Environmental determinants correlated to Vibrio harveyi-mediated death of marine gastropods

Vibrio harveyi is an emerging pathogen that causes mass mortality in a wide variety of marine animal species; however, it is still unclear which environmental determinants correlate V. harveyi dynamics and the bacterium-mediated death of marine animal life. We conducted a correlation analysis over a 5-year period (2003–2007) analysing the following data: V. harveyi abundance, marine animal mortality and environmental variables (seawater temperature, salinity, pH, chlorophyll a , rainfall and total viable bacterial counts). The samples were collected from a coastal area in northern Japan, where deaths of a marine gastropod species ( Haliotis discus hannai ) have been reported. Our analysis revealed significant positive correlations between average seawater temperature and average V. harveyi abundance ( R  = 0.955; P  < 0.05), and between average seawater temperature and V. harveyi -mediated abalone death ( R  = 0.931; P  < 0.05). Based on the regression model, n °C rise in seawater temperature gave rise to a 21 ⁿ -fold increase in the risk of mortality caused by V. harveyi infection. This is the first report providing evidence of the strong positive correlation between seawater temperature and V. harveyi -mediated death of marine species.     

Isolation and characterization of a cDNA encoding mitochondrial chaperonin 10 from Arabidopsis thaliana by functional complementation of an Escherichia coli groES mutant

Chaperonin (Cpn) is one of the molecular chaperones. Cpn10 is a co-factor of Cpn60, which regulates Cpn60-mediated protein folding. It is known that Cpn10 is located in mitochondria and chloroplasts in plant cells. The Escherichia coli homologue of Cpn10 is called GroES. A cDNA for the Cpn10 homologue was isolated from Arabidopsis thaliana by functional complementation of the E. coli groES mutant. The cDNA was 647 bp long and encoded a polypeptide of 98 amino acids. The deduced amino acid sequence showed approximately 50% identity to mammalian mitochondrial Cpn10s and 30% identity to GroES. A Northern blot analysis revealed that the mRNA for the Cpn10 homologue was expressed uniformly in various organs and was markedly induced by heat-shock treatment. The Cpn10 homologue was constitutively expressed in transgenic tobaccos. Immunogold and immunoblot analyses following the subcellular fractionation of leaves from transgenic tobaccos revealed that the Cpn10 homologue was localized in mitochondria and accumulated at a high level in transgenic tobaccos.     

Stereoselectivity and enantiomer-enantiomer interactions in the binding of ibuprofen to human serum albumin

Binding of ibuprofen (IB) enantiomers to human serum albumin (HSA) was studied using a chiral fluorescent derivatizing reagent, which enabled the measurement of IB enantiomers at a concentration as low as 5 × 10⁻⁸ M. Scatchard analyses revealed that there were two classes of binding sites for both enantiomers. For the high affinity site, the number of the binding sites was one for both enantiomers, and the binding constant of R-IB was 2.3-fold greater than that of S-IB. The difference in the affinity at the high affinity site may result in the stereoselective binding of IB enantiomers at therapeutic concentrations. It was confirmed that the high affinity site of IB enantiomers is Site II (diazepam binding site) by using site marker ligands. Also, significant enantiomer-enantiomer interactions were observed in the binding. The binding data were quantitatively analyzed and a binding model with an assumption of competitive interactions only at the high affinity site simulated the binding characteristics of IB enantiomers fairly well. Chirality 9:643–649, 1997. © 1997 Wiley-Liss, Inc.     

Structure of acidic phospholipase A2 for the venom of Agkistrodon halys blomhoffii at 2.8 A resolution.

The crystal structure of acidic phospholipase A2 from the venom of Agkistrodon halys blomhoffii has been determined by molecular replacement methods based on the known structure of Crotalus atrox PLA2, a same group II enzyme. The overall structures, except the calcium-binding regions, are very similar to each other. A calcium ion is pentagonally ligated to two carboxylate oxygen atoms of Asp-49 and each carbonyl oxygen atoms of Tyr-28, Gly-30 and Ala-31. A reason why the former enzyme functions as monomeric form, while the latter one does as dimer, could be presumed by the structural comparison of these calcium-binding regions. Although Gly-32 is usually participated as a ligand in the coordination with calcium ion in group I PLA2, it is characteristically replaced to Ala-31 in the present structure, and thus the coordination geometry of calcium ion is rather different from the usually observed one.     

Arabidopsis vacuolar sorting mutants (green fluorescent seed) can be identified efficiently by secretion of vacuole-targeted green fluorescent protein in their seeds

Two Arabidopsis thaliana genes have been shown to function in vacuolar sorting of seed storage proteins: a vacuolar sorting receptor, VSR1/ATELP1, and a retromer component, MAIGO1 (MAG1)/VPS29. Here, we show an efficient and simple method for isolating vacuolar sorting mutants of Arabidopsis. The method was based on two findings in this study. First, VSR1 functioned as a sorting receptor for beta-conglycinin by recognizing the vacuolar targeting signal. Second, when green fluorescent protein (GFP) fusion with the signal (GFP-CT24) was expressed in vsr1, mag1/vps29, and wild-type seeds, both vsr1and mag1/vps29 gave strongly fluorescent seeds but the wild type did not, suggesting that a defect in vacuolar sorting provided fluorescent seeds by the secretion of GFP-CT24 out of the cells. We mutagenized transformant seeds expressing GFP-CT24. From approximately 3,000,000 lines of M2 seeds, we obtained >100 fluorescent seeds and designated them green fluorescent seed (gfs) mutants. We report 10 gfs mutants, all of which caused missorting of storage proteins. We mapped gfs1 to VSR1, gfs2 to KAM2/GRV2, gfs10 to the At4g35870 gene encoding a novel membrane protein, and the others to different loci. This method should provide valuable insights into the complex molecular mechanisms underlying vacuolar sorting of storage proteins.     

Quantitative evaluation of each catalytic subsite of cathepsin B for inhibitory activity based on inhibitory activity-binding mode relationship of epoxysuccinyl inhibitors by X-ray crystal structure analyses of complexes

To quantitatively estimate the inhibitory effect of each substrate-binding subsite of cathepsin B (CB), a series of epoxysuccinyl derivatives with different functional groups bound to both carbon atoms of the epoxy ring were synthesized, and the relationship between their inhibitory activities and binding modes at CB subsites was evaluated by the X-ray crystal structure analyses of eight complexes. With the common reaction in which the epoxy ring of inhibitor was opened to form a covalent bond with the SgammaH group of the active center Cys29, the observed binding modes of the substituents of inhibitors at the binding subsites of CB enabled the quantitative assessment of the inhibitory effect of each subsite. Although the single blockage of S1' or S2' subsite exerts only the inhibitory effect of IC50 = approximately 24 microM (k2 = approximately 1250 M(-1) s(-1)) or approximately 15 microM (k2 = approximately 1800 M(-1) s(-1)), respectively, the synchronous block of both subsites leads to IC50 = approximately 23 nM (k2 = 153,000 - 185,000 M(-1) s(-1)), under the condition that (i) the inhibitor possesses a P1' hydrophobic residue such as Ile and a P2' hydrophobic residue such as Ala, Ile or Pro, and (ii) the C-terminal carboxyl group of a P2' residue is able to form paired hydrogen bonds with the imidazole NH of His110 and the imidazole N of His111 of CB. The inhibitor of a Pn' > or = 3' substituent was not potentiated by collision with the occluding loop. On the other hand, it was suggested that the inhibitory effects of Sn subsites are independent of those of Sn' subsites, and the simultaneous blockage of the funnel-like arrangement of S2 and S3 subsites leads to the inhibition of IC50 = approximately 40 nM (k2 = approximately 66,600 M(-1) s(-1)) regardless of the lack of Pn' substituents. Here we present a systematic X-ray structure-based evaluation of structure-inhibitory activity relationship of each binding subsite of CB, and the results provide the structural basis for designing a more potent CB-specific inhibitor.     

Genomic characterization of closely related species in the Rumoiensis clade infers ecogenomic signatures to non-marine environments

Members of the family Vibrionaceae are generally found in marine and brackish environments, playing important roles in nutrient cycling. The Rumoiensis clade is an unconventional group in the genus Vibrio, currently comprising six species from different origins including two species isolated from non-marine environments. In this study, we performed comparative genome analysis of all six species in the clade using their complete genome sequences. We found that two non-marine species, Vibrio casei and Vibrio gangliei, lacked the genes responsible for algal polysaccharide degradation, while a number of glycoside hydrolase genes were enriched in these two species. Expansion of insertion sequences was observed in V. casei and Vibrio rumoiensis, which suggests ongoing genomic changes associated with niche adaptations. The genes responsible for the metabolism of glucosylglycerate, a compound known to play a role as compatible solutes under nitrogen limitation, were conserved across the clade. These characteristics, along with genes encoding species-specific functions, may reflect the habit expansion which has led to the current distribution of Rumoiensis clade species. Genome analysis of all species in a single clade give us valuable insights into the genomic background of the Rumoiensis clade species and emphasize the genomic diversity and versatility of Vibrionaceae.     

Synthesis and absolute configuration of hormone alpha1

An important biological event in phytopathogens of the genus Phytophthora is sexual reproduction, which is conducted by two mating types, A1 and A2. A factor known as hormone alpha1 is secreted by the A1 mating type and induces the formation of sexual spores (oospores) in the A2 mating type. Here we describe the asymmetric synthesis and assignment of the absolute configuration of hormone alpha1 by oospore-inducing assays of the synthesized isomers.     

Modified multiplex PCR methods for comprehensive detection of Pectinatus and beer-spoilage cocci

Specific PCR primers were designed based on the 16S rRNA genes of recently proposed beer-spoilage species, Pectinatus haikarae, Megasphaera sueciensis, and M. paucivorans, and two sets of our previously reported multiplex PCR methods for Pectinatus spp. and beer-spoilage cocci were reconstructed. Each modified multiplex PCR method was found specifically to detect beer-spoilage species of Pectinatus and cocci, including new species.