Molecular cloning of cDNA encoding a cyclin-selective ubiquitin carrier protein (E2-C) from Carassius auratus (goldfish) and expression analysis of the cloned gene

Destruction of cyclin B is required for exit from mitosis and meiosis. A cyclin-specific ubiquitinating system, including cyclin-selective ubiquitin carrier protein (E2-C), is thought to be responsible for cyclin B destruction. Here we present the cloning, sequencing and expression analysis of goldfish, Carassius auratus, E2-C which encodes the cyclin-selective ubiquitin carrier protein from goldfish ovary. The cloned cDNA is 677 bp long and encodes 172 amino acids. The deduced amino acid sequence is highly homologous to E2-C from other species. Recombinant goldfish E2-C possesses ubiquitinating activity against cyclin B. The expression of mRNA for E2-C was similar to that of mRNA for cyclin B, occurring at very high level in the ovary. The similarity of the expression pattern of E2-C and cyclin B suggests that E2-C mediates a cyclin-specific ubiquitination.     

Inhibitory effect of a SALMFamide neuropeptide on secretion of gonad-stimulating substance from radial nerves in the starfish Asterina pectinifera

In starfish, the peptide hormone gonad-stimulating substance (GSS) secreted from nervous tissue stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) production by ovarian follicle cells. The SALMFamide family is also known to an echinoderm neuropeptide. The present study examined effect of SALMFamide 1 (S1) on oocyte maturation of starfish Asterina pectinifera. Unlike GSS, S1 did not induce spawning in starfish ovary. In contrast, S1 was found to inhibit GSS secretion from radial nerves by treatment with high K+ concentration. Fifty percent inhibition was obtained by 0.1 mM S1. S1 did not have any effect on GSS- and 1-MeAde-induced oocyte maturation. Following incubation with a S1 antibody and subsequently with rhodamine-conjugated second antibody, neural networks were observed in ovaries. The networks were restricted mainly to their surface with little evidence of immunoreactivity inside the basement membranes. This indicates that neural networks are distributed in the ovarian wall. The result further suggests that S1 plays a role in oocyte maturation to regulate GSS secretion from the nervous system.     

Chloroplast phylogeny indicates that bryophytes are monophyletic

Opinions on the basal relationship of land plants vary considerably and no phylogenetic tree with significant statistical support has been obtained. Here, we report phylogenetic analyses using 51 genes from the entire chloroplast genome sequences of 20 representative green plant species. The analyses, using translated amino acid sequences, indicated that extant bryophytes (mosses, liverworts, and hornworts) form a monophyletic group with high statistical confidence and that extant bryophytes are likely sisters to extant vascular plants, although the support for monophyletic vascular plants was not strong. Analyses at the nucleotide level could not resolve the basal relationship with statistical confidence. Bryophyte monophyly inferred using amino acid sequences has a good statistical foundation and is not rejected statistically by other data sets. We propose bryophyte monophyly as the currently best hypothesis.     

4-Coumarate:coenzyme A ligase in black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) catalyses the conversion of sinapate to sinapoyl-CoA

4-Coumarate:coenzyme A (CoA) ligase (4CL, EC 6.2.1.12) in crude enzyme preparation from the developing xylem of black locust (Robinia pseudoacacia) converted sinapate to sinapoyl CoA. The sinapate-converting activity was not inhibited by other cinnamate derivatives, such as p-coumarate, caffeate or ferulate, in the mixed-substrate assay. The crude extract prepared from the developing xylem was separated by anion-exchange chromatography into three different 4CL isoforms. The isoform 4CL1 had a strong substrate preference for p-coumarate, but lacked the activity for ferulate and sinapate. On the other hand, 4CL2 and 4CL3 displayed activity toward sinapate and also possessed high activity toward caffeate as well as p-coumarate. The crude extract from the shoots exhibited a very similar substrate preference to that of the developing xylem; therefore, 4CL2 may be a major isoform in both crude enzyme preparations. These results support the hypothesis that sinapate-converting 4CL isoform is constitutively expressed in lignin-forming cells.     

Maillard reaction rate in various glassy matrices

The Maillard Reaction (MR) rate below the glass transition temperature (T(g)) for various model glassy food systems was studied at temperatures between 40 degrees C and 70 degrees C. As a sample, freeze-dried glucose and lysine systems embedded in various glassy matrices (e.g., polyvinylpyrrolodone and trehalose) were used, and the MR rate below the T(g) was compared among the various glassy matrices. The extent of MR was estimated spectrophotometrically from the optical density at 280 nm (OD(280)), and the MR rate (k(280)) was determined as a pseudo zero order reaction rate from the time course of OD(280). Although k(280) was described by the Arrhenius plot, the temperature dependence of k(280) was almost the same and the intercept was different among the matrices. From the comparison of k(280), it was suggested that the MR rate in glassy matrix was affected not only by the T(g), but also by the hydrogen bonding between MR reactants and glassy matrix.     

The enhancing action of D-galactosamine on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS.     

High-frequency gene replacement in cyanobacteria using a heterologous rps12 gene

Multiple targeted gene replacements are often required for functional analyses of cyanobacterial genomes. For this purpose, we previously devised a simple genetic method, termed rps12-mediated gene replacement, in a cyanobacterium Synechococcus elongatus PCC 7942 for construction of mutants free from drug resistance markers. Here, we improved the method by employing a heterologous rps12 gene encoding a ribosomal protein S12 from Synechocystis sp. PCC 6803. Dominant streptomycin-sensitive phenotype of the Synechocystis rps12 gene was manifested only when it was expressed under the strong promoter of psbAI gene in S. elongatus PCC 7942 bearing a streptomycin-resistant rps12 allele. Transformation of the rps12 heteroallelic strains with non-replicating template plasmids permitted the selection of recombinants with gene replacement at frequencies up to 50% among streptomycin-resistant progeny.