Many alkalophilic bacteria were found to produce inhibitors of protein kinases. We isolated a novel inhibitor of protein kinase from an alkalophilic strain of Bacillus species. This substance was A heat-stable peptide with a molecular weight of 13, 000 daltons. It was found to be a selective inhibitor of cyclic AMP-dependent protein kinase (A kinase). The inhibition of a kinase by this substance was non-competitive with histone or ATP. It behaved distinctly; other known inhibitors such as H-7, H-8, Staurosprine, K-252 and Erbstatine inhibit protein kinase less selectively and their functions are competitive with either substrate or ATP. This inhibitor was found to bind to the regulatory subunits of A kinase and markedly inhibited the separation of the catalitic subunits from A kinase induced by the binding of cAMP despite of no effect on the binding of cAMP. Thus, the activation step of A kinase was influenced by this inhibitor. This molecule had no effect on the inhibition by cAMP of CHO cell proliferation. This may have been due to the inability of this molecule to reach the target in the cell. Modification of the molecule itself or the administration method is needed for cellular or animal application.
A mycolic acid-containing glycolipid, trehalose-2, 3, 6'-trimycolate (GaGM), derived from Gordona aurantiaca, an acid-fast bacteria closely related taxonomically to Mycobacterium, was investigated for its immune adjuvant activity in vitro. The liposomes containing GaGM showed strong mitogenic effects on murine spleen cells at the doses used (25-100μg/ml), but not on T-cell-depleted spleen cells or macrophage-depleted spleen cells. These results suggest that the mitogenic property of liposomes containing GaGM differs from that of such as lipopolysaccharide, a B-cell mitogen and that its mitogenic effects depend on the presence of macrophages. In addition, liposomes containing GaGM augmented the mixed lymphocyte reaction (MLR) and in vitro induction of cytotoxic T-lymphocytes (CTLs) against allogeneic tumor cells. These results suggest that liposomes containing GaGM have immune adjuvant properties in vitro and the adjuvant activity may be related to such cytokines as interleukin-1 and -2.
Vibrio anguillarum, one of the causative agents of fish vibriosis, is serologically and biochemically divided into three groups (A, B and C). The chemical composition and molecular architecture of lipopolysaccharide (LPS) isolated from V. anguillarum PT514, which belongs to serogroup B, were investigated. The LPS contained glucose (Glc), fructose (Fru), L-glycero-D-mannoheptose (L-D Hep), glucosamine (GlcN) and 4-amino-4, 6-dideoxyglucose as sugar constituents in molar ratios of 8.9:0.7:3.0:1.1:1.6. Sephadex G-50 gel-chromatography of a degraded polysaccharide fraction separated from the LPS by 5% acetic acid hydrolysis suggested that the O-specific polysaccharide region consists of, in average, as much as 29 moles of Glc per 3 moles of L-D Hep, while the core polysaccharide contains at least Glc, L-D Hep and GlcN in molar ratios of 3.2:3.0:0.2. Fru and 4-amino-4, 6-dideoxyglucose components were released from LPS on weak-acid hydrolysis, indicating that PT514 LPS is distinguishable from those of Vibrio anguillarum belonging to the other serogroups. 2-Keto-3-deoxyoctonate (KDO), a common sugar constituent of gram-negative bacterial LPS, was not detected by Weissbach's color reaction under the conventional hydrolysis condition, but O-phosphoryl KDO was found in the strong-acid hydrolysate (4M HCl, 100C, 45min). This substance was identical, at least in high-voltage paper electrophoresis, to 5-O-phosphoryl KDO. Lipid A isolated from the LPS contained C12:0, C14:0, C16:0, C16:1, C18:1, 3-OHC12:0 and 3-OHC14:0. The LPS showed endotoxic activity in limulus lysate assay to the same extent as those of V. cholerae NIH 41 (Ogawa) and 569B (Inaba).
The membrane filter technique with AC agar medium supplemented with 0.04% NaN3 and 0.00015% 2, 3, 5-triphenyltetrazolium chloride for enumeration of enterococci in water is described. An appropriate volume of a water sample was filtered through the membrane filter. The membrane filter was put on an AC agar plate (designated as the AC⋅MF technique), which was incubated at 37C for 18hr and further at 45C for 24hr. By this AC⋅MF technique, all the colonies grown on the membrane filters were identified as enterococci, and the count of enterococci obtained by the AC⋅MF technique was similar to that by the AC⋅MPN technique. The AC⋅MF technique may be useful for accurate and rapid enumeration of enterococci in water and serve as a simple method for determining the sanitary quality of water.
Several species of mycoplasmas including M. pneumoniae, the causative agent of human respiratory infection, were investigated for tumor necrosis factor-alpha (TNF-α) induction. The cytotoxic activity to Meth A cells of peritoneal macrophages purified from BALB/c mice was enhanced markedly when cultured with either viable or nonviable mycoplasmas. The supernatant of macrophage culture mixed with mycoplasmas, M. pneumoniae or A. laidlawii, showed a potent cytotoxic activity to TNF-α-sensitive but not to TNA-α-insensitive L cells. Addition of anti-TNA-α antiserum inhibited completely the cytotoxic activity of the supernatant, indicating that the cytotoxic activity is due mostly to TNF-α. These results strongly suggest that mycoplasmas possess an activity to induce TNF-α, which enhances the cytotoxic activity of macrophages and prevent infection with mycoplasmas in vivo.