Highly pathogenic H5N1 influenza virus causes coagulopathy in chickens

Severe hemorrhage at multiple organs is frequently observed in chickens infected with highly pathogenic avian influenza (HPAI) A viruses. In this study we examined whether HPAI virus infection leads to coagulation disorder in chickens. Pathological examinations showed that the fibrin thrombi were formed in arterioles at the lung, associated with the viral antigens in endothelial cells of chickens infected intravenously with HPAI virus. Hematological analyses of peripheral blood collected from the chickens revealed that coagulopathy was initiated at early stage of infection when viral antigens were detected only in the endothelial cells and monocytes/macrophages. Furthermore, gene expression of the tissue factor, the main initiator of blood coagulation, was upregulated in the spleen, lung, and brain of HPAI virus-infected chickens. These results suggest that dysfunction of endothelial cells and monocytes/macrophages upon HPAI virus infection may induce hemostasis abnormalities represented by the excessive blood coagulation and consumptive coagulopathy in chickens.     

Light activates the degradation of PIL5 protein to promote seed germination through gibberellin in Arabidopsis

Angiosperm seeds integrate various environmental signals, such as water availability and light conditions, to make a proper decision to germinate. Once the optimal conditions are sensed, gibberellin (GA) is synthesized, triggering germination. Among environmental signals, light conditions are perceived by phytochromes. However, it is not well understood how phytochromes regulate GA biosynthesis. Here we investigated whether phytochromes regulate GA biosynthesis through PIL5, a phytochrome-interacting bHLH protein, in Arabidopsis. We found that pil5 seed germination was inhibited by paclobutrazol, the ga1 mutation was epistatic to the pil5 mutation, and the inhibitory effect of PIL5 overexpression on seed germination could be rescued by exogenous GA, collectively indicating that PIL5 regulates seed germination negatively through GA. Expression analysis revealed that PIL5 repressed the expression of GA biosynthetic genes (GA3ox1 and GA3ox2), and activated the expression of a GA catabolic gene (GA2ox) in both PHYA- and PHYB-dependent germination assays. Consistent with these gene-expression patterns, the amount of bioactive GA was higher in the pil5 mutant and lower in the PIL5 overexpression line. Lastly, we showed that red and far-red light signals trigger PIL5 protein degradation through the 26S proteasome, thus releasing the inhibition of bioactive GA biosynthesis by PIL5. Taken together, our data indicate that phytochromes promote seed germination by degrading PIL5, which leads to increased GA biosynthesis and decreased GA degradation.     

Discovery of SMP-304, a novel benzylpiperidine derivative with serotonin transporter inhibitory activity and 5-HT1A weak partial agonistic activity showing the antidepressant-like effect

We report the discovery of a novel benzylpiperidine derivative with serotonin transporter (SERT) inhibitory activity and 5-HT_1A receptor weak partial agonistic activity showing the antidepressant-like effect. The 3-methoxyphenyl group and the phenethyl group of compound 1, which has weak SERT binding activity, but potent 5-HT_1A binding activity, were optimized, leading to compound 35 with potent and balanced dual SERT and 5-HT_1A binding activity, but also potent CYP2D6 inhibitory activity. Replacement of the methoxy group in the left part of compound 35 with a larger alkoxy group, such as ethoxy, isopropoxy or methoxy-ethoxy group ameliorated CYP2D6 inhibition, giving SMP-304 as a candidate. SMP-304 with serotonin uptake inhibitory activity and 5-HT_1A weak partial agonistic activity, which could work as a 5-HT_1A antagonist, displayed faster onset of antidepressant-like effect than a representative SSRI paroxetine in an animal model.     

Synthesis and characterization of a novel reagent containing dansyl group, which specifically alkylates sulfhydryl group: an example of application for protein chemistry

We have synthesized a novel reagent containing dansyl group, iodoacethyl dansylcadaverine (IADC), which specifically alkylates sulfhydryl groups. The carboxyl group of iodoacetic acid was activated with dicyclohexylcarbodiimide and was condensed with amino group of dansylcadaverine. Purity and chemical structure of IADC was confirmed with mass spectrometry (MS) and NMR. IADC alkylated GSH but not GSSG, which was confirmed by MS. The reactivity of IADC with proteins was also investigated with Western blotting using anti-dansyl antibody. IADC reacted only with sulfhydryl-containing proteins. The specificity of the interaction of IADC with sulfhydryl groups in proteins was confirmed by adding excessive amount of a well-known sulfhydryl-specific reagent, 5, 5'-dithiobis(2-nitrobenzoic acid), which led to a complete inhibition. To show the usefulness of IADC, the cysteines in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from chicken muscle were modified with this reagent, and GAPDH was then digested by lysyl endopeptidase. The peptides generated from digestion of IADC-incorporated GAPDH were applied to an anti-dansyl immunoaffinity column. The peptide fragments bound and eluted from the column were separated by HPLC, and the amino acid sequence of each peptide was analyzed, and peptide was identified as the one containing a Cys residue(s). These data showed that IADC is a useful reagent to specifically identify the positions of a Cys residue(s) in proteins.     

Capsaicin Modifies Responses of Rat Chorda Tympani Nerve Fibers to NaCl

Single-fiber preparations of the rat chorda tympani (CT) nervewere used to study the mechanism of action of capsaicin on salt-tastetransduction. Capsaicin selectively suppressed the responsesto NaCl of the CT nerve fibers (N-fibers) that are sodium-specific(insensitive or poorly sensitive to potassium). Among the morebroadly responsive, cation-sensitive fibers (E-fibers) thereare two subtypes, both of which responded to capsaicin but indifferent ways (‘enhanced’ type and ‘suppressed’type). In both N- and E-fibers, 5% ethanol (the vehicle forcapsaicin) slightly reduced the response to 100 mM NaCl. Thesuppressive effect of capsaicin on the response of the N-typefibers to 100 mM NaCl was significantly stronger than the effectof 5% ethanol. The suppression lasted for at least 20 s afterthe simultaneous application of 100 p.p.m. capsaicin-100 mMNaCl. These results indicate that 100 p.p.m. capsaicin can modifythe response of CT fibers to NaCl. The observed effect of capsaicinon gustatory fibers could be the net result of opposite suppressiveand enhancing processes in the taste buds cells and excitedintra- or extragemmal trigeminal nerve endings. Chem. Senses22: 249–255, 1997. ^*These authors contributed equally to this study     

Up-regulation of adipogenin, an adipocyte plasma transmembrane protein, during adipogenesis

Until now, the various proteins highly expressed in adipose tissues have been identified and characterized by traditional gene cloning techniques. However, methods of computer analysis have been developed to compare the levels of expression among various tissues, and genes whose expression levels differ significantly between tissues have been found. Among these genes, we report on the possible function of a new adipose-specific gene, showed higher expression in adipose tissue through ‘Search Expression’ on Genome Institute of Norvartis Research Foundation (GNF) SymAtlas v0.8.0. This database has generated and analyzed gene expression of each gene in diverse samples of normal tissues, organs, and cell lines. This newly discovered gene product was named adipogenin because of its role in stimulating adipocyte differentiation and development. Adipogenin mRNA was highly expressed in four different fat depots, and exclusively expressed in adipocytes isolated from adipose tissues. The level of adipogenin mRNA was up-regulated in the subcutaneous and visceral adipose tissues of mice fed a high-fat diet compared to those on the control diet. The expression of adipogenin mRNA is dramatically elevated during adipocyte differentiation of 3T3-L1 cells. Troglitazone, which up-regulated peroxisome proliferators-activated receptor γ2 (PPAR-γ2) expression, increased adipogenin mRNA expression, although this gene was down-regulated by retinoic acid. Confocal image analyses of green-fluorescent protein-adipogenin (pEGFP-adipogenin) transiently expressed in 3T3-L1 adipocytes showed that adipogenin was strictly localized to membranes and was absent from the cytosol. Moreover, small interfering RNA (siRNA) mediated a reduction of adipogenin mRNA in 3T3-L1 cells and blocked the process of adipocyte differentiation. These results indicate that adipogenin, an adipocyte-specific membrane protein, may be involved with adipogenesis, as one of the regulators of adipose tissue development.Yeon-Hee Hong and Daisuke Hishikawa contributed equally to this work     

Development of a series of gateway binary vectors possessing a tunicamycin resistance gene as a marker for the transformation of Arabidopsis thaliana

We made two series of Gateway binary vectors, pGWBs and R4pGWBs, possessing a UDP-N-acetylglucosamine: dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene driven by the nopaline synthase promoter (Pnos) as a tunicamycin resistance marker for the transformation of Arabidopsis thaliana. The reporters and tags employed in this system are sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, TagRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7, and TAP. Selection of transformants was successful on plates containing 0.15 mg/L of tunicamycin. These vectors were compatible with existing pGWB and R4pGWB vectors for kanamycin, hygromycin B, and BASTA? selection, and are useful new tools for making transgenic Arabidopsis.     

Role of claudin species-specific dynamics in reconstitution and remodeling of the zonula occludens

Tight-junction strands, which are organized into the beltlike cell-cell adhesive structure called the zonula occludens (TJ), create the paracellular permselective barrier in epithelial cells. The TJ is constructed on the basis of the zonula adherens (AJ) by polymerized claudins in a process mediated by ZO-1/2, but whether the 24 individual claudin family members play different roles at the TJ is unclear. Here we established a cell system for examining the polymerization of individual claudins in the presence of ZO-1/2 using an epithelial-like cell line, SF7, which lacked endogenous TJs and expressed no claudin but claudin-12 in immunofluorescence and real-time PCR assays. In stable SF7-derived lines, exogenous claudin-7, -14, or -19, but no other claudins, individually reconstituted TJs, each with a distinct TJ-strand pattern, as revealed by freeze-fracture analyses. Fluorescence recovery after photobleaching (FRAP) analyses of the claudin dynamics in these and other epithelial cells suggested that slow FRAP-recovery dynamics of claudins play a critical role in regulating their polymerization around AJs, which are loosely coupled with ZO-1/2, to form TJs. Furthermore, the distinct claudin stabilities in different cell types may help to understand how TJs regulate paracellular permeability by altering the paracellular flux and the paracellular ion permeability.