular proteases by ME142 also exhibited conditional deficiency, possibly due to pleiotropic effects of the sporulation deficiency. The production of protease was 2-10% that of the wild-type level in the presence of erythromycin. ME142 showed poor competence for transformation even in the absence of erythromycin; however, derivatives of ME142 were isolated which had the same Eryr phenotype but which exhibited normal competence. One such mutant, ME162, was used as a host for the secretion of Escherichia coli beta-lactamase. The amount of beta-lactamase in the culture supernatants of ME162 increased significantly when the cells were cultured with erythromycin, suggesting that proteolysis of the beta-lactamase in the supernatants of ME162 was greatly reduced as compared to that in the supernatants of the wild-type strain.     

The effect of fatty acids on the developmental direction of Strongyloides ratti first-stage larvae

The effect of fatty acids was studied on the developmental direction of Strongyloides ratti first-stage larvae (L1). The proportion of third-stage infective larvae increased markedly when L1 were cultured in faeces with added fatty acids such as palmitic (C16), stearic (C18), oleic (C18:1) and linoleic (C18:2) acids. Unsaturated fatty acids were more effective than saturated ones. Moreover, the proportion of infective larvae increased with quantity of linoleic acid but the triacylglycerols of any fatty acid had no effect. These results suggest that these free fatty acids cause physiological changes that determine the developmental course of L1 of S. ratti in nature.     

Inhibitory effect of anti-class II antibody on the spontaneous activation of B cells in patients with systemic lupus erythematosus. Analysis with IL-1 production and IL-1 receptor expression

The mechanism of the spontaneous activation of B cells in patients with SLE was analyzed from the standpoint of the production of IL-1 from B cells and the expression of IL-1R on B cells. SLE B cells spontaneously produced IL-1-like factors which stimulated murine thymocyte proliferative responses. Their m.w. was about 17,000 and their isoelectric point was 4.8. The IL-1-like activity produced by B cells was absorbed with rabbit anti-IL-1 alpha antibody, but not with anti-IL-1 beta antibody. The differentiation of SLE B cells was enhanced by rIL-1 alpha, beta or IL-1-like factors produced by SLE B cells in a concentration-dependent manner. SLE B cells expressed large number of IL-1R detected by FITC-conjugated IL-1 alpha. By a Percoll gradient density centrifugation, IL-1-producing cells and B cells responsive to IL-1 were enriched in a higher density fraction, but were reduced in a lower density fraction. IL-1R-positive B cells were enriched in the lower density fraction, but were depleted in the higher density fraction. However, the expression of IL-1R on the lower density B cells was reduced by 2-day culture. The expression of IL-1R on the higher density B cells was increased during a 2-day culture. Anti-class II antibody inhibited the production of IL-1R on the higher density B cells. These results suggest that the cellular interaction among B precursor cells mediated by class II Ag induces the production of IL-1 and the expression of its receptors on their surface and the interaction between IL-1 and its receptors stimulates B precursor cells to spontaneously differentiate into Ig-producing cells as an autocrine mechanism in patients with SLE.