Immuno-Gold Localization of Nitrogenase in Root Nodules of Elaeagnus pungens Thunb

The molybdenum-iron component of nitrogenase (Mo-Fe component)was purified from soybean nodule bacteroids and antibody wasraised against it in rabbits. Antibody raised against the 53kDa polypeptide which was the major protein in the Mo-Fe componentfraction of soybean nitrogenase was confirmed to be specificto the nitrogenase by immunodiffusion and immunotitration. Thenitrogenase from root nodules of Elaeagnus pungens cross-reactedwith the antibody and appeared from the results of the immunodiffusionto be partially identical to soybean nitrogenase. Using the antibody, we examined intracellular localization ofnitrogenase in root nodules of Elaeagnus pungens, in which Frankiais present as a symbiont, by immuno-gold labelling. Thin sectionsof nodules of Elaeagnus pungens were first treated with anti-nitrogenasespecific antibody and then with colloidal gold-protein A asa marker. The gold particles were observed to be concentratedin the vesicles of the endophyte Frankia. This provides strongsupport for the existence E of nitrogenase in the vesicles.Furthermore, our results suggested that nitrogenase localizesin the hyphae of the endophyte Frankia in Elaeagnus pungensnodules. ¹Present address: Iwata Experiment Station, Japan Tobacco Inc.,Iwata-gun, Shizuoka 438, Japan. (Received March 9, 1988; Accepted July 28, 1988)     

Rapid profiling of bacterial quinones by two-dimensional thin-layer chromatography

Bacterial quinones were analysed by two-dimensional thin-layer chromatography with ready-made, multi-phase silica gel plates which allowed good separation of complicated quinone mixtures. A combination of this method and silver-ion-modified thin-layer chromatography made it possible to identify partially hydrogenated quinones.     

Light and electron microscopic studies on experimental nocardia-toxicosis in mice

An exotoxin (HS-6) produced by Nocardia otitidiscaviarum isolated from certain lesions of cutaneous nocardiosis of a male 82-year-old patient induced severe injuries in the pancreas, liver, stomach, small intestine, heart, thymus and kidney of male ICR mice. Mice given Nocardia-free preparation of HS-6 at a dose of 1 mg/kg of body weight developed several autophagic vacuoles in the pancreas and liver within 20 min after the i.p. injection. Thereafter, the autophagic vacuoles increased in number and size with time. About 24 hr after the administration of HS-6, the liver showed marked accumulation of fat droplets in the cytoplasm of the hepatocytes. Although they contained abundant autophagic vacuoles in the regions of RER, there were no lipomatoses in the acinar cells of the pancreas, those of the chief cells and smooth muscle cells of the stomach, Paneth cells, goblet cells, smooth muscle cells of the small intestine, and plasma cells in the digestive tract. Biochemical examinations revealed that HS-6 had no significant effect on the protein synthesis of reticulocytes. Inoculation of the Nocardia into the mouse peritoneal cavities caused marked granulomatoses in the pancreas, liver and regional lymph nodes, but did not develop autophagic vacuoles in RER regions of these organs.     

Distribution and possible intergradation betweenMacaca tonkeana andM. ochreata at the borderland of the species in Sulawesi

Morphological observations of pet and wild monkeys were made in the area that was inferred to be the borderland betweenMacaca tonkeana andM. ochreata in Sulawesi. Almost all individual monkeys could be classified into one of two species by their external characteristics. The possible borderland was estimated to extend from the La River in the east and to around Karaena River in the west. These two species may make contact in the forest in the western area of the borderland. Some external characteristics exhibited wide individual variations in the two species. Some monkeys originating from the borderland showed external characteristics that were intermediate between those of the two species. The possible intergradation between these two species is discussed in terms of the morphological variations found in the two species.     

Silence of streptomycin 6-phosphotransferase gene derived by incubation at a high temperature inStreptomyces griseus

Summary The bald mutants from streptomycin (SM)-producingStreptomyces griseus 2247 obtained by incubation at high temperature (36° C), designated as HT strains, lost resistance to their own antibiotic and scarcely produced the antibiotic. Although SM susceptibility in the mutant was due to loss of SM 6-phosphotransferase activity produced in the cell, the gene coding for the enzyme cloned from an HT strain was surely expressed inS. lividans 1326 as a host. Northern blot analysis showed that the corresponding RNA is not detected in the mutant, indicating that though the gene encoding SM 6-phosphotransferase, at least, the structural gene is not deleted in the cell, the expression is silent.     

A hyperthermostable pullulanase produced by an extreme thermophile,Bacillus flavocaldarius KP 1228, and evidence for the proline theory of increasing protein thermostability

Summary A cell-associated pullalanase (-dextrin 6-glucanohydrolase, EC 3.2.1.41) of an extreme thermophile, Bacillus flavocaldarius KP 1228, was purified to homogeneity. The molecular weight and isoelectric point were estimated to be about 55 000 and 7.0, respectively. The N-terminal sequence was Ala-Try-Tyr-Glu-Gly-Ala-Phe-Phe-Tyr-Gln-Ile-Phe-Pro-Asp-Tyr-Phe-Phe-Tyr-Ala-Gly-. The enzyme was most active at pH 6.3. The activities for 5% pullulan and 5% soluble starch were maximal at 75–80° C and at 80–85° C, respectively. The enzyme was stable up to 90° C for 10 min at pH 6.8. The enzyme had no antigenic determinants shared with pullulanases from the mesophiles Klebsiella pneumoniae and B. acidopullulyticus NCIB 11647. A comparison of amino acid composition demonstrated that the proline content increased greatly in a linear fashion with the rise in thermostability in the order K. pneumoniae B. acidopullulyticus B. flavocaldarius enzymes, as found with Bacillus oligo-1,6-glucosidases.Presented in part at the Annual Meeting of the Agricultural Chemical Society of Japan at Tokyo, April 2, 1987 (Abstracts, p 91)Offprint requests to: Y. Suzuki     

Simultaneous formation of menaquinones and demethylmenaquinones byMicrococcus varians IAM 12146 depending on cell growth media

Changes in the isoprenoid quinone composition ofMicrococcus varians IAM 12146 in response to growth in different media were investigated. When the bacterium was growth in an ordinary complex medium, it produced menaquinones as the sole quinones, with a dihydrogenated menaquinone with seven isoprene units as the major component, at all growth stages. On the other hand, cells grown in a chemically defined medium containing glutamate and pyruvate as carbon sources produced both menaquinones and demethylmenaquinones. The major demethylmenaquinone homologs produced were the unsaturated and dihydrogenated types with seven isoprene units. The demethylmenaquinone/menaquinone ratio in cells varied during a batch growth in the chemically defined medium. The highest ratio was found in cells at the mid-exponential phase of growth.     

Endothelin stimulates c-fos and c-myc expression and proliferation of vascular smooth muscle cells

Recently, a potent vasoconstrictor peptide, endothelin (EDT), was isolated from vascular endothelial cells. We examined its effect on rat vascular smooth muscle cells (VSMCs). EDT induced the elevation of intracellular calcium, which was dependent on extracellular calcium and inhibited by a calcium-channel antagonist in a competitive manner. EDT caused a rapid and transient increase in the c-fos and c-myc mRNA levels and stimulated the DNA synthesis of VSMCs in a dose-dependent manner. This effect of EDT on the proliferation of VSMCs might be related to the development of atherosclerosis.     

Selective potentiation of 5-methyl-2-(1-methylcyclohexyl)-4-oxazoleacetic acid (AD-4610) on glucose-induced insulin secretion

In the perfused pancreas from normal SD rats, AD-4610 (0.01-0.1 mM) potentiated biphasic insulin secretion induced by 7.5 mM of glucose. The concentration-response curve of insulin secretion to glucose was shifted leftwards with AD-4610 (0.1 mM) without altering either the threshold concentration of glucose to induce insulin secretion or the maximal insulin response to glucose, indicating increased sensitivity of the pancreatic B-cells to glucose. On the other hand, AD-4610 was 10-fold less effective in altering insulin secretion induced by arginine and glyceraldehyde. The effect of AD-4610 on insulin secretion and glucose metabolism was compared with that of tolbutamide in vivo. AD-4610 (100 mg/kg) potentiated insulin secretion induced by an intravenous glucose load, and also accelerated glucose metabolism without altering basal insulin secretion in normal rats. On the other hand, tolbutamide (20 mg/kg) increased basal insulin secretion, but slightly decreased glucose-induced insulin secretion. In yellow KK mice with hyperglycemia, AD-4610 (10-100 mg/kg) had a dose-dependent hypoglycemic action, but tolbutamide did not. Thus, AD-4610 stimulated insulin secretion in a glucose-dependent fashion and enhanced glucose metabolism in vivo. These results suggest that AD-4610 selectively potentiates glucose-induced insulin secretion by increasing the sensitivity of pancreatic B-cells to glucose and may be useful for treating human NIDDM through a different mechanism than that of tolbutamide.