Mechanistic studies of O2-resistant N2-fixation in N2-fixing Escherichia coli

Escherichia coli carrying the entire nif gene cluster from Klebsiella pneumoniae on a multicopy plasmid becomes more O2-resistant in a N-free medium as a result of the integration of the nif gene cluster into the chromosome and the loss of the plasmid (H.Iwahashi and J.Someya, Biochem. Biophys. Res. Comm. 1990, 168: 288–294). Our purpose is to characterize the physiological reason why the strain became O2-resistant by measuring the levels of nif proteins in cells under microaerobic conditions. The O2-resistant strain had a higher amount of NifH and a lower amount of NifL under microaerobic conditions (compared to that under anaerobic conditions), while the parent strain showed the opposite characteristics. Thus, the biochemical mechanism of the O2-resistant strain is attributed to the strain's ability to synthesize and maintain a high amount of NifH and a low amount of NifL under microaerobic conditions. © Rapid Science Ltd. 1998     

Genetic structure of island populations of <Emphasis Type="Italic">Prunus lannesiana</Emphasis> var. <Emphasis Type="Italic">speciosa</Emphasis> revealed by chloroplast DNA,AFLP and nuclear SSR loci analyses

The wild flowering cherry Prunus lannesiana var. speciosa is highly geographically restricted, being confined to the Izu Islands and neighboring peninsulas in Japan. In an attempt to elucidate how populations of this species have established we investigated the genetic diversity and differentiation in seven populations (sampling 408 individuals in total), using three kinds of genetic markers: chloroplast DNA (cpDNA), amplified fragment length polymorphisms (AFLPs), and 11 nuclear SSR polymorphic loci. Eight haplotypes were identified based on the cpDNA sequence variations, 64 polymorphic fragments were scored for the AFLP markers, and a total of 154 alleles were detected at the 11 nuclear SSR loci. Analysis of molecular variance showed that among-population variation accounted for 16.55, 15.04 and 7.45% of the total detected variation at the cpDNA, AFLPs, and SSR loci, respectively. Thus, variation within populations accounted for most of the genetic variance for all types of markers, although the genetic differentiation among populations was also highly significant. For cpDNA variation, no clear structure was found among the populations, except that of the most distant island, although an “isolation by distance” pattern was found for each marker. Both neighbor-joining trees and structure analysis indicate that the genetic relationships between populations reflect geological variations between the peninsula and the islands and among the islands. Furthermore, hybridization with related species may have affected the genetic structure, and some genetic introgression is likely to have occurred.     

Crystal structure of the superfamily 1 helicase from Tomato mosaic virus

The genomes of the Tomato mosaic virus and many other plant and animal positive-strand RNA viruses of agronomic and medical importance encode superfamily 1 helicases. Although helicases play important roles in viral replication, the crystal structures of viral superfamily 1 helicases have not been determined. Here, we report the crystal structure of a fragment (S666 to Q1116) of the replication protein from Tomato mosaic virus. The structure reveals a novel N-terminal domain tightly associated with a helicase core. The helicase core contains two RecA-like α/β domains without any of the accessory domain insertions that are found in other superfamily 1 helicases. The N-terminal domain contains a flexible loop, a long α-helix, and an antiparallel six-stranded β-sheet. On the basis of the structure, we constructed deletion mutants of the S666-to-Q1116 fragment and performed split-ubiquitin-based interaction assays in Saccharomyces cerevisiae with TOM1 and ARL8, host proteins that are essential for tomato mosaic virus RNA replication. The results suggested that both TOM1 and ARL8 interact with the long α-helix in the N-terminal domain and that TOM1 also interacts with the helicase core. Prediction of secondary structures in other viral superfamily 1 helicases and comparison of those structures with the S666-to-Q1116 structure suggested that these helicases have a similar fold. Our results provide a structural basis of viral superfamily 1 helicases.     

Autophagy Induced by Calcium Phosphate Precipitates Involves Endoplasmic Reticulum Membranes in Autophagosome Biogenesis

Calcium can play an important role in the regulation of autophagy. We previously reported that exogenously introduced calcium in the form of calcium phosphate precipitates (CPP) induces autophagy. Here we showed that CPP-induced autophagy required the classical autophagic machinery, including the autophagosome initiating molecules FIP200 and Beclin 1, as well as molecules involved in the autophagosome membrane extension, Atg4, Atg5 and Atg3. On the other hand, Atg9 seemed to place a restriction on CPP-induced autophagy. Loss of Atg9 led to enhanced LC3 punctation and enhanced p62 degradation. CPP-induced autophagy was independent of mTOR and reactive oxygen species. It also did not affect MAP kinase activation and ER stress. DFCP1 is an ER-resident molecule that binds to phosphatidylinositol 3-phosphate. CPP activated DFCP1 punctation in a class III phosphatidylinositol-3-kinase and calcium dependent manner, and caused the association of DFCP1 puncta with the autophagosomes. Consistently, ER membranes, but not Golgi or mitochondrial membranes, colocalized with CPP-induced LC3 positive autophagosomes. These data suggest that CPP-induced autophagosome formation involves the interaction with the ER membrane.     

Cloning of FLOWERING LOCUS C ortholog in Wasabia japonica (Matsum.)

Wasabi (Wasabia japonica) is a commercially important crop in Japan. We isolated a FLC ortholog from wasabi and named as WjFLC. Predicted amino acid sequence encoded by WjFLC showed 89% identity with FLC of arabidopsis and conserved the MADS box motif. WjFLC was expressed in young and mature leaves, apical region of lateral bud, rhizome, and root. The expression of WjFLC was high in October and reduced in November when flower buds are formed in wasabi. WjFLC may be useful in monitoring the flowering response in wasabi.     

Estimation of DNA Base Composition by High Performance Liquid Chromatography of Its Nuclease PI Hydrolysate

The Saccharomyces diastaticus glucoamylase encoded by ST A1 contains two signal sequences for potent secretion of the enzyme, a hydrophobic leader peptide (HL), and a tract consisting of threonine- and serine-rich sequences (TS); hybrid proteins of Escherichia coli β-galactosidase carrying both HL and TS are secreted through the cytoplasmic membrane to the cell-surface fraction of yeast cells, but those carrying either HL or TS are not. To investigate the molecular mechanisms for these signal sequences, we have isolated a dominant mutation, SSD1, which suppresses a secretory defect caused by deletion of these sequences. Yeast cells harboring the mutation secreted hybrid β-galactosidase proteins carrying either HL or TS into the cell-surface fraction. Even β-ga!actosidase itself was secreted to the cell surface in the mutant. These results suggest that HL and TS interact with a wild- type ssd1⁺ gene product to promote protein secretion.     

Ezetimibe enhances macrophage reverse cholesterol transport in hamsters: Contribution of hepato–biliary pathway

Reverse cholesterol transport (RCT) is pivotal in the return of excess cholesterol from peripheral tissues to the liver for excretion in bile and eventually feces. RCT from macrophages is a critical anti-atherogenicity mechanism of HDL. As the cholesterol absorption inhibitor ezetimibe promoted RCT in mice, which lack cholesterol ester transfer protein (CETP), we investigated its effects in hamsters, which have CETP.     

Preparation and Derivatives of Optically Inactive Isopulegols

The unknown three isomers of optically inactive isopulegol, (±)-isopulegol(II), (±)-neo-isopulegol(III), and (±)-iso-isopulegol(IV) were isolated; then 3,5-dinitrobenzoates and p-nitrobenzoates were prepared from them.