Genome sequence of Oceanobacillus iheyensis isolated from the Iheya Ridge and its unexpected adaptive capabilities to extreme environments

Oceanobacillus iheyensis HTE831 is an alkaliphilic and extremely halotolerant Bacillus-related species isolated from deep-sea sediment. We present here the complete genome sequence of HTE831 along with analyses of genes required for adaptation to highly alkaline and saline environments. The genome consists of 3.6 Mb, encoding many proteins potentially associated with roles in regulation of intracellular osmotic pressure and pH homeostasis. The candidate genes involved in alkaliphily were determined based on comparative analysis with three Bacillus species and two other Gram-positive species. Comparison with the genomes of other major Gram-positive bacterial species suggests that the backbone of the genus Bacillus is composed of approximately 350 genes. This second genome sequence of an alkaliphilic Bacillus-related species will be useful in understanding life in highly alkaline environments and microbial diversity within the ubiquitous bacilli.     

Characterization of an anti-decorin monoclonal antibody,and its utility

6B6 is a monoclonal antibody raised against a purified small dermatan sulfate proteoglycan from human ovarian fibroma capsule, has Although it been widely used as an anti-decorin monoclonal antibody, its epitope has not yet been characterized at the molecular level. Here, we show that 6B6 is specific to decorin. The antibody recognized human, mouse, and bovine decorin core protein, but not biglycan. Using recombinant decorin domains, we determined that the epitope lies within the region of amino acid residues 50-65, termed the cysteine cluster region. Cross-reactivity among species further narrowed it down to a primary sequence of residues 57-65. We also established the conditions for immunostaining. 6B6 stained both frozen and fixed sections. Whereas the glycosaminoglycan chain of decorin inhibited access of the antibody in immunoblotting, pretreatment of tissue sections with chondrotinase ABC did not affect the intensity of staining, suggesting that the glycosaminoglycan chain is integrated and the Cys cluster region oriented outside of the collagen fibrils in the tissue. When 6B6 was applied to enzyme-linked immunosorbent assay, a concentration as low as 0.5 microg/ml of decorin was detectable by either direct or sandwich ELISA. 6B6 is thus a sensitive and reliable antibody to study functions of decorin from various aspects.     

Dominant-negative connexin43-EGFP inhibits calcium-transient synchronization of primary neonatal rat cardiomyocytes.

Recent studies using mice with genetically engineered gap junction protein connexin (Cx) genes have provided evidence that reduced gap-junctional coupling in ventricular cardiomyocytes predisposes to ventricular arrhythmia. However, the pathological processes of arrhythmogenesis due to abnormalities in gap junctions are poorly understood. We have postulated a hypothesis that dysfunction of gap junctions at the single-cell level may affect synchronization of calcium transients among cardiomyocytes. To examine this hypothesis, we developed a novel system in which gap-junctional intercellular communication in primary neonatal rat cardiomyocytes was inhibited by a mutated (Delta130-137) Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP), and calcium transients were imaged in real time while the mutated Cx43-EGFP-expressing cardiomyocytes were identified. The mutated Cx43-EGFP inhibited dye coupling not only in the liver epithelial cell line IAR 20 but also in primary neonatal rat cardiomyocytes in a dominant-negative manner, whereas wild-type Cx43-EGFP made functional gap junctions in otherwise communication-deficient HeLa cells. The mutated Cx43-EGFP induced desynchronization of calcium transients among cardiomyocytes with significantly higher frequency than wild-type Cx43-EGFP. These results suggest that dysfunction of gap-junctional intercellular communication at the single-cell level could hamper synchronous beating among cardiomyocytes as a result of desynchronization of calcium transients.     

Homologous Desensitization of Serotonin 5-HT2 Receptor-Stimulated Intracellular Calcium Mobilization in C6BU-1 Glioma Cells via a Mechanism Involving a Calmodulin Pathway

Abstract: Serotonin 5-HT₂ receptor-mediated intracellular Ca²⁺ mobilization was investigated in rat glioma C6BU-1 cells. The receptors became desensitized after previous exposure to 5-HT in a time-and concentration-dependent manner. The desensitization of 5-HT₂ receptor-mediated intracellular signaling appeared to be homologous because previous exposure to 5-HT did not alter the response to other transmitters such as thrombin or isoproterenol and because previous exposure to thrombin or isoproterenol did not diminish the response to 5-HT. The desensitization induced by pretreatment with 5-HT was potently prevented by the naphthalenesulfonamide derivative W-7, a calmodulin antagonist, when it was cosupplied with 5-HT. Furthermore, the preventive effect of W-7 was greater than that of W-5, a weak analogue of W-7, and than that of H-7, a nonselective inhibitor of protein kinases. These results suggest that 5-HT₂ receptor-mediated Ca²⁺ mobilization can be desensitized homologously after prolonged exposure to 5-HT in a calmodulin-dependent manner in rat glioma C6BU-1 cells.     

Ultrastructure and morphometry of testicular Leydig cells and the interstitial components correlated with testosterone in aging rats

The ultrastructure of testicular interstitium in young and aged adult rats was analysed using morphometric methods, and the plasma testosterone concentration was measured. With increasing age there was an augumentation in the volume of collagen fibrils in the intercellular matrix and in blood vessels. During the aging process (approximately two years) the average volume of the Leydig cell decreased from 1364 m³ to 637 m³, but the number of Leydig cells in paired testes increased from 53x10⁶ to 113x10⁶. The absolute volume of smooth surfaced endoplasmic reticulum (SER) per Leydig cell amounted in aged rats to 78% of that in young adult rats. The total amount of SER in paired testes increased by 62% with aging. The present analysis suggests that the ability of SER to maintain peripheral testosterone concentration decreases with age. In young adult rats the absolute volume of peroxisomes per Leydig cell correlated significantly with the concentration of testosterone in blood and also with the absolute volume of SER per Leydig cell. These results combined with ultrastructural observations of close apposition of peroxisomes and SER suggest that peroxisomes have a role in testosterone secretion by Leydig cells.Visiting scientist to Laboratory of Electron Microscopy (Director: Prof. L.J. Pelliniemi)     

Thermoadaptation trait revealed by the genome sequence of thermophilic Geobacillus kaustophilus

We present herein the first complete genome sequence of a thermophilic Bacillus-related species, Geobacillus kaustophilus HTA426, which is composed of a 3.54 Mb chromosome and a 47.9 kb plasmid, along with a comparative analysis with five other mesophilic bacillar genomes. Upon orthologous grouping of the six bacillar sequenced genomes, it was found that 1257 common orthologous groups composed of 1308 genes (37%) are shared by all the bacilli, whereas 839 genes (24%) in the G.kaustophilus genome were found to be unique to that species. We were able to find the first prokaryotic sperm protamine P1 homolog, polyamine synthase, polyamine ABC transporter and RNA methylase in the 839 unique genes; these may contribute to thermophily by stabilizing the nucleic acids. Contrasting results were obtained from the principal component analysis (PCA) of the amino acid composition and synonymous codon usage for highlighting the thermophilic signature of the G.kaustophilus genome. Only in the PCA of the amino acid composition were the Bacillus-related species located near, but were distinguishable from, the borderline distinguishing thermophiles from mesophiles on the second principal axis. Further analysis revealed some asymmetric amino acid substitutions between the thermophiles and the mesophiles, which are possibly associated with the thermoadaptation of the organism.     

Synthesis and inhibition mechanism of Delta lac-acetogenins, a novel type of inhibitor of bovine heart mitochondrial complex I

We have synthesized Deltalac-acetogenins that are new acetogenin mimics possessing two n-alkyl tails without an alpha,beta-unsaturated gamma-lactone ring and suggested that their inhibition mechanism may be different from that of common acetogenins [Hamada et al. (2004) Biochemistry 43, 3651-3658]. To elucidate the inhibition mechanism of Deltalac-acetogenins in more detail, we carried out wide structural modifications of original Deltalac-acetogenins and characterized the inhibitory action with bovine heart mitochondrial complex I. In contrast to common acetogenins, both the presence of adjacent bis-THF rings and the stereochemistry around the hydroxylated bis-THF rings are important structural factors required for potent inhibition. The inhibitory potency of a derivative possessing an n-butylphenyl ether structure (compound 7) appeared to be superior to that of the original Deltalac-acetogenins and equivalent to that of bullatacin, one of the most potent natural acetogenins. Double-inhibitor titration of steady-state complex I activity showed that the extent of inhibition of compound 7 and bullatacin is not additive, suggesting that the binding sites of the two inhibitors are not identical. Competition tests using a fluorescent ligand indicated that the binding site of compound 7 does not overlap with that of other complex I inhibitors. The effects of compound 7 on superoxide production from complex I are also different from those of other complex I inhibitors. Our results clearly demonstrate that Deltalac-acetogenins are a novel type of inhibitor acting at the terminal electron-transfer step of bovine complex I.     

Cyclic AMP potentiates vascular endothelial cadherin-mediated cell-cell contact to enhance endothelial barrier function through an Epac-Rap1 signaling pathway

Cyclic AMP (cAMP) is a well-known intracellular signaling molecule improving barrier function in vascular endothelial cells. Here, we delineate a novel cAMP-triggered signal that regulates the barrier function. We found that cAMP-elevating reagents, prostacyclin and forskolin, decreased cell permeability and enhanced vascular endothelial (VE) cadherin-dependent cell adhesion. Although the decreased permeability and the increased VE-cadherin-mediated adhesion by prostacyclin and forskolin were insensitive to a specific inhibitor for cAMP-dependent protein kinase, these effects were mimicked by 8-(4-chlorophenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate, a specific activator for Epac, which is a novel cAMP-dependent guanine nucleotide exchange factor for Rap1. Thus, we investigated the effect of Rap1 on permeability and the VE-cadherin-mediated cell adhesion by expressing either constitutive active Rap1 or Rap1GAPII. Activation of Rap1 resulted in a decrease in permeability and enhancement of VE-cadherin-dependent cell adhesion, whereas inactivation of Rap1 had the counter effect. Furthermore, prostacyclin and forskolin induced cortical actin rearrangement in a Rap1-dependent manner. In conclusion, cAMP-Epac-Rap1 signaling promotes decreased cell permeability by enhancing VE-cadherin-mediated adhesion lined by the rearranged cortical actin.     

Mapping QTLs for grain dormancy on wheat chromosome 3A and the group 4 chromosomes,and their combined effect

A major QTL for grain dormancy, QPhs.ocs-3A.1, derived from the highly dormant wheat Zenkoujikomugi (Zen), has been identified in a study made under a controlled environment. Further investigations were needed to dissect the precise position and expression of QPhs.ocs-3A.1 under different field conditions because the ability to detect genetic loci for grain dormancy traits is compromised by environmental effects and genotype/environment interactions. Group 4 chromosomes have also been shown to be possible sites of QTLs for grain dormancy. The objectives of this study were (1) to locate additional molecular markers in the QPhs.ocs-3A.1 region, (2) to identify QTLs on the group 4 chromosomes and (3) to elucidate their combined effects. We examined the recombinant inbred lines (RILs) from a cross between Chinese Spring (CS) and Zen over a 3-year period in one location and 1 year in a different location. In an interval mapping study QPhs.ocs-3A.1 was mapped to within the 4.6 cM region flanked by Xbarc310 and Xbcd907 at the proximal end of the short arm of chromosome 3A. QPhs.ocs-3A.1 was confirmed to be the predominant dormancy QTL since it explained a large portion (11.6–44.8%) of the phenotypic variation, and was strongly displayed under dormancy-breaking conditions or at low germination temperatures. For QPhs.ocs-4A.1, identified on the long arm of chromosome 4A, and QPhs.ocs-4B.1, on the centromeric region of the long arm of Chr 4B, the LOD peak positions and the desirable allele were consistent between the trials, while the LOD scores and contribution to the phenotypic variation varied. Transgressive segregants were observed among the 125 RILs and most of them had a combination of the three alleles conferring a higher dormancy: the Zen alleles at QPhs.ocs-3A.1 and QPhs.ocs-4A.1 and the CS allele at QPhs.ocs-4B1. This demonstrated a combined effect of the desirable alleles on accelerating grain dormancy, with their total effect being superior to that of Zen.     

In vitro hydrolysis of poly(l-lactide) crystalline residues as extended-chain crystallites: II. Effects of hydrolysis temperature

The effects of hydrolysis temperature on the hydrolysis behavior and mechanism of poly(l-lactide) crystalline residues or extended-chain crystallites were investigated in phosphate-buffered solution (50-97 degrees C), using gel permeation chromatography and differential scanning calorimetry (DSC). The hydrolysis of the crystalline residues proceeded from their surface composed of very short chains with a free end along the chain direction, irrespective of hydrolysis temperature, but the hydrolysis from their lateral surface could not be traced. The activation energy of hydrolysis for the crystalline residues (extended-chain crystallites) was evaluated to be 18.0 kcal mol(-1) (75.2 kJ mol(-1)). The monotonic melting temperature (T(m)) and crystallinity decreases occurred after their initial very small increases, excluding the monotonic crystallinity decrease at 97 degrees C with no initial increase. The T(m) decrease reflects the decreased thickness of the crystalline residues. The equilibrium T(m) of the crystalline residues (extended-chain crystallites) was estimated to be 464.5-464.9 K. The free energy values for the surface composed of very short chains with a free end, which are neighboring the air (or nitrogen during DSC scanning), were calculated to be 55.6-56.4 erg cm(-2) for heat of fusion per unit mass = 135 J g(-1). The obtained surface free energy values are significantly higher than that for the surface composed of folding chains, tie chains, and the chains with a free end, which are neighboring the same kind of amorphous chains (39.9 erg cm(-2)).