Exogenous and endogenous ghrelin counteracts GLP-1 action to stimulate cAMP signaling and insulin secretion in islet β-cells

We studied interactive effects of insulinotropic GLP-1 and insulinostatic ghrelin on rat pancreatic islets. GLP-1 potentiated glucose-induced insulin release and cAMP production in isolated islets and [Ca(2+)](i) increases in single β-cells, and these potentiations were attenuated by ghrelin. Ghrelin suppressed [Ca(2+)](i) responses to an adenylate cyclase activator forskolin. Moreover, GLP-1-induced insulin release and cAMP production were markedly enhanced by [D-lys(3)]-GHRP-6, a ghrelin receptor antagonist, in isolated islets. These results indicate that both exogenous and endogenous islet-derived ghrelin counteracts glucose-dependent GLP-1 action to increase cAMP production, [Ca(2+)](i) and insulin release in islet β-cells, positioning ghrelin as a modulator of insulinotropic GLP-1.     

PB2 Protein of a Highly Pathogenic Avian Influenza Virus Strain A/chicken/Yamaguchi/7/2004 (H5N1) Determines Its Replication Potential in Pigs

It has been shown that not all but most of the avian influenza viruses replicate in the upper respiratory tract of pigs (H. Kida et al., J. Gen. Virol. 75:2183-2188, 1994). It was shown that A/chicken/Yamaguchi/7/2004 (H5N1) [Ck/Yamaguchi/04 (H5N1)] did not replicate in pigs (N. Isoda et al., Arch. Virol. 151:1267-1279, 2006). In the present study, the genetic basis for this host range restriction was determined using reassortant viruses generated between Ck/Yamaguchi/04 (H5N1) and A/swine/Hokkaido/2/1981 (H1N1) [Sw/Hokkaido/81 (H1N1)]. Two in vivo-generated single-gene reassortant virus clones of the H5N1 subtype (virus clones 1 and 2), whose PB2 gene was of Sw/Hokkaido/81 (H1N1) origin and whose remaining seven genes were of Ck/Yamaguchi/04 (H5N1) origin, were recovered from the experimentally infected pigs. The replicative potential of virus clones 1 and 2 was further confirmed by using reassortant virus (rg-Ck-Sw/PB2) generated by reverse genetics. Interestingly, the PB2 gene of Ck/Yamaguchi/04 (H5N1) did not restrict the replication of Sw/Hokkaido/81 (H1N1), as determined by using reassortant virus rg-Sw-Ck/PB2. The rg-Sw-Ck/PB2 virus replicated to moderate levels and for a shorter duration than parental Sw/Hokkaido/81 (H1N1). Sequencing of two isolates recovered from the pigs inoculated with rg-Sw-Ck/PB2 revealed either the D256G or the E627K amino acid substitution in the PB2 proteins of the isolates. The D256G and E627K mutations enhanced viral polymerase activity in the mammalian cells, correlating with replication of virus in pigs. These results indicate that the PB2 protein restricts the growth of Ck/Yamaguchi/04 (H5N1) in pigs.     

Chromosaponin I stimulates the sugar taste receptor cells of the blowfly, Phormia regina

Chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea and other leguminous plants, stimulates the growth of roots in a variety of plants. In the present work, we introduce CSI as a sugar taste substance for the blowfly, Phormia regina. The blowfly has taste chemosensilla on the labellum. The sensory receptor cells in the chemosensillum are highly specialized for the tastes of sugar, salt and water, respectively. Application of CSI induced the feeding response of blowflies including full proboscis extension. CSI also induced impulses of the sugar taste receptor cell in the LL-type sensillum. The optimum concentration of CSI in these responses was 0.1 mM which is much lower than that of sucrose. Based on the comparison of dose-response relationships, CSI is 100 times more effective than sucrose in stimulating the sugar taste receptor cells. CSI-induced impulses appeared after a significant latency compared with sucrose. As far as we know, this is the first report describing that a natural saponin induces sugar responses in insects. CSI is a unique saponin because of its bifunctional property in plants and insects.     

Crystal structure of alkaline cellulase K: insight into the alkaline adaptation of an industrial enzyme

The crystal structure of the catalytic domain of alkaline cellulase K was determined at 1.9 A resolution. Because of the most alkaliphilic nature and it's highest activity at pH 9.5, it is used commercially in laundry detergents. An analysis of the structural bases of the alkaliphilic character of the enzyme suggested a mechanism similar to that previously proposed for alkaline proteases, that is, an increase in the number of Arg, His, and Gln residues, and a decrease in Asp and Lys residues. Some ion pairs were formed by the gained Arg residues, which is similar to what has been found in the alkaline proteases. Lys-Asp ion pairs are disfavored and partly replaced with Arg-Asp ion pairs. The alkaline adaptation appeared to be a remodeling of ion pairs so that the charge balance is kept in the high pH range.     

Wheat germ agglutinin-induced platelet activation via platelet endothelial cell adhesion molecule-1: involvement of rapid phospholipase C gamma 2 activation by Src family kinases.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a 130K transmembrane glycoprotein that belongs to the immunoglobulin gene superfamily and is expressed on the surface of hematological or vascular cells, including platelets and endothelial cells. Although the importance of this adhesion molecule in various cell-cell interactions is established, its function in platelets remains ill-defined. In the process of clarifying the mechanism by which the lectin wheat germ agglutinin (WGA) activates platelets, we unexpectedly discovered that PECAM-1 is involved in signal transduction pathways elicited by this N-acetyl-D-glucosamine (NAGlu)-reactive lectin. WGA, which is a very potent platelet stimulator, elicited a rapid surge in Syk and phospholipase C (PLC)-gamma 2 tyrosine phosphorylation and the resultant intracellular Ca(2+) mobilization; collagen, as reported, induced these responses, but in a much slower and weaker manner. WGA strongly induced tyrosine phosphorylation of a 130-140K protein, which was confirmed to be PECAM-1 by immunoprecipitation and immunodepletion studies. WGA-induced PECAM-1 tyrosine phosphorylation occurred rapidly, strongly and in a manner independent of platelet aggregation or cell-cell contact; these characteristics of PECAM-1 phosphorylation were not mimicked at all by receptor-mediated platelet agonists. In addition, WGA was found to associate with PECAM-1 itself, and anti-PECAM-1 antibody, as well as NAGlu, specifically inhibited WGA-induced platelet aggregation. In PECAM-1 immunoprecipitates, Src family tyrosine kinases existed, and a kinase activity was detected, which increased upon WGA stimulation. Furthermore, the Src family kinase inhibitor PP2 inhibited WGA-induced platelet aggregation, Ca(2+) mobilization, and PLC-gamma 2 tyrosine phosphorylation. Finally, WGA induced PECAM-1 tyrosine phosphorylation and cytoskeletal reorganization in vascular endothelial cells. Our results suggest that (i) PECAM-1 is involved in WGA-induced platelet activation, (ii) PECAM-1 clustering by WGA activates unique and strong platelet signaling pathways, leading to a rapid PLC activation via Src family kinases, and (iii) WGA is a useful tool for elucidating PECAM-1-mediated signaling with wide implications not confined to platelets.     

Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae

Summary Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae R11340 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding -glucuronidase (GUS) downstream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.     

Synthesis of Oligodeoxyribonucleotide Bearing 2′-S-Alkyl Residue and its Effect on the Duplex Stability

Abstract

2′-Deoxy-2′-S-hexyluridine derivative was synthesized from 2,2′-anhydrouridine and 1-hexanethiol and incorporated into an oligodeoxyribonucleotide. The thermal stability of the duplexes formed by the 2′-S-hexyl modified ODN with either the complementary DNA or RNA strand was decreased compared to the unmodified counterparts.     

Isolation and Characterization of 2-Nitroimidazole Produced by Streptomyces Species as an Inhibitor of Both Carbonic Anhydrase and Shell Formation in the Barnacle Balanus amphitrite

Carbonic anhydrase is thought to be involved in the process of calcium carbonate deposition in calcified tissues of many organisms. Barnacles form hard calcified shells for protection against predation, and represent a class of marine-fouling animals. In order to inhibit barnacle growth by inhibiting shell formation, we searched for carbonic anhydrase inhibitors from microbial secondary metabolites. A simple assay for assessing carbonic-anhydrase-inhibiting activity was developed. Screening of many microorganisms isolated from soil with this assay resulted in a microbial strain that produced a carbonic anhydrase inhibitor. This strain was identified as Streptomyces eurocidicus mf294. The inhibitor was isolated through 4 purification steps and identified as 2-nitroimidazole on the basis of spectroscopic data. 2-Nitroimidazole inhibited barnacle carbonic anhydrase dose-dependently and complete inhibition was reached at the concentration of 1 x 10(-5) M. 2-Nitroimidazole did not affect settlement or metamorphosis of barnacle larvae, but inhibited shell formation at concentrations higher than 1 x 10(-4) M. These findings strongly support the idea that carbonic anhydrase is involved in calcification.