RAGE and soluble RAGE: potential therapeutic targets for cardiovascular diseases

Receptor for advanced glycation end-products (RAGE) is known to be involved in microvascular complications in diabetes. RAGE is also profoundly associated with macrovascular complications in diabetes through regulation of atherogenesis, angiogenic response, vascular injury, and inflammatory response. The potential significance of RAGE in the pathogenesis of cardiovascular disease appears not to be confined solely to nondiabetic rather than diabetic conditions. Numerous truncated forms of RAGE have recently been described, and the C-terminally truncated soluble form of RAGE has received much attention. Soluble RAGE consists of several forms, including endogenous secretory RAGE (esRAGE), which is a spliced variant of RAGE, and a shedded form derived from cell-surface RAGE. These heterogeneous forms of soluble RAGE, which carry all of the extracellular domains but are devoid of the transmembrane and intracytoplasmic domains, bind ligands including AGEs and can antagonize RAGE signaling in vitro and in vivo. ELISA systems have been developed to measure plasma esRAGE and total soluble RAGE, and the pathophysiological roles of soluble RAGE have begun to be unveiled clinically. In this review, we summarize recent findings regarding pathophysiological roles in cardiovascular disease of RAGE and soluble RAGE and discuss their potential usefulness as therapeutic targets and biomarkers for the disease.     

The beta-1,3-exoglucanase gene exgA (exg1) of Aspergillus oryzae is required to catabolize extracellular glucan, and is induced in growth on a solid surface

The biological role of ExgA (Exg1), a secretory beta-1,3-exoglucanase of Aspergillus oryzae, and the expression pattern of the exgA (exg1) gene were analyzed. The exgA disruptant and the exgA-overexpressing mutant were constructed, and phenotypes of both mutants were compared. Higher mycelial growth rate and conidiation efficiency were observed for the exgA-overexpressing mutant than for the exgA disruptant when beta-1,3-glucan was supplied as sole carbon source. On the other hand, no difference in phenotype was observed between them in the presence or absence of the inhibitors of cell wall beta-glucan remodeling when grown with glucose. exgA Expression was induced in growth on solid surfaces such as filter membrane and onion inner skin. A combination of poor nutrition and mycelial attachment to a hydrophobic solid surface appears to be an inducing factor for exgA expression. These data suggest that ExgA plays a role in beta-glucan utilization, but is not much involved in cell wall beta-glucan remodeling.     

Substrate specificities of wild and mutated farnesyl diphosphate synthases from Bacillus stearothermophilus with artificial substrates

To determine the substrate specificities of wild and mutated types of farnesyl diphosphate (FPP) synthases from Bacillus stearothermophilus, we examined the reactivities of 8-hydroxygeranyl diphosphate (HOGPP) and 8-methoxygeranyl diphosphate (CH(3)OGPP) as allylic substrate homologs. The wild-type FPP synthase reaction of HOGPP (and CH(3)OGPP) with isopentenyl diphosphate (IPP) gave hydroxyfarnesyl- (and methoxyfarnesyl-) diphosphates that stopped at the first stage of condensation. On the other hand, with mutated type FPP synthase (Y81S), the former gave hydroxygeranylgeranyl diphosphate as the main double-condensation product together with hydroxyfarnesyl diphosphate as a single-condensation product and a small amount of hydroxygeranylfarnesyl diphosphate as a triple-condensation product. Moreover, the latter gave a double-condensation product, methoxygeranylgeranyl diphosphate, as the main product and only a trace of methoxyfarnesyl diphosphate was obtained.     

Influence of maceration temperature in red wine vinification on extraction of phenolics from berry skins and seeds of grape (Vitis vinifera)

The extraction of phenolics from berry skins and seeds of the grape, Vitis vinifera cv. Cabernet Sauvignon, during red wine maceration and the influence of different temperature conditions (cold soak and/or heating at the end of maceration) were examined. Phenolics contained mainly in berry skins, viz., anthocyanin, flavonol, and epigallocatechin units within proanthocyanidins, were extracted during the early stage of maceration, whereas those in seeds, viz., gallic acid, flavan-3-ol monomers, and epicatechin-gallate units within proanthocyanidins, were gradually extracted. In addition to their localization, the molecular size and composition of the proanthocyanidins possibly influenced the kinetics of their extraction. Cold soak reduced the extraction of phenolics from the seeds. Heating at the end of maceration decreased the concentration of proanthocyanidins. Thus, modification of the temperature condition during maceration affected the progress of the concentration of phenolics, resulting in an alteration of their make-up in the finished wine.     

Cell biology of deep-sea multicellular organisms

Establishing tissue cultures derived from deep-sea multicellular organisms has been extremely difficult because of the serious damage they sustain upon decompression and exposure to the high temperature of surface seawater. We developed a novel pressure-stat aquarium system for the study of living deep-sea multicellular organisms under pressure. Using this system, we have succeeded in maintaining a variety of deep-sea multicellular organisms under pressure and atmospheric conditions after gradual, slow decompression. Furthermore, we successfully cultivated and freeze-stocked pectoral fin cells of the deep-sea eel Simenchelys parasiticus collected at a depth of 1,162 m under atmospheric pressure conditions. This review describes novel capture and maintenance devices for deep-sea organisms and cell culture studies of the organisms under atmospheric and pressure conditions.     

Diversity of microbial eukaryotes in sediment at a deep-sea methane cold seep: surveys of ribosomal DNA libraries from raw sediment samples and two enrichment cultures

Recent culture-independent surveys of eukaryotic small-subunit ribosomal DNA (SSU rDNA) from many environments have unveiled unexpectedly high diversity of microbial eukaryotes (microeukaryotes) at various taxonomic levels. However, such surveys were most probably biased by various technical difficulties, resulting in underestimation of microeukaryotic diversity. In the present study on oxygen-depleted sediment from a deep-sea methane cold seep of Sagami Bay, Japan, we surveyed the diversity of eukaryotic rDNA in raw sediment samples and in two enrichment cultures. More than half of all clones recovered from the raw sediment samples were of the basidiomycetous fungus Cryptococcus curvatus. Among other clones, phylotypes of eukaryotic parasites, such as Apicomplexa, Ichthyosporea, and Phytomyxea, were identified. On the other hand, we observed a marked difference in phylotype composition in the enrichment samples. Several phylotypes belonging to heterotrophic stramenopiles were frequently found in one enrichment culture, while a phylotype of Excavata previously detected at a deep-sea hydrothermal vent dominated the other. We successfully established a clonal culture of this excavate flagellate. Since these phylotypes were not identified in the raw sediment samples, the approach incorporating a cultivation step successfully found at least a fraction of the “hidden” microeukaryotic diversity in the environment examined. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A molecular mechanism that confines the activity pattern of miR165 in Arabidopsis leaf primordia

In Arabidopsis leaf primordia, the expression of HD‐Zip III, which promotes tissue differentiation on the adaxial side of the leaf primordia, is repressed by miRNA165/166 (miR165/166). Small RNAs, including miRNAs, can move from cell to cell. In this study, HD‐Zip III expression was strikingly repressed by miR165/166 in the epidermis and parenchyma cells on the abaxial side of the leaf primordia compared with those on the adaxial side. We also found that the MIR165A locus, which was expressed in the abaxial epidermis, was sufficient to establish the rigid repression pattern of HD‐Zip III expression in the leaf primordia. Ectopic expression analyses of MIR165A showed that the abaxial‐biased miR165 activity in the leaf primordia was formed neither by a polarized distribution of factors affecting miR165 activity nor by a physical boundary inhibiting the cell‐to‐cell movement of miRNA between the adaxial and abaxial sides. We revealed that cis‐acting factors, including the promoter, backbone, and mature miRNA sequence of MIR165A, are necessary for the abaxial‐biased activity of miR165 in the leaf primordia. We also found that the abaxial‐determining genes YABBYs are trans‐acting factors that are necessary for the miR165 activity pattern, resulting in the rigid determination of the adaxial–abaxial boundary in leaf primordia. Thus, we proposed a molecular mechanism in which the abaxial‐biased patterning of miR165 activity is confined.     

Elevated expression of TcHMA3 plays a key role in the extreme Cd tolerance in a Cd-hyperaccumulating ecotype of Thlaspi caerulescens

Cadmium (Cd) is a highly toxic heavy metal for plants, but several unique Cd-hyperaccumulating plant species are able to accumulate this metal to extraordinary concentrations in the aboveground tissues without showing any toxic symptoms. However, the molecular mechanisms underlying this hypertolerance to Cd are poorly understood. Here we have isolated and functionally characterized an allelic gene, TcHMA3 (heavy metal ATPase 3) from two ecotypes (Ganges and Prayon) of Thlaspi caerulescens contrasting in Cd accumulation and tolerance. The TcHMA3 alleles from the higher (Ganges) and lower Cd-accumulating ecotype (Prayon) share 97.8% identity, and encode a P(1B)-type ATPase. There were no differences in the expression pattern, cell-specificity of protein localization and transport substrate-specificity of TcHMA3 between the two ecotypes. Both alleles were characterized by constitutive expression in the shoot and root, a tonoplast localization of the protein in all leaf cells and specific transport activity for Cd. The only difference between the two ecotypes was the expression level of TcHMA3: Ganges showed a sevenfold higher expression than Prayon, partly caused by a higher copy number. Furthermore, the expression level and localization of TcHMA3 were different from AtHMA3 expression in Arabidopsis. Overexpression of TcHMA3 in Arabidopsis significantly enhanced tolerance to Cd and slightly increased tolerance to Zn, but did not change Co or Pb tolerance. These results indicate that TcHMA3 is a tonoplast-localized transporter highly specific for Cd, which is responsible for sequestration of Cd into the leaf vacuoles, and that a higher expression of this gene is required for Cd hypertolerance in the Cd-hyperaccumulating ecotype of T. caerulescens.