Some properties and occurrence of cytochrome c-552 in the aerobic photosynthetic bacterium Roseobacter denitrificans

Characteristics and occurrence of cytochrome c-552 from an aerobic photosynthetic bacterium, Roseobacter denitrificans, were described.Relative molecular mass of the cytrochrome was 13.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 15,000 by gel filtration. This cytochrome was a acidic protein having a pI of 5.6 and E_m was +215 mV at pH 7.0. Absorption peaks were at 278, 408 and 524 nm in the oxidized form and 416, 523 and 552 nm in the reduced form.Amino acid composition and N-terminal amino acid sequence of cytochrome c-552 determined for 24 residues had low similarities to those of cytochrome c-551 of this bacterium, which is homologous to cytochrome c ₂, although the physico-chemical properties of these two cytochromes were similar to each other.Cytochrome c-552 was maximally synthesized in the light under aerobic conditions but not in the dark. The synthesis also occurred in the presence of alternative acceptors such as trimethylamine N-oxide (TMAO) and nitrate under anaerobic conditions. Our results suggest that cytochrome c-552 is involved in TMAO respiration and denitrification in R. denitrificans, although the effect of light remains to be solved.Abbreviations E_m Midpoint redox potential - PAGE Polyacrylamide ge electrophoresis - SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis - TMAO Trimethylamine N-oxide     

Characterization of Borrelia Species Isolated from Ixodid Ticks,Ixodes ovatus

Thirty-two Borrelia isolates were obtained from the adult stage of ixodid ticks, Ixodes ovatus, collected in various localities in Japan. Borrelial isolates were cultivated and analyzed by polyacrylamide gel electrophoresis, with monoclonal antibodies, by pulsed field gel electrophoresis, and by genomic Southern hybridization. All borrelial isolates showed similar protein profiles and monoclonal antibody reactivities, while plasmid profiles were rather diverse. Genomic hybridization using rRNA gene probes demonstrated the genetic similarities of those isolates. We found no significant differences among the borrelial isolates tested, and the restriction fragment length polymorphism patterns of I. ovatus isolates were quite distinct from those of borrelial strains associated with Lyme disease. Therefore, the isolates of Borrelia obtained from I. ovatus were thought to fall into different genospecies.     

Accumulation of multiacetylated forms of histones by trichostatin A and its developmental consequences in early starfish embryos

Summary External application of 10 rig/ml (R)-trichostatin A (TSA), a potent and specific inhibitor of mammalian histone deacetylase, to the embryo of the starfish Asterina pectinifera inhibited development during the early gastrula stage before formation of mesenchyme cells. The TSA-sensitive period was limited to the mid-blastula stage before hatching. The pulse-chase experiment clearly demonstrated that TSA induced an accumulation of acetylated histone species in blastulae through inhibition of historic deacetylation. Similar blockage of development at the early gastrula stage was observed with n-butyrate, which has been known as a weak inhibitor of historic deacetylase. These results suggest an intimate role for historic acetylation-deacetylation equilibria in starfish development. Correspondence to: S. Ikegami     

Courtship Activity of Wandering and Burrow-holding Male Uca arcuata

The male of Uca (Deltuca) arcuata, a vertical claw-waving fiddler in the Indo-Pacific, is used to court females by approaching them from his burrow or while wandering. Differences in the rate of encounters with females between burrow-holding males and burrowless wandering males are found not to be significant. Burrow-holding males less often cause displacement of wandering by females than wandering males do. Wandering of females caused by wandering males occurs as often as wandering of males caused by other males. Thus, burrow-holding males tend not to reduce the number of their potential mates in the neighborhood. Burrow dwelling males are apt to start wandering after decreased encounters with females. Wandering males experience more interactions with other males than burrow-holding males do. Most wandering males that displace burrow owners descend the burrows one or more times after displacing the owners. The extra fighting for temporary burrows is responsible for no increase of the encounter rate with females during wandering.     

Identification and characterization of ispA, a Shigella flexneri chromosomal gene essential for normal in vivo cell division and intercellular spreading

The virulent phenotype of Shigella requires loci on the chromosome as well as on the large virulence plasmid, and is regulated via a complex web of interactions amongst various chromosomal and large plasmid genes. To further investigate the role of chromosomal loci in virulence, we performed random Tn 10 mutagenesis in Shigella flexneri YSH6000T, and isolated an avirulent mutant (V3404) incapable of spreading throughout an epithelial cell monolayer. Although V3404 initially spread intercellularly at the same rate as the wild-type, it gradually slowed down and ceased spreading as a result of increasing defects in cell division, leading to the formation of long filamentous bacteria lacking septa, trapped within cells. In addition, the mutation affected the ability of V3404 to polymerize actin, a prerequisite for intra- and inter-cellular spreading ability. Sequencing of Tn 10 -flanking DNA revealed that the mutated gene, designated ispA (intracellular septation), was equivalent to a previously sequenced but uncharacterised gene of Escherichia coli located between trp and tonB . Using E. coli sequence data, we cloned the ispA gene from the YSH6000T chromosome and found that it complemented the V3404 mutation. Nucleotide sequencing and in vitro expression experiments revealed that ispA coded for a small (21 kDa), very hydrophobic protein. These results thus show that ispA is an essential virulence gene affecting several functions of the virulence process.     

Tauropine dehydrogenase from the sandwormArabella iricolor (Polychaeta: Errantia): Purification and characterization

This is the first report of the purification of tauropine dehydrogenase (NAD: tauropine oxidoreductase) from a polychaete worm. In the sandwormArabella iricolor Montagu (Polychaet: Errantia), two forms of TaDH were detected: major component (pl = 7.5) and minor one (pI = 6.4). The major TaDH component was purified to homogeneity by means of (NH₄)₂SO₄ precipitation, anion-exchange, affinity, chromatofocusing and hydrophobic chromatography, and characterized. From the molecular mass of 43.7 kDa obtained by rapid gel-filtration and that of 43.5 kDa by SDS-PAGE, the sandworm enzyme appeared to be a monomeric protein. Maximum rates of reduction of pyruvate and oxidation of tauropine were observed at pH 7.0 and 8.5, respectively. Pyruvate and taurine were preferred substrate for the enzyme. Apparent Km values determined using constant co-substrate concentrations were: 35.7 mM, 0.34 mM, and 0.036 mM for taurine, pyruvate and NADH, respectively, in the tauropine synthesizing reaction; and 4.8 mM and 0.051 mM for tauropine and NAD⁺, respectively, in the tauropine oxidizing reaction. The tauropine synthesizing reaction was subject to substrate inhibition by pyruvate: maximum rate was observed at 2.5–3.0 mM (inhibitory range of pyruvate concentration producing half-maximal rate was 26.8 mM).     

Intra- and inter-specific variations in Lens revealed by RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate intra- and interspecific variations in the genus Lens (lentil). Twenty cultivars of L. culinaris ssp. culinaris, including 11 microsperma (small-seeded) and nine macrosperma (large-seeded) types, and 16 wild relatives (four accessions each of L. culinaris ssp. orientalis, L. odemensis, L. nigricans and L. ervoides), were evaluated for genetic variability using a set of 40 random 10-mer primers. Fifty reproducibly scorable DNA bands were observed from ten of the primers, 90% of which were polymorphic. Genetic distances between each of the accessions were calculated from simple matching coefficients. A dendrogram showing genetic relationships between them was constructed by an unweighted pair-group method with arithmetical averages (UPGMA). This study revealed that (1) expect for L. ervoides, the level of intraspecific variation in cultivated lentil is lower than that in wild species, (2) L. culinaris ssp. orientalis is the most likely candidate for a progenitor of the cultivated species, and (3) microsperma and macrosperma cultivars were indistinguishable by the RAPD markers identified here.     

DIVERSITY OF HALOGENATED SECONDARY METABOLITES IN THE RED ALGA LAURENCIA NIPPONICA (RHODOMELACEAE,CERAMIALES)1,2

Many morphologically similar, but chemically distinct, populations have been found in the marine red alga Laurencia nipponica Yamada (Rhodomelaceae, Ceramiales) growing in Japan. Each chemical type is characterized by a specific end-product of halogenated secondaly metabolite synthesis: chamigrane-type sesquiterpenoids such as prepacifenol and halochamigrene epoxide and C₁₅ bromoethers such as laurencin, laureatin, isoprelaurefucin, epilaurallene, and kumausallene. These seven types of secondary metabolite syntheses remained the same in the wild and under various culture conditions. Because bromoethers and terpenoids are probably synthesized by different metabolic pathways, it is virtually certain that different sets of enzymes participate in their synthesis. Prepacifenol- and laureatin-producing populations were selected as representatives of terpenoid and bromoether groups, respectively. F₁ tetrasporophytes derived from crosses between reciprocal, female and male gametophytes of prepacifenol- and laureatin-producing strains bore both types of metabolites, suggesting that the genes Producing these enzyme systems are encoded by nuclear genomes. The F₁ gametophytes resulting from the reciprocal crosses produced either prepacifenol or laureatin, and the four individuals derived from spore tetrads (a set of tetraspores derived from a single tetrasporangium) produced either prepacifenol or laureatin in a 1:1 ratio, indicating that genes participating in terpenoid synthsis and those participating in bromoether synthesis are on different loci of homologous chromosomes and are segregated at meiosis (tetrasporogenesis). One individual of this interpopulational F₁ gamtophyte produced both parental types of metabolite, perhaps indicating the occurrence of a recombination type. Natural hybrid individuals, including such recombination-type gametophytes, were found in a sympatric locality at which these two chemical types occur. F₁ tetrasporophytes derived from crosses between respective prepacifenol- and laureatin-producing strains and their F₁ gametohytes produced only parental-type metabolite-producing plants. These results indicate that the diverse chemical types can be referred to as races (chemical races).     

Involvement of Ca2+ signalling in the sugar-inducible expression of genes coding for sporamin and β-amylase of sweet potato

Genes coding for sporamin and β-amylase of sweet potato are inducible not only by high levels of metabolizable sugars, such as sucrose, but also by a low concentration of polygalacturonic acid (PGA). Calmodulin inhibitors and EGTA inhibited both the PGA-inducible and the sucrose-inducible accumulation of mRNAs for sporamin and β-amylase in sweet potato. Calmodulin inhibitors, EGTA and La³⁺, also inhibited the sucrose-inducible expression, in leaves of transgenic tobacco, of a fusion gene, β-Amy:GUS, which consists of the promoter of the β-amylase gene and the coding sequence for β-glucuronidase. The sucrose-inducible expression of the β-Amy:GUS fusion gene was also inhibited by two inhibitors of Ca²⁺ channels, diltiazem and nicardipine. These results suggest that the sugar-inducible expression of genes for sporamin and β-amylase involves, at least in part, Ca²⁺-mediated signalling, and that the cytosolic free Ca²⁺ may mediate cross-talk between signals related to carbohydrate metabolism and other stimuli. Treatment of coelenterazine-loaded leaf discs of tobacco expressing a Ca²⁺-binding photoprotein, aequorin, with 0.2 M sucrose for 24 h significantly reduced the level of luminescence that could be induced by cold shock, as compared to cold shock-induced luminescence in coelenterazine-loaded leaf discs treated with water. Repression of cold shock-induced luminescence was due to the conversion of holoaequorin to apoaequorin during the treatment with sucrose. Treatment of coelenterazine-loaded leaf discs with a 0.2 M solution of glucose or fructose, but not of mannitol or sorbitol, also reduced the cold shock-induced luminescence. It is suggested that non-synchronous increases in cytosolic level of free Ca²⁺ occur in leaf discs during treatment with high levels of metabolizable sugars.