No abstract

Cryptochromes regulate the circadian clock in animals and plants. Humans and mice have two cryptochrome (Cry) genes. A previous study showed that mice lacking the Cry2 gene had reduced sensitivity to acute light induction of the circadian gene mPer1 in the suprachiasmatic nucleus (SCN) and had an intrinsic period 1 hr longer than normal. In this study, Cry1 ؊͞؊ and Cry1 ؊͞؊ Cry2 ؊͞؊ mice were generated and their circadian clocks were analyzed at behavioral and molecular levels. Behaviorally, the Cry1 ؊͞؊ mice had a circadian period 1 hr shorter than wild type and the Cry1 ؊͞؊ Cry2 ؊͞؊ mice were arrhythmic in constant darkness (DD). Biochemically, acute light induction of mPer1 mRNA in the SCN was blunted in Cry1 ؊͞؊ and abolished in Cry1 ؊͞؊ Cry2 ؊͞؊ mice. In contrast, the acute light induction of mPer2 in the SCN was intact in Cry1 ؊͞؊ and Cry1 ؊͞؊ Cry2 ؊͞؊ animals. Importantly, in double mutants, mPer1 expression was constitutively elevated and no rhythmicity was detected in either 12-hr light͞12-hr dark or DD, whereas mPer2 expression appeared rhythmic in 12-hr light͞12-hr dark, but nonrhythmic in DD with intermediate levels. These results demonstrate that Cry1 and Cry2 are required for the normal expression of circadian behavioral rhythms, as well as circadian rhythms of mPer1 and mPer2 in the SCN. The differential regulation of mPer1 and mPer2 by light in Cry double mutants reveals a surprising complexity in the role of cryptochromes in mammals.gene targeting ͉ photoreceptor ͉ suprachiasmatic nucleus C ircadian rhythms are oscillations with daily periodicities in physiological and behavioral functions of organisms (1-3). The rhythms are generated by a cell-autonomous circadian oscillator (4) that is synchronized with the environment by light. Recently, it was proposed that, in mammals, the nonopsin pigments, cryptochrome blue-light photoreceptors (5, 6), may be the photoactive pigments that synchronized the molecular oscillator and, ultimately, the organismic circadian rhythm with the daily light-dark cycle (7,8). In humans and mice there are two genes encoding the apoproteins of the cryptochromes: CRY1 and CRY2 in humans and Cry1 and Cry2 in mice (6,8,9). Both genes are expressed throughout the body (8-11). Of particular interest, both Cry1 and Cry2 are expressed at high levels in the ganglion cells and the inner nuclear layer of the retina, which are known to be important for circadian photoreception, and Cry1 is expressed with a robust circadian rhythm in the suprachiasmatic nucleus (SCN) (8).In a previous study (12), we found that mice lacking a functional Cry2 gene (i) had reduced sensitivity to acute light induction of the clock gene mPer1 (mouse period gene 1) in the SCN, (ii) had an intrinsic circadian period about 1 hr longer than normal, and (iii) exhibited high amplitude phase shifts in response to light pulses administered at circadian time (CT) 17. These data, and related findings in cryptochrome mutants of Drosophila melanogaster (13) and Arabidopsis thaliana (14), supported the notion that cr...

Phospholipase A2 (PLA2) comprises a superfamily of enzymes that hydrolyse the ester bond of phospholipids at the sn-2 position. Among the members of this superfamily, cytosolic PLA2 has attracted attention because it preferentially hydrolyses arachidonoyl phospholipids and is activated by submicromolar concentrations of Ca2+ ions and by phosphorylation by mitogen-activated protein kinases (MAP kinases). Here we investigate the function of cytosolic PLA2 in vivo by using homologous recombination to generate mice deficient in this enzyme. These mice showed a marked decrease in their production of eicosanoids and platelet-activating factor in peritoneal macrophages. Their ovalbumin-induced anaphylactic responses were significantly reduced, as was their bronchial reactivity to methacholine. Female mutant mice failed to deliver offspring, but these could be rescued by administration of a progesterone-receptor antagonist to the mother at term. Considered together with previous findings, our results indicate that cytosolic PLA2 plays a non-redundant role in allergic responses and reproductive physiology.

We have developed a technique for cultivation of chemolithoautotrophs under high hydrostatic pressures that is successfully applicable to various types of deep-sea chemolithoautotrophs, including methanogens. It is based on a glass-syringe-sealing liquid medium and gas mixture used in conjunction with a butyl rubber piston and a metallic needle stuck into butyl rubber. By using this technique, growth, survival, and methane production of a newly isolated, hyperthermophilic methanogen Methanopyrus kandleri strain 116 are characterized under high temperatures and hydrostatic pressures. Elevated hydrostatic pressures extend the temperature maximum for possible cell proliferation from 116°C at 0.4 MPa to 122°C at 20 MPa, providing the potential for growth even at 122°C under an in situ high pressure. In addition, piezophilic growth significantly affected stable carbon isotope fractionation of methanogenesis from CO 2. Under conventional growth conditions, the isotope fractionation of methanogenesis by M. kandleri strain 116 was similar to values (؊34‰ to؊27‰) previously reported for other hydrogenotrophic methanogens. However, under high hydrostatic pressures, the isotope fractionation effect became much smaller (<؊12‰), and the kinetic isotope effect at 122°C and 40 MPa was ؊9.4‰, which is one of the smallest effects ever reported. This observation will shed light on the sources and production mechanisms of deep-sea methane.carbon isotope fractionation ͉ deep-sea hydrothermal vent ͉ hyperthermophile ͉ methanogenesis ͉ piezophilic M icrobial methanogenesis in the deep sea is a key process in the carbon cycle of Earth. It contributes to the CH 4 pool (free gas and methane hydrate), a potential energy source and alternative to petroleum (1, 2) as well as a strong greenhouse gas with a potential for rapid release (3), in deep-sea and subseafloor sediments. Methanogens are known to have several methanogenic types using different substrates of H 2 , acetate, methanol, CO, and so on. Hyperthermophilic hydrogenotrophic methanogens play a major role in primary production of ecosystems in deep-sea hydrothermal areas in the present Earth (4, 5) and may represent the most ancient type of microorganisms flourishing in the Archean Earth (6-10).Despite the significance of methanogens in the deep-sea and subseafloor ecosystems, the ecophysiological and biogeochemical characteristics of their in situ habitats have been little understood. It has been quite difficult to incorporate high hydrostatic pressures into experiments involving gaseous substrates such as H 2 and CO 2 . If this difficulty can be overcome by any specific apparatus (11,12), the subsequent handling of microbiological experiments under high hydrostatic pressures remains a great technical barrier. Thus, growth characterization of only thermophilic methanogens Methanocaldococcus jannaschii and Methanothermococcus thermolithotrophicus under high pressures has been successfully achieved, and only their piezophilic responses of growth and methane production have been inv...

Hadal oceans at water depths below 6,000 m are the least-explored aquatic biosphere. The Challenger Deep, located in the western equatorial Pacific, with a water depth of ∼11 km, is the deepest ocean on Earth. Microbial communities associated with waters from the sea surface to the trench bottom (0 ∼10,257 m) in the Challenger Deep were analyzed, and unprecedented trench microbial communities were identified in the hadal waters (6,000 ∼10,257 m) that were distinct from the abyssal microbial communities. The potentially chemolithotrophic populations were less abundant in the hadal water than those in the upper abyssal waters. The emerging members of chemolithotrophic nitrifiers in the hadal water that likely adapt to the higher flux of electron donors were also different from those in the abyssal waters that adapt to the lower flux of electron donors. Species-level niche separation in most of the dominant taxa was also found between the hadal and abyssal microbial communities. Considering the geomorphology and the isolated hydrotopographical nature of the Mariana Trench, we hypothesized that the distinct hadal microbial ecosystem was driven by the endogenous recycling of organic matter in the hadal waters associated with the trench geomorphology.hadal | trench | niche separation | nitrification | Challenger Deep

GPR40, one of the G protein-coupled receptors predominantly expressed in pancreatic β-cells, mediates enhancement of glucose-stimulated insulin secretion by free fatty acids. A potent and selective GPR40 agonist is theorized to be a safe and effective antidiabetic drug with little or no risk of hypoglycemia. Cyclization of the phenylpropanoic acid moiety of lead compound 1 produced fused phenylalkanoic acids with favorable in vitro agonist activities and pharmacokinetic profiles. Further optimization led to the discovery of dihydrobenzofuran derivative 9a ([(3S)-6-({2',6'-dimethyl-4'-[3-(methylsulfonyl)propoxy]biphenyl-3-yl}methoxy)-2,3-dihydro-1-benzofuran-3-yl]acetic acid hemi-hydrate, TAK-875) as a potent, selective, and orally bioavailable GPR40 agonist, with a pharmacokinetic profile enabling long-acting drug efficacy. Compound 9a showed potent plasma glucose-lowering action and insulinotropic action during an oral glucose tolerance test in female Wistar fatty rats with impaired glucose tolerance. Compound 9a is currently in clinical trials for the treatment of type 2 diabetes mellitus.

Hepatocyte growth factor (HGF) plays an important role in many biological events such as angiogenesis, cell proliferation, anti-fibrosis and antiapoptosis. It is well known that HGF promotes tumor progression and suppresses development of fibrosis after tissue injury. In contrast, its role in immune-mediated disorders has not been fully clarified. In the present study, we examined the role of HGF in Ag-specific immune response using in vitro studies and an experimental model of allergic airway inflammation. We first confirmed that dendritic cells (DCs) expressed the receptor for HGF, c-met, which was not expressed in T cells. Treatment with HGF both in vitro and in vivo potently suppressed DC functions such as Ag-presenting capacity, thus down-regulating Ag-induced Th1- and Th2-type immune responses. Exogenous administration of the HGF expression plasmid into Ag-primed mice markedly suppressed the development of airway eosinophilia and airway hyperresponsiveness, which was induced by Ag inhalation, with suppression of the Ag-presenting capacity of DCs in the lung. HGF exhibited these immunosuppressive effects without up-regulation of IL-10 or TGF-β. We also found that expression of endogenous HGF in the lung significantly increased following Ag sensitization and inhalation challenges. Finally, neutralization of endogenous HGF in vivo significantly increased airway eosinophilia and airway hyperresponsiveness with up-regulation of the Ag-presenting capacity of DCs in the lung. These results demonstrated a novel, significant, and possibly therapeutic role of HGF as a potent regulator in immune-mediated disorders such as asthma.

Normal T cell repertoire contains regulatory T cells that control autoimmune responses in the periphery. One recent study demonstrated that CD4+CD25+ T cells were generated from autoreactive T cells without negative selection. However, it is unclear whether, in general, positive selection and negative selection of autoreactive T cells are mutually exclusive processes in the thymus. To investigate the ontogeny of CD4+CD25+ regulatory T cells, neo-autoantigen-bearing transgenic mice expressing chicken egg OVA systemically in the nuclei (Ld-nOVA) were crossed with transgenic mice expressing an OVA-specific TCR (DO11.10). Ld-nOVA × DO11.10 mice had increased numbers of CD4+CD25+ regulatory T cells in the thymus and the periphery despite clonal deletion. In Ld-nOVA × DO11.10 mice, T cells expressing endogenous TCR αβ chains were CD4+CD25− T cells, whereas T cells expressing autoreactive TCR were selected as CD4+CD25+ T cells, which were exclusively dominant in recombination-activating gene 2-deficient Ld-nOVA × DO11.10 mice. In contrast, in DO11.10 mice, CD4+CD25+ T cells expressed endogenous TCR αβ chains, which disappeared in recombination-activating gene 2-deficient DO11.10 mice. These results indicate that part of autoreactive T cells that have a high affinity TCR enough to cause clonal deletion could be positively selected as CD4+CD25+ T cells in the thymus. Furthermore, it is suggested that endogenous TCR gene rearrangement might critically contribute to the generation of CD4+CD25+ T cells from nonautoreactive T cell repertoire, at least under the limited conditions such as TCR-transgenic models, as well as the generation of CD4+CD25− T cells from autoreactive T cell repertoire.