Thrombomodulin is a clock-controlled gene in vascular endothelial cells

Cardiovascular diseases are closely related to circadian rhythm, which is under the control of an internal biological clock mechanism. Although a biological clock exists not only in the hypothalamus but also in each peripheral tissue, the biological relevance of the peripheral clock remains to be elucidated. In this study we searched for clock-controlled genes in vascular endothelial cells using microarray technology. The expression of a total of 229 genes was up-regulated by CLOCK/BMAL2. Among the genes that we identified, we examined the thrombomodulin (TM) gene further, because TM is an integral membrane glycoprotein that is expressed primarily in vascular endothelial cells and plays a major role in the regulation of intravascular coagulation. TM mRNA and protein expression showed a clear circadian oscillation in the mouse lung and heart. Reporter analyses, gel shift assays, and chromatin immunoprecipitation analyses using the TM promoter revealed that a heterodimer of CLOCK and BMAL2 binds directly to the E-box of the TM promoter, resulting in TM promoter transactivation. Indeed, the oscillation of TM gene expression was abolished in clock mutant mice, suggesting that TM expression is regulated by the clock gene in vivo. Finally, the phase of circadian oscillation of TM mRNA expression was altered by temporal feeding restriction, suggesting TM gene expression is regulated by the peripheral clock system. In conclusion, these data suggest that the peripheral clock in vascular endothelial cells regulates TM gene expression and that the oscillation of TM expression may contribute to the circadian variation of cardiovascular events.     

Population dynamics of the harmful diatom Eucampia zodiacus Ehrenberg causing bleachings of Porphyra thalli in aquaculture in Harima-Nada, the Seto Inland Sea, Japan

The diatom Eucampia zodiacus Ehrenberg is one of the harmful diatom species which indirectly cause bleachings of Nori (Porphyra thalli) in aquaculture through competitive utilizing of nutrients (especially nitrogen) and resultant nutrient depletion in water columns during the bloom events. The seasonal changes in environmental factors, cell density and cell size of E. zodiacus were investigated for 4 years (April 2002–December 2005) to understand the population ecology of this diatom in Harima-Nada, the eastern part of the Seto Inland Sea, Japan. Vegetative cells of E. zodiacus were usually detected year-round. Total cell densities of E. zodiacus annually peaked from mid-February to early April, and high cell densities were observed in the whole water columns during the bloom-period. Nutrient concentrations decreased with the increase of cell density of E. zodiacus, and low nutrients concentrations continued throughout the E. zodiacus bloom-period. The average cell size (length of apical axis) of E. zodiacus populations ranged from 10.8 μm to 81.2 μm, and the restoration of cell size occurred once in autumn every year just after reaching the minimum cell size. In addition, its great seasonal regularity was confirmed by the decrease and restoration of its cell size through 4-year study period. Temperature and nutrients were suitable in autumn for the growth of E. zodiacus, its blooms never occur in that season. These results strongly suggest that E. zodiacus did not have a resting stage, and it spends autumn for size restoration and starts to bloom thereafter in Harima-Nada in winter and spring, causing fishery damage to Nori aquaculture by resulting nutrient deprivation.     

Gliadain, a gibberellin-inducible cysteine proteinase occurring in germinating seeds of wheat, Triticum aestivum L., specifically digests gliadin and is regulated by intrinsic cystatins

We cloned a new cysteine proteinase of wheat seed origin, which hydrolyzed the storage protein gliadin almost specifically, and was named gliadain. Gliadain mRNA was expressed 1 day after the start of seed imbibition, and showed a gradual increase thereafter. Gliadain expression was suppressed when uniconazol, a gibberellin synthesis inhibitor, was added to germinating seeds. Histochemical detection with anti-gliadain serum indicated that gliadain was present in the aleurone layer and also that its expression intensity increased in sites nearer the embryo. The enzymological characteristics of gliadain were investigated using recombinant glutathione S-transferase (GST)-progliadain fusion protein produced in Escherichia coli. The GST-progliadain almost specifically digested gliadin into low molecular mass peptides. These results indicate that gliadain is produced via gibberellin-mediated gene activation in aleurone cells and secreted into the endosperm to digest its storage proteins. Enzymologically, the GST-progliadain hydrolyzed benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin (Z-Phe-Arg-NH(2)-Mec) at K(m) = 9.5 microm, which is equivalent to the K(m) value for hydrolysis of this substrate by cathepsin L. Hydrolysis was inhibited by two wheat cystatins, WC1 and WC4, with IC(50) values of 1.7 x 10(-8) and 5.0 x 10(-8) m, respectively. These values are comparable with those found for GST-progliadain inhibition by E-64 and egg-white cystatin, and are consistent with the possibility that, in germinating wheat seeds, gliadain is under the control of intrinsic cystatins.     

Effects of instantaneous and growth CO2 levels and abscisic acid on stomatal and mesophyll conductances

C₃ photosynthesis is often limited by CO₂ diffusivity or stomatal (g_s) and mesophyll (g_m) conductances. To characterize effects of stomatal closure induced by either high CO₂ or abscisic acid (ABA) application on g_m, we examined g_s and g_m in the wild type (Col‐0) and ost1 and slac1‐2 mutants of Arabidopsis thaliana grown at 390 or 780 μmol mol^?1 CO₂. Stomata of these mutants were reported to be insensitive to both high CO₂ and ABA. When the ambient CO₂ increased instantaneously, g_m decreased in all these plants, whereas g_s in ost1 and slac1‐2 was unchanged. Therefore, the decrease in g_m in response to high CO₂ occurred irrespective of the responses of g_s. g_m was mainly determined by the instantaneous CO₂ concentration during the measurement and not markedly by the CO₂ concentration during the growth. Exogenous application of ABA to Col‐0 caused the decrease in the intercellular CO₂ concentration (C_i). With the decrease in C_i, g_m did not increase but decreased, indicating that the response of g_m to CO₂ and that to ABA are differently regulated and that ABA content in the leaves plays an important role in the regulation of g_m.     

Population genetic structure and demography of Magnolia kobus: variety borealis is not supported genetically

Species delimitations by morphological and by genetic markers are not always congruent. Magnolia kobus consists of two morphologically different varieties, kobus and borealis. The latter variety is characterized by larger leaves than the former. For the conservation of M. kobus genetic resources in natural forests, the relationships between morphological and genetic variation should be clarified. We investigated variations in nuclear microsatellites, chloroplast DNA (cpDNA) sequences and leaf morphological traits in 23 populations of M. kobus over the range of species. Two genetically divergent lineages, northern and southern were detected and their geographical boundary was estimated to be at 39°N. The northern lineage consisted of two genetic clusters and a single cpDNA haplotype, while the southern one had multiple genetic clusters and cpDNA haplotypes. The northern lineage showed significantly lower genetic diversity than the southern. Approximate Bayesian computation indicated that the northern and southern lineages had experienced, respectively, population expansion and long-term stable population size. The divergence time between the two lineages was estimated to be 565,000 years ago and no signature of migration between the two lineages after divergence was detected. Ecological niche modeling showed that the potential distribution area in northern Japan at the last glacial maximum was very small. It is thus considered that the two lineages have experienced different population histories over several glacial-inter-glacial cycles. Individuals of populations in the central to northern part of Honshu on the Sea of Japan side and in Hokkaido had large leaf width and area. These leaf characteristics corresponded with those of variety borealis. However, the delimitation of the northern and southern lineages detected by genetic markers (39°N) was not congruent with that detected by leaf morphologies (36°N). It is therefore suggested that variety borealis is not supported genetically and the northern and southern lineages should be considered separately when identifying conservation units based not on morphology but on genetic markers.

     

Comparison Study of Allelochemicals and Bispyribac-Sodium on the Germination and Growth Response of Echinochloa crus-galli L.

The phytotoxic effects of two allelochemicals (trans-cinnamic acid and syringaldehyde) at different concentrations (1000, 100, 10, and 1 µM) on seed germination, seedling growth, and physiological and biochemical changes of Echinochloa crus-galli L. were tested by comparison to a commercial herbicide ‘Nominee’ (that is, 100 g/L bispyribac-sodium). trans-Cinnamic acid and the herbicide inhibited seed germination completely at 100 µM, whereas for syringaldehyde, complete inhibition required 1000 µM. However, with 100 µM syringaldehyde, the seed germination of the test species was 53% of the control. Allelochemicals and the herbicide delayed seed germination and significantly affected the speed of germination index (S), speed of cumulative germination index (AS), and coefficient of germination rate (CRG). The roots were more affected when nutrients were not added to the growth bioassay. In general, with the increasing concentration of allelochemicals from 100 to 1000 µM, the inhibitory effects increased. Via microscopy analysis, we found leaf blade wilting and necrosis at concentrations above 100 µM in allelochemical-treated plants. Roots of E. crus-galli treated with 1000 µM allelochemicals had black points on root nodes but had no root hairs. The anatomy of roots treated with allelochemicals (1000 µM) showed contraction or reduction of root pith cells as well as fewer and larger vacuoles compared to the control. The allelochemicals also showed remarkable effects on seedling growth, SPAD index, chlorophyll content, and free proline content in a pot culture bioassay, indicating that trans-cinnamic acid and syringaldehyde are potent inhibitors of E. crus-galli growth and can be developed as herbicides for future weed management strategies.

     

Localization of ghrelin-producing cells in the stomach of the rainbow trout (Oncorhynchus mykiss)

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), was isolated from the rat stomach and determined to be n-octanoylated 28-amino-acid peptide. In this study, we studied the distribution of ghrelin-producing cells (ghrelin cells) in the gastrointestinal tract of male and female rainbow trout (Oncorhynchus mykiss) by immunohistochemistry using N-terminal region-recognizing antibody and also by in situ hybridization using a trout ghrelin-specific cRNA probe. Ghrelin cells were found in the mucosal layer of the stomach but not in the myenteric plexus, and no ghrelin cells were observed in other regions of the gastrointestinal tract. Ghrelin cells could be classified into two types: closed- and opened-type cells. The density of ghrelin cells increased gradually in the direction from the cardiac to pyloric portions of the stomach in both sexes. The number of ghrelin cells per unit area seemed to be higher in females than in males. In conclusion, trout ghrelin cells exist in the stomach and are classified into two types of cells, closed- and opened-type cells.     

Regulatory molecules for coronary expressions of VEGF and its angiogenic receptor KDR in hypoestrogenic middle-aged female rats

We studied the effects of estrogen deprivation and replacement on the protein and gene expression levels molecules that can be considered to be essential for coronary angiogenesis in middle-aged female rats. The animals were subjected to sham operation, ovariectomy, or ovariectomy with estrogen replacement therapy (ERT). Following ovariectomy, protein and gene expressions of vascular endothelial growth factor (VEGF) and its angiogenic receptor (KDR) showed a marked decline in coronary vessels, as determined by immunohistochemistry and in situ hybridization. ERT resulted in restoration of the ovariectomy-induced changes to intact levels. The coronary expression level of basic fibroblast growth factor was unaffected by estrogen deprivation or treatment. The changes in VEGF and KDR expressions were strongly associated with those in endothelial nitric oxide synthase (eNOS) expression in coronary vessels. Moreover, the age- and gender-dependent accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) protein appeared to be a determinant molecule of VEGF expression in middle-aged female rats. We reached a conclusion that the VEGF-KDR system plays a key role in coronary angiogenesis in hypoestrogenic elderly women and is critically regulated by estrogen, eNOS and HIF-1alpha.     

Isolation and cDNA cloning of ovarian cortical rod protein in kuruma prawn Marsupenaeus japonicus (Crustacea: Decapoda: Penaeidae)

Two cortical rod proteins having molecular weights of 28.6 kDa and 30.5 kDa were isolated from the mature ovary of Marsupenaeus japonicus using gel filtration and reversed-phase HPLC. Analysis of the N-terminal amino acid sequence of the 28.6 kDa molecule revealed that amino acid residues 1-21 corresponded to residues 9-29 of the 30.5 kDa molecule. Examination of homology using BLAST showed that 21 amino acids out of 29 residues of the 28.6 kDa molecule, and 14 out of 29 residues of the 30.5 kDa molecule were identical to that of the ovarian cortical rod proteins of Penaeus semisulcatus. Positive immunohistochemical reaction to antiserum raised against the 28.6 kDa protein was observed on cortical rods forming around the periphery of oocytes at the maturation stages. Western blotting analysis revealed that both the 28.6 kDa and 30.5 kDa molecules stained with the anti-28.6 kDa antiserum. Furthermore, the 28.6 kDa and 30.5 kDa proteins were both glycosylated, as evidenced by positive carbohydrate staining using Concanavalin A and production of positive PAS reaction. These results indicate that the cortical rods are comprised of the 28.6 kDa and 30.5 kDa molecules. We subsequently cloned two full-length cDNAs based on the N-terminal sequences of the 28.6 kDa and 30.5 kDa molecules. The open reading frame of 28.6 kDa and 30.5 kDa encoded 276 amino acid residues. Comparison analysis of the two cDNAs revealed that the location of the processing site and sequence of signal peptides differed, indicating that the two cDNAs are products of two separate genes and encode the 28.6 kDa molecule and 30.5 kDa molecule, respectively. Both proteins possessed one potential N-linked glycosylation site. It is considered that both molecules are components of the cortical rods, forming a jelly layer after fertilization.     

Balneomonas flocculans gen. nov., sp. nov., a new cellulose-producing member of the alpha-2 subclass of Proteobacteria

A new bacterial strain capable of producing cellulose was isolated from a hot spring. The isolate was Gram-negative, aerobic, and rod-shaped. The optimum temperature for growth was 40-45 degrees C. Methanol, glucose and other common carbohydrates were not utilized as sole growth substrates. Thiosulfate was not oxidized. The G+C content of the DNA was determined to be 64.0 mol%. Comparative 16S rDNA analysis indicated that Bosea thiooxidans and some strains of the genus Methylobacterium were the nearest relatives. The isolate can be distinguished from these relatives by its defectiveness in methanol utilization and thiosulfate oxidation. On the basis of its phenotypic properties and phylogeny, it is proposed that the isolate be designated Balneomonas flocculans gen. nov., sp. nov. The type strain is TFBT (= JCM 11936T, = KCTC 12101T, = IAM 15034T, = ATCC BAA-817T).