Filamentous bacteriophages of vibrios are integrated into the dif-like site of the host chromosome

The dif site is located in the replication terminus region of bacterial chromosomes, having a function of resolving dimeric chromosomes formed during replication. We demonstrate that filamentous bacteriophages of vibrios, such as f237 (Vibrio parahaemolyticus) and CTXphi (V. cholerae), are integrated into the dif-like site of host chromosome.     

Rad51 suppresses gross chromosomal rearrangement at centromere in Schizosaccharomyces pombe

Centromere that plays a pivotal role in chromosome segregation is composed of repetitive elements in many eukaryotes. Although chromosomal regions containing repeats are the hotspots of rearrangements, little is known about the stability of centromere repeats. Here, by using a minichromosome that has a complete set of centromere sequences, we have developed a fission yeast system to detect gross chromosomal rearrangements (GCRs) that occur spontaneously. Southern and comprehensive genome hybridization analyses of rearranged chromosomes show two types of GCRs: translocation between homologous chromosomes and formation of isochromosomes in which a chromosome arm is replaced by a copy of the other. Remarkably, all the examined isochromosomes contain the breakpoint in centromere repeats, showing that isochromosomes are produced by centromere rearrangement. Mutations in the Rad3 checkpoint kinase increase both types of GCRs. In contrast, the deletion of Rad51 recombinase preferentially elevates isochromosome formation. Chromatin immunoprecipitation analysis shows that Rad51 localizes at centromere around S phase. These data suggest that Rad51 suppresses rearrangements of centromere repeats that result in isochromosome formation.     

Differences in Ca2+ mobilization induced by alpha-adrenergic agonist and phosphatidic acid in cultured hepatocytes

In an attempt to elucidate the relationship between phosphatidylinositol breakdown and alpha-adrenergic responses, effects of phosphatidic acid and phosphatidylinositol related metabolites on Ca²⁺ mobilization and glucose output in cultured hepatocytes were examined. Norepinephrine induced the net ⁴⁵Ca²⁺ efflux from preloaded cells and stimulated glucose output via alpha-adrenergic receptor stimulation, whereas phosphatidic acid caused ⁴⁵Ca²⁺ uptake to cells and did not stimulate glucose output. Myo-inositol-monophosphate, diglyceride and arachidonic acid, which are released by phosphatidylinositol breakdown, had no effect on ⁴⁵Ca²⁺ efflux and glucose output in cells. These results suggest that phosphatidic acid and phosphatidylinositol related metabolites can not mimic the alpha-adrenergic actions in cultured hepatocytes.     

Molecular Characterization of a Deep-Sea Methanotrophic Mussel Symbiont that Carries a RuBisCO Gene

In our previous investigation on the genes of 1,5-ribulose bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39) in deep-sea chemoautotrophic and methanotrophic endosymbioses, the gene encoding the large subunit of RuBisCO form I (cbbL) had been detected in the gill of a mussel belonging to the genus Bathymodiolus from a western Pacific back-arc hydrothermal vent. This study further examined the symbiont source of the RuBisCO cbbL gene along with the genes of 16S ribosomal RNA (16S rDNA) and particulate methane monooxygenase (EC 1.14.13.25; pmoA) and probed for the presence of the ATP sulfurylase gene (EC 2.7.7.4; sopT). The 16S rDNA sequence analysis indicated that the mussel harbors a monospecific methanotrophic Gammaproteobacterium. This was confirmed by amplification and sequencing of the methanotrophic pmoA, while thiotrophic sopT was not amplified from the same symbiotic genome DNA. Fluorescence in situ hybridization demonstrated simultaneous occurrence of the symbiont-specific 16S rDNA, cbbL and pmoA, but not sopT, in the mussel gill. This is the first molecular and visual evidence for a methanotrophic bacterial endosymbiont that bears the RuBisCO cbbL gene relevant to autotrophic CO₂ fixation.     

Structural characterization of a novel mono-sulfated gangliotriaosylceramide containing a 3-O-sulfatedN-acetylgalactosamine from rat kidney

A novel mono-sulfated glycosphingolipid based on the gangliotriaose core structure was isolated from rat kidney. The isolation procedure involved extraction of lipids with chloroform/methanol, mild alkaline methanolysis, column chromatographies with anion exchangers and silica beads. The structure was characterized by compositional analysis, FTIR spectroscopy, methylation analysis,¹H-NMR spectroscopy and negative-ion liquid secondary ion mass spectrometry (LSIMS) using the intact glycolipid and its desulfation product. The two dimensional chemical shift correlated spectroscopy provided information on the sugar sequence as well as anomeric configurations, and indicated the presence of a 3-O-sulfatedN-acetylgalactosamine within the molecule. Negative-ion LSIMS with high- and low-energy collision-induced dissociation defined the sugar sequence and ceramide composition, confirming the presence of a sulfatedN-acetylgalactosamine at the non-reducing terminus. From these results, the complete structure was proposed to be HSO₃-3GalNAc1-4Gal1-4Glc1-1Cer (Gg₃Cer III³-sulfate, SM2b). Abbreviations: Abbreviations for sulfated glycolipids [17] follow the modifications of the nomenclature system of Svennerholm for gangliosides [37], and the designation of the other glycosphingolipids follows the IUPAC-IUB recommendations [38]. Cer, ceramide; LacCer, lactosylceramide, Gal1-4Glc1-1Cer; Gg₃Cer, gangliotriaosylceramide, GalNAc1-4Gal1-4Glc1-1Cer; Gg₄Cer, gangliotetraosylceramide, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; iGb₄Cer, isoglobotetraosylceramide, GalNAc1-3Gal1-3Gal1-4Glc1-1Cer; Gb₄Cer, globotetraosylceramide, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; SM4s, galactosylceramide sulfate, GalCer I³-sulfate; SM3, lactosylceramide sulfate, LacCer II³-sulfate; SM2a, Gg₃Cer II³-sulfate; SM2b, Gg₃Cer III³-sulfate; SB2, Gg₃Cer II³,III³-bis-sulfate; SM1a, Gg₄Cer II³-sulfate; SM1b, Gg₄Cer IV³-sulfate; SB1a, Gg₄Cer II³,IV³-bissulfate; GLC, gas-liquid chromatography; GC-MS, gas chromatography-mass spectrometry; DQF, double quantum filtered; COSY, chemical-shift-correlated spectroscopy; LSIMS, liquid secondary ion mass spectrometry; CID, collision-induced dissociation; MS/MS, tandem mass spectrometry.     

Generation of a purely clonal defective interfering influenza virus

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.     

Age-independent constancy of mineral contents in human ribs

On age relationships of mineral contents in human bones, the contents of the sixth rib and a piece of its compact bone were determined by inductively coupled plasma atomic emission spectrometry (ICPS). The ribs were resected from 21 subjects (14 men and 7 women) who died in age ranging from 65 to 93 yr. There were no age-dependent decreases in Ca and P contents of the ribs in the age range on ICPS. It was found that there were no age-dependent decreases in Ca and P in compact bones of ribs.     

Synthesis and conformational characterization of the epidermal growth factor-like domain of blood coagulation factor IX carrying xylosyl-glucose

Solid-phase synthesis of glycopeptide generally requires the protection of both peptide side chains and hydroxyl groups of the carbohydrate portion. However, if the mild coupling conditions are used, the protection of the carbohydrate portion can be omitted. In this paper, we demonstrated it by the synthesis of Fmoc-serine carrying unmasked xylosyl glucose followed by the solid-phase synthesis of epidermal growth factor (EGF)-like domain of factor IX (45-87) using the unit. The product was well characterized by enzymatic digestion, amino acid analysis and mass spectrometry. The secondary structure of the product as well as glucosylated and non-glycosylated EGF-like domain was characterized by circular dichroism (CD) spectroscopy.     

Methylation assay by nucleotide incorporation: a quantitative assay for regional CpG methylation density

Although the aberrant methylation in CpG islands is of great interest as a causative role in human malignancies, it has been very difficult to accurately determine methylation density. Here we report a novel microplate-based quantitative methylation assay, designated MANIC, for a region containing a number of CpG sites based on incorporation of hapten-labeled dCTP at cytosine sites where the methylated cytosines have not been converted to uracil by the bisulfite treatment. Validation using control DNAs revealed that the method was sensitive enough to detect < 1.25% methylated DNA and that calibration curve was linear. With this approach, we determined relative methylation density of O6-methylguanine-DNA methyltransferase gene promoter containing 12 CpG sites among the 12 colorectal cancers and corresponding normal mucosal tissues. Consequently, MANIC showed a high concordance with results by a quantitative method, bisulfite PCR single-stranded conformational polymorphism (BiPS). MANIC is a technique that avoids cumbersome procedures such as electrophoresis or the use of radiolabeling and is applicable to any sequence regardless of the total number of CpG sites or heterogeneity in methylation status.