The Changes of Lysine- and Ornithine-lipids in Streptomyces sioyaensis

Lysine-lipid (siolipin A) and ornithine-lipid (siolipin B) were found at the same time in Streptomyces sioyaensis. They were also found in mutant strains (Lys^?, Met^?, Try^?, His^?) of Streptomyces sioyaensis. Ratio of siolipin A to siolipin B differed, depending on the culture conditions. The young mycelium contained siolipin A predominantly, while the aged mycelium did much more siolipin B. They also varied according to pH of the broth. As the whole, the effect was more conspicuous in the mycelium from jar fermenter than that from Sakaguchi flasks.     

Gene Expression Patterns in the Outer Mantle Epithelial Cells Associated with Pearl Sac Formation

For pearl culture, nucleus and mantle grafts are implanted into the gonad of the host oyster. The epithelial cells of the implanted mantle graft elongate and surround the nucleus, and a pearl sac is formed. Shell matrix proteins secreted by the pearl sac play an important role in pearl formation. We studied the gene expression patterns of six shell matrix proteins (msi60, n16, nacrein, msi31, prismalin-14, and aspein) in the epithelial cells associated with pearl sac formation. There were differences in the expression patterns of the six genes in the epithelial cells, and the relative expression levels for msi60 and aspein differed between the mantle graft and pearl sac (48 days after implantation). Therefore, the gene expression patterns of the epithelial cells were genetically undetermined, and changed between before and after pearl sac formation. The gene expression patterns of the epithelial cells of the pearl sac may be regulated by the host oysters.     

A multifaceted genomics approach allows the isolation of the rice Pia-blast resistance gene consisting of two adjacent NBS-LRR protein genes

The Oryza sativa (rice) resistance gene Pia confers resistance to the blast fungus Magnaporthe oryzae carrying the AVR-Pia avirulence gene. To clone Pia, we employed a multifaceted genomics approach. First, we selected 12 R-gene analog (RGA) genes encoding nucleotide binding site-leucine rich repeats (NBS-LRRs) proteins from a region on chromosome 11 that shows linkage to Pia. By using seven rice accessions, we examined the association between Pia phenotypes and DNA polymorphisms in the 10 genes, which revealed three genes (Os11gRGA3-Os11gRGA5) exhibiting a perfect association with the Pia phenotypes. We also screened ethyl methane sulfonate (EMS)-treated mutant lines of the rice cultivar 'Sasanishiki' harboring Pia, and isolated two mutants that lost the Pia phenotype. DNA sequencing of Os11gRGA3-Os11gRGA5 from the two mutant lines identified independent mutations of major effects in Os11gRGA4. The wild-type 'Sasanishiki' allele of Os11gRGA4 (SasRGA4) complemented Pia function in both mutants, suggesting that SasRGA4 is necessary for Pia function. However, when the rice cultivar 'Himenomochi' lacking Pia was transfected with SasRGA4, the Pia phenotype was not recovered. An additional complementation study revealed that the two NBS-LRR-type R genes, SasRGA4 and SasRGA5, that are located next to each other and oriented in the opposite direction are necessary for Pia function. A population genetics analysis of SasRGA4 and SasRGA5 suggests that the two genes are under long-term balancing selection.     

Site-specific recombination system based on actinophage TG1 integrase for gene integration into bacterial genomes

Phage integrases are enzymes that catalyze unidirectional site-specific recombination between the attachment sites of phage and host bacteria, attP and attB, respectively. We recently developed an in vivo intra-molecular site-specific recombination system based on actinophage TG1 serine-type integrase that efficiently acts between attP and attB on a single plasmid DNA in heterologous Escherichia coli cells. Here, we developed an in vivo inter-molecular site-specific recombination system that efficiently acted between the att site on exogenous non-replicative plasmid DNA and the corresponding att site on endogenous plasmid or genomic DNA in E. coli cells, and the recombination efficiencies increased by a factor of ~10^1–3 in cells expressing TG1 integrase over those without. Moreover, integration of attB-containing incoming plasmid DNA into attP-inserted E. coli genome was more efficient than that of the reverse substrate configuration. Together with our previous result that purified TG1 integrase functions efficiently without auxiliary host factors in vitro, these in vivo results indicate that TG1 integrase may be able to introduce attB-containing circular DNAs efficiently into attP-inserted genomes of many bacterial species in a site-specific and unidirectional manner. This system thus may be beneficial to genome engineering for a wide variety of bacterial species.     

Purification, characterization, and sugar binding specificity of an N-Glycolylneuraminic acid-specific lectin from the mushroom Chlorophyllum molybdites

A carbohydrate-binding protein was isolated from the carpophores of the mushrooms and designated the Chlorophyllum molybdites lectin (CML) based on its origin. The molecular mass of CML was 32 kDa, and it was composed of two 16-kDa monomers with no disulfide bonds. Monosaccharide analysis indicated that 12% of the mass of CML was carbohydrate and consisted of GlcNAc:GalNAc:Gal:Man:l-Fuc in a molar ratio of 1.5:1.9: 4.4:4.8:1.0. In the hemagglutination inhibition assay, CML exhibited the strongest binding specificity toward N-glycolylneuraminic acid (NeuGc) among the monosaccharides tested, whereas NeuAc did not inhibit the hemagglutination at all. GalNAc and Mealpha-GalNAc were also inhibitory at much higher concentrations than NeuGc. Among the glycoproteins, asialobovine submaxillary mucin (BSM) and porcine stomach mucin (PSM) showed strong inhibitory effects. In surface plasmon resonance analysis, asialo-BSM and PSM exhibited the strongest binding affinity. After co-injection of CML and NeuGc or GalNAc onto the asialo-BSM- or PSM-immobilized chip, the dissociation of CML from the immobilized PSM was accelerated by NeuGc and GalNAc, but the dissociation of CML from the immobilized asialo-BSM was only promoted by GalNAc. These results and the other surface plasmon resonance experiments allowed us to conclude that the binding of asialo-BSM to CML was because of an interaction between the lectin and the GalNAc residues of asialo-BSM, and both the NeuGc and GalNAc residues were responsible for the binding of PSM to CML. The results also suggested that CML had two different carbohydrate binding domains, one specific for NeuGc and the other for GalNAc.     

Anticoagulant activity of M-LAO,L-amino acid oxidase purified from Agkistrodon halys blomhoffii,through selective inhibition of factor IX

One of haemorrhagic toxins present in snake venoms is L-amino acid oxidase (LAO), which catalyzes the oxidative deamination of L-amino acids with the generation of hydrogen peroxide. Although it is widely accepted that LAO alters platelet function, the effects of LAO on human blood coagulation remain largely unknown. The present study demonstrated, for the first time, that M-LAO, LAO purified from the venom of Agkistrodon halys blomhoffii (Japanese mamushi), possesses an anticoagulant activity. Thrombelastography (TEG) showed that M-LAO significantly delayed the onset and the progress of the coagulation process. In addition, the enzyme prolonged the activated partial thromboplastin time (aPTT) dose-dependently, but had little effect on the prothrombin time (PT), suggesting that its principal activity was mediated in the intrinsic coagulation pathway. Furthermore, M-LAO reduced factor IX procoagulant activity in a dose-dependent manner and did not affect other coagulation factors. These results indicate that M-LAO has an anticoagulant activity that impairs the intrinsic clotting by inhibiting factor IX.     

Major patterns of higher teleostean phylogenies: a new perspective based on 100 complete mitochondrial DNA sequences

A recent preliminary study using complete mitochondrial DNA sequences from 48 species of teleosts has suggested that higher teleostean phylogenies should be reinvestigated on the basis of more intensive taxonomic sampling. As a second step towards the resolution of higher teleostean phylogenies, which have been described as the "(unresolved) bush at the top of the tree," we reanalyzed their relationships using mitogenomic data from 100 purposefully chosen species that fully represented all of the higher teleostean orders, except for the Batrachoidiformes. Unweighted and weighted maximum parsimony analyses were conducted with the data set that comprised concatenated nucleotide sequences from 12 protein-coding genes (excluding 3rd codon positions) and 21 transfer RNA (tRNA) genes (stem regions only) from each species. The resultant trees were well resolved and largely congruent, with most internal branches being supported by high statistical values. All major, comprehensive groups above ordinal level as currently defined in higher teleosts (with the exception of the Neoteleostei and several monotypic groups), such as the Eurypterygii, Ctenosquamata, Acanthomorpha, Paracanthopterygii, Acanthopterygii, and Percomorpha, appeared to be nonmonophyletic in the present tree. Such incongruities largely resulted from differences in the placement and/or limits of the orders Ateleopodiformes, Lampridiformes, Polymixiiformes, Ophidiiformes, Lophiiformes, Beryciformes, Stephanoberyciformes, and Zeiformes, long-standing problematic taxa in systematic ichthyology. Of these, the resulting phylogenetic positions of the Ophidiiformes and Lophiiformes were totally unexpected, because, although they have consistently been considered relatively primitive groups within higher teleosts (Paracanthopterygii), they were confidently placed within a crown group of teleosts, herein called the Percomorpha. It should be noted that many unexpected, but highly supported relationships were found within the Percomorpha, being highly promising for the next investigative step towards resolution of this remarkably diversified group of teleosts.     

Effects of diacylglycerol on glucose, lipid metabolism, and plasma serotonin levels in lean Japanese

Objective: Diacylglycerol (DAG)‐rich oil has been suggested to suppress postprandial hyperlipidemia and promote negative caloric balance by increasing energy expenditure (EE), due to small intestine physiochemical dynamics that differ from triacylglycerol (TAG). We studied the effect of DAG on postprandial glucose/insulin metabolism by loading of carbohydrate with oil. Further, to reveal the mechanism for increased EE by DAG, we measured plasma serotonin, which is mostly present in the small intestine and mediates peripheral sympathetic thermogenesis. Methods and Procedures: Randomized crossover study with 2‐week wash‐out interval between differing fat ingestion. Seven male, lean, Japanese students ingested DAG or TAG oil with 40 g of carbohydrate. Measurements of metabolic parameters were performed before and at 2, 4, and 6 h after fat ingestion. Plasma serotonin levels and cholesterol concentration in each lipoprotein were measured using high‐performance liquid chromatography (HPLC). Results: The substitution of DAG for TAG decreased very‐low‐density lipoprotein‐cholesterol (VLDL‐C) by 45.6% at 2 h, and decreased serum insulin by 41.3% at 4 h after ingestion. The incremental area under the curve (IAUC) for VLDL‐C was positively correlated with the IAUC for insulin. Concurrently, DAG elevated plasma serotonin levels by 47.3% at 2 h, while TAG did not influence. Discussion: This study indicates that the substitution of DAG for TAG suppresses the postprandial increase in serum VLDL‐C and insulin. This study also demonstrates that DAG ingestion increases plasma serotonin, proposing a possible mechanism for a postprandial increase in EE by DAG.     

Morphogenetic pathway of bacteriophage φ6: A flow analysis of subviral and viral particles in infected cells

Three types of virus-specific particles of double-stranded RNA bacteriophage φ6 were isolated and characterized by pulse-label and pulse-chase experiments on φ6-infected Pseudomonas phaseolicola. The first particle was “previrion I”, which consisted of early proteins P1, P2, P4 and P7, and had no RNA. It was detected immediately after labeling of proteins and the radioactivity was chased into the second structure, designated previrion II, after ten minutes. Previrion II contained three segments of double-stranded RNA in addition to the component of previrion I, and had RNA polymerase activity that produced messenger RNA species coding for late proteins. The RNA polymerase activity in the cell extract emerged nearly in parallel with the synthesis of late proteins, and this activity of previrion II was supposed to be responsible for late protein synthesis in infected cells. Via previrions I and II, the third radioactive particle was observed in infected cells after late protein synthesis started. This particle was identified as the intact virion, because it had infectivity as well as all of the viral components, including lipids. This intact virion was accumulated in the infected cell before bursting the cell.