A procedure to remove protease activities from Bacillus subtilis sporulating cells and their crude extracts.

A simple procedure was developed to remove both extracellular and intracellular proteases associated with aporulating Bacillus subtilis cells. Cells are washed four times with 1 m KCl before breakage and their crude extracts are treated with 2 mm diisopropylfluorophosphate before passage through a hemoglobin-Sepharose affinity column. RNA polymerase in crude B. subtilis extracts treated by this procedure was stable, functionally and structurally, for more than 1 month at 4°C. This process for removing all proteases should work essentially with any crude extracts containing proteolytic activities.     

Accumulation of O-methyl-L-homoserine by methanol-utilizing bacteria

Summary Several classes of mutants derived from facultative methylotrophs, Microcyclus eburneus and Protaminobacter ruber, accumulate 0.5–3.8 mg/ml of O-methyl-L-homoserine (OMH) in a medium containing methanol as sole carbon source. The wild-type parent strains also accumulate OMH when the medium is supplemented with L-homoserine; the accumulation of OMH by the mutants is ascribed to an increased L-homoserine pool.     

A photochemical fuel cell system using Anabaena N-7363

Summary A blue-green algae, Anabaena N-7363, was immobilized in 2% agar gel. The hydrogen productivity of the immobilized algae was three times higher than that of free algae. The maximum hydrogen production rate by the immobilized blue-green algae was 0.52 moles h^–1 g^–1 (of wet gel) in the medium without nitrogen sources under illumination (10,000 lux). The oxygen evolved was then removed by a reactor containing aerobic bacteria. A photo-current of 15–20 mA was continuously produced for 7 days by the photochemical fuel cell system consisting of the immobilized Anabaena reactor, the oxygen-removing reactor and the hydrogen-oxygen fuel cell. The conversion ratio of hydrogen to current was from 80% to 100%.     

Determination of serum unconjugated estrone, estradiol-17β and estriol during pregnancy by selected ion monitoring

A simple, rapid and highly specific method by selected ion monitoring (SIM), using 9α,11α-[²H₂]estrone, [2,4-²H₂]estradiol-17β and 2,4-[²H₂]estriol as internal standards, was developed for the determination of serum estrogens during pregnancy. Serum samples were submitted to a simple extraction procedure and were analysed after formation of the trifluoroacetic anhydride derivative. The inter-assay coefficients of variation for estrone, estradiol-17β and estriol were 3.73%, 3.42% and 3.49%, respectively. The results obtained by SIM were compared with analysis performed using radioimmunoassay.     

Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.     

Subcellular localization of glycolytic key enzymes in germinating castor bean endosperm

Phosphofructokinase and pyruvate kinase activities in castorbean endosperm increased during germination. Subcellular localizationof pyruvate kinase and phosphofructokinase in germinating endospermtissues was studied by differential and sucrose density gradientcentrifugation techniques. Eighty five percent or more of thepyruvate kinase and phosphofructokinase activities were locatedin cytosol. The remaining activities were mainly detected inproplastids. (Received June 30, 1977; )     

Cloning and functional expression of a novel endoprotease involved in prohormone processing at dibasic sites.

We cloned and sequenced a cDNA from a library of mouse pituitary AtT-20 cells which are known to cleave an endogenous and various foreign prohormones at dibasic sites. This cDNA encodes a novel 753-residue protein, named PC3, which is structurally related to the yeast Kex2 protease involved in precursor cleavage at dibasic sites and to recently identified mammalian Kex2-like proteins, furin and PC2. Among examined cell lines and tissues, PC3 mRNA was only detected in AtT-20 cells. The substrate specificity of PC3 expressed in mammalian cells was similar to that observed in AtT-20 cells. We conclude that PC3 is a resident prohormone processing endoprotease in AtT-20 cells.     

Adhesion of mouse mast cells to fibroblasts: adverse effects of steel (Sl) mutation

Mouse bone marrow-derived cultured mast cells proliferate on +/+ mouse embryo-derived 3T3 fibroblasts, but not on Sl/Sld mouse embryo-derived 3T3 fibroblasts, in the absence of IL-3 and IL-4 (Fujita et al: Proc. Natl. Acad. Sci. U.S.A. 86:2888-2891, 1989). To further characterize the mast cell-fibroblast interactions and the effects of Sl mutation, we tried to analyze the adhesion of cultured mast cells to 3T3 fibroblasts in vitro. Mast cells plated onto NIH/3T3 fibroblasts showed marked adhesion within 30 min, which reached a plateau after 3 h. The numbers of adhered mast cells were linear over the range of 10(3) to 5 x 10(5) cells inoculated into each (2 cm2) of 24 multiwells. Adhesion required active energy production and the presence of divalent cations. It was not inhibited by an RGD-containing peptide, an anti-LFA-1 antibody, or asialofetuin. Mast cells adhered efficiently to the eight 3T3 cell lines derived from +/+ mouse embryos, but not to the eight 3T3 cell lines derived from Sl/Sld mouse embryos. Adhesion to +/+ mouse spleen-derived fibroblasts lacking mast cell-supporting activity was comparable to that to Sl/Sld/3T3 cells. The failure of mast cells to adhere to fibroblasts with the Sl mutations was not due to a production of a diffusible inhibitor by the latter. These results indicate that production of wild type Sl gene product by fibroblasts is mandatory for adhesion/migration, as well as for proliferation of mast cells on them, and that the coculture system should be useful for the biochemical and molecular analysis of these interactions.     

A possible contribution of endogenous atrial natriuretic peptide to proteinuria in patients with chronic renal failure.

Plasma levels of immunoreactive atrial natriuretic peptide (IR-ANP) were measured with a specific radioimmunoassay in 19 undialysed patients with chronic renal failure. At the beginning, an extremely high level of plasma hANP (50 fmol/ml) seen in a patient was rejected with Smirnov's test and was excluded from further statistics. The plasma IR-ANP levels in these patients were significantly higher than those of 19 normal subjects matched with age and sex (10.9 +/- 1.6 vs 5.3 +/- 0.6 fmol/ml, mean +/- SEM, p less than 0.01), and positively correlated with mean blood pressure (r = 0.44, p less than 0.05) and the cardiothoracic ratio (r = 0.65, p less than 0.01), but did not correlate with creatinine clearance (r = -0.38, n.s.). Further, a significant correlation was observed between plasma IR-ANP and urinary protein output (r = 0.47, p less than 0.05). On the other hand, urinary protein output did not correlate significantly with variables such as mean blood pressure, the cardiothoracic ratio or creatinine clearance. Since it has been suggested that ANP enhances glomerular capillary permeability, increased ANP responding to volume overload in those patients may play an important role in increasing urinary protein excretion.