Purification and properties of ATPase from an alkalophilic Bacillus

ATPase was purified from an alkalophilic Bacillus. The enzyme has a molecular weight of 410,000 and consists of five types of subunits of molecular weights of 60,000 (α), 58,000 (β), 34,000 (γ), 14,000 (δ), and 11,000 (?). The subunit structure is suggested to be α₃β₃γδ?. The enzyme is activated by Mg²⁺ and Ca²⁺. The pH optima of the enzyme with 0.1 and 2.0 mm Mg²⁺ are 9 and 6, and those with 1 and 10 mm Ca²⁺ are 8–9 and 7, respectively. Ca²⁺-ATPase hydrolyzes only ATP, whereas Mg²⁺-ATPase hydrolyzes GTP and, to a lesser extent, ATP. The values of V and K_m of the enzyme with ATP in the presence of 10 mm Ca²⁺ or 0.6 mm Mg²⁺ at pH 7.2 are 17 or 0.5 units/mg protein and 1.2 or 0.3 mm, respectively. The enzyme with Mg²⁺ is appreciably activated by HCO^?₃. Relationship of the ATPase to the active transport system in the bacterium is suggested.     

Gray and Red Fragments of the Egg of the Ascidian Ciona savignyi: Preferential Development of Muscle Cells from Gray Fragments

The egg of the ascidian Ciona savignyi is pinkish red with brownish myoplasm that contains the putative determinants responsible for differentiation of muscle cells. When dechorionated unfertilized eggs were centrifuged at moderate speed, eggs were divided into centripetal, small gray fragments and centrifugal, large red fragments. The former contained the female pronucleus and clear cytoplasm, while most of the latter was filled with yolk granules. An antibody raised against the myoplasm of C. intestinalis eggs extensively stained the cortical region of gray fragments, while the antibody stained only small regions of the red fragments. After insemination, both fragments cleaved and gave rise to partial embryos. When development of muscle and epidermal cells in the partial embryos was examined with specific antibodies, muscle development was conspicuous in gray partial embryos, while epidermal differentiation was extensive in red partial embryos. Furthermore, when expression of markers of differentiation was examined in cleavage-arrested gray and red fragments, the number of arrested gray fragments exhibiting the muscle marker was about three-fold greater than in controls. These results suggest that putative muscle determinants are concentrated into gray fragments.     

A new shuttle vector forBacillus stearothermophilus andEscherichia coli

Summary A new shuttle vector was constructed by inserting a 3.1 kbp-DNA fragment from thermophilicBacillus sp. plasmid pIH41 intoEscherichia coli plasmid pUC18. The resultant hybrid replicates in bothE. coli andB. stearothermophilus. This vector has ten unique restriction sites within a part oflacZ gene. Insertion of foreign DNA into these sites can be readily detected by a coloration method.     

Adenine depurination and inactivation of plant ribosomes by an antiviral protein of Mirabilis jalapa (MAP)

Mirabilis antiviral protein (MAP) is a single-chain ribosome-inactivating protein (RIP) isolated from Mirabilis jalapa L. It depurinates the 28S-like rRNAs of prokaryotes and eukaryotes. A specific modification in the 25S rRNA of M. jalapa was found to occur during isolation of ribosomes by polyacrylamide/agarose composite gel electrophoresis. Primer extension analysis revealed the modification site to be at the adenine residue corresponding to A⁴³²⁴ in rat 28S rRNA. The amount of endogenous MAP seemed to be sufficient to inactivate most of the homologous ribosomes. The adenine of wheat ribosomes was also found to be removed to some extent by an endogenous RIP (tritin). However, the amount of endogenous tritin seemed to be insufficient for quantitative depurination of the homologous ribosomes.Endogenous MAP could shut down the protein synthesis of its own cells when it spreads into the cytoplasm through breaks of the cells. Therefore, we speculate that MAP is a defensive agent to induce viral resistance through the suicide of its own cells.     

Effect of fatty acids and monoglycerides on permeability of lipid bilayer

The effect of fatty acids and monoglycerides on barrier properties of liposomal membranes prepared from egg phosphatidylcholine was investigated. The incorporation of these lipids as liposomal membrane components induced the alteration of the permeability to less permeable liposomally entrapped drugs, sulfanilic acid and procainamide ethobromide (PAEB). Monoolein caused greatly increased permeability of both drugs and unsaturated fatty acids markedly enhanced the release rate of PAEB, while saturated fatty acids caused a small increase in the release rate.Electron spin resonance (ESR) investigation with 5-nitroxide stearic acid showed that fatty acids disordered the hydrophobic region of the lipid bilayer and the disordering effect of unsaturated fatty acids was greater than that of saturated ones. It was demonstrated that the incorporated fatty acids and monoglycerides interacted with the polar region of the membranes by ESR study with cholestane label and ¹H-NMR study. These results indicated that the increase in the membrane permeability caused by fatty acids and monoglycerides associated with the disorder in the membranes' interior and the interaction of the incorporated lipid with the polar head group of phospholipid.     

Light and electron microscopical combined staining by immunohistochemical and enzyme histochemical methods using rat prolactin-secreting pituitary tumours

Summary Combined immunohistochemical staining (IHCS) and enzyme histochemical staining (EHCS) methods for light microscopy (LM) and electron microscopy (EM) are reported, using oestrogeninduced rat pituitary tumours. For LM, combined staining for alkaline phosphatase and acid phosphatase by EHCS, using the azo dye method, and for prolactin and ACTH by IHCS, using the enzyme-labelled antibody method, gave the best results on 1 m glycol methacrylate sections. For EM, combined staining by EHCS on 30 m tissue sections followed by IHCS for prolactin on ultrathin Epon sections (enzyme-labelled antibody method) provided acceptable results. By these combined staining methods, the neoplastic prolactin cells were shown to have close affinity to rich alkaline phosphatase-positive capillaries and to possess an alkaline phosphatase-positive cell membrane. Furthermore, they revealed acid phosphatase-positive lysosomal and secretory granules. These combined staining methods may be valuable in studies on the actual functional status of cells.     

Effect of the pH of culture medium on the alkalophilicity of a species of Bacillus

The amino acid incorporation and -aminoisobutyric acid (AIB) uptake of an alkalophilic Bacillus grown at pH 8.2 (the pH 8-bacteria) were much less pH dependent (less alkalophilic) than those of the organisms grown at pH 10.0 (the pH 10-bacteria), respectively. The rate of AIB uptake of the pH 10-bacteria was almost the same as that of the pH 8-bacteria, while the rate of amino acid incorporation of the pH 10-bacteria was higher than that of the pH 8-bacteria in alkaline environments. The colloidal titration with clupein showed that the amount of negative charge on the pH 10-bacteria was greater than that of the pH 8-bacteria in alkaline environments. Considerable difference in protein composition was observed between the membranes of the pH 8-and 10-bacteria, while no difference was observed in phospholipid composition.Abbreviations AIB Amino-isobutyric acid     

Dietary GABA induces endogenous synthesis of a novel imidazole peptide homocarnosine in mouse skeletal muscles

Amino Acids - Carnosine (β-alanyl-l-histidine) is an imidazole dipeptide present at high concentrations in skeletal muscles, where it plays a beneficial role. However, oral intake of carnosine...     

Two ANGUSTIFOLIA genes regulate gametophore and sporophyte development in Physcomitrella patens

In Arabidopsis thaliana the ANGUSTIFOLIA (AN) gene regulates the width of leaves by controlling the diffuse growth of leaf cells in the medio‐lateral direction. In the genome of the moss Physcomitrella patens, we found two normal ANs (PpAN1‐1 and 1‐2). Both PpAN1 genes complemented the A. thaliana an‐1 mutant phenotypes. An analysis of spatiotemporal promoter activity of each PpAN1 gene, using transgenic lines that contained each PpAN1‐promoter– uidA (GUS) gene, showed that both promoters are mainly active in the stems of haploid gametophores and in the middle to basal region of the young sporophyte that develops into the seta and foot. Analyses of the knockout lines for PpAN1‐1 and PpAN1‐2 genes suggested that these genes have partially redundant functions and regulate gametophore height by controlling diffuse cell growth in gametophore stems. In addition, the seta and foot were shorter and thicker in diploid sporophytes, suggesting that cell elongation was reduced in the longitudinal direction, whereas no defects were detected in tip‐growing protonemata. These results indicate that both PpAN1 genes in P. patens function in diffuse growth of the haploid and diploid generations but not in tip growth. To visualize microtubule distribution in gametophore cells of P. patens, transformed lines expressing P. patens α‐tubulin fused to sGFP were generated. Contrary to expectations, the orientation of microtubules in the tips of gametophores in the PpAN1‐1/1‐2 double‐knockout lines was unchanged. The relationships among diffuse cell growth, cortical microtubules and AN proteins are discussed.     

Eighteen Coral Genomes Reveal the Evolutionary Origin of Acropora Strategies to Accommodate Environmental Changes

The genus Acropora comprises the most diverse and abundant scleractinian corals (Anthozoa, Cnidaria) in coral reefs, the most diverse marine ecosystems on Earth. However, the genetic basis for the success and wide distribution of Acropora are unknown. Here, we sequenced complete genomes of 15 Acropora species and 3 other acroporid taxa belonging to the genera Montipora and Astreopora to examine genomic novelties that explain their evolutionary success. We successfully obtained reasonable draft genomes of all 18 species. Molecular dating indicates that the Acropora ancestor survived warm periods without sea ice from the mid or late Cretaceous to the Early Eocene and that diversification of Acropora may have been enhanced by subsequent cooling periods. In general, the scleractinian gene repertoire is highly conserved; however, coral- or cnidarian-specific possible stress response genes are tandemly duplicated in Acropora. Enzymes that cleave dimethlysulfonioproprionate into dimethyl sulfide, which promotes cloud formation and combats greenhouse gasses, are the most duplicated genes in the Acropora ancestor. These may have been acquired by horizontal gene transfer from algal symbionts belonging to the family Symbiodiniaceae, or from coccolithophores, suggesting that although functions of this enzyme in Acropora are unclear, Acropora may have survived warmer marine environments in the past by enhancing cloud formation. In addition, possible antimicrobial peptides and symbiosis-related genes are under positive selection in Acropora, perhaps enabling adaptation to diverse environments. Our results suggest unique Acropora adaptations to ancient, warm marine environments and provide insights into its capacity to adjust to rising seawater temperatures.