Quinotrierixin inhibited ER stress-induced XBP1 mRNA splicing through inhibition of protein synthesis

Quinotrierixin was isolated from microbes as an inhibitor of ER stress-induced XBP1 mRNA splicing, but its mode of action was unclear. We found that quinotrierixin is an inhibitor of protein synthesis, and that the required dose range of quinotrierixin to inhibit ER stress-induced XBP1 mRNA splicing was similar to that to inhibit protein synthesis. Furthermore, we also found that quinotrierixin inhibited the ER stress-induced increases of unfolded protein response-related genes such as GRP78, CHOP, EDEM, ERdj4, and p58(IPK). Thus, we showed that quinotrierixin inhibited the ER stress-induced unfolded protein response, possibly due to its inhibitory activity of protein synthesis.     

Re-examination of ultrastructures of the stellate chloroplast organization in brown algae: Structure and development of pyrenoids

Some taxa of brown algae have a so‐called ‘stellate’ chloroplast arrangement composed of multiple chloroplasts arranged in a stellate configuration, or else a single chloroplast with radiating lobes. The fine structures of chloroplasts and pyrenoids have been studied, but the details of their membrane configurations as well as pyrenoid ontogeny have not been well understood. The ultrastructure of the single stellate chloroplast in Splachnidium rugosum and Scytothamnus australis were re‐examined in the present study, as well as the stellate arrangement of chloroplasts in Asteronema ferruginea and Asterocladon interjectum, using freeze‐substitution fixation. It was confirmed that the chloroplast envelope invaginated into the pyrenoid in Splachnidium rugosum, Scytothamnus australis and Asteronema ferruginea, but chloroplast endoplasmic reticulum (CER) remained on the surface of the chloroplast. The space between the invaginated chloroplast envelope and CER was filled with electron‐dense material. In Asteronema ferruginea, CER surrounding each pyrenoid was closely appressed to the neighboring CER over the pyrenoids, so that the chloroplasts formed a stellate configuration; however, in the apical cells chloroplasts formed two or more loose groups, or were completely dispersed. The pyrenoids of Asterocladon interjectum did not have any invagination of the chloroplast envelope, but a unique membranous sac surrounded the pyrenoid complex and occasionally other organelles (e.g. mitochondria). Immunolocalization of β‐1,3‐glucans showed that the membranous sac in Asterocladon interjectum did not contain photosynthetic products such as chrysolaminaran. Observations in the dividing cells of Splachnidium rugosum and Scytothamnus australis indicated that the pyrenoid in the center of the chloroplast enlarged and divided into two before or during chloroplast division.     

Mutational analysis of predicted extracellular domains of human growth hormone secretagogue receptor 1a

The Class A family of guanine nucleotide-binding protein (G protein)-coupled receptors that includes receptors for motilin, ghrelin, and growth hormone secretagogue (GHS) has substantial potential importance as drug targets. Understanding of the molecular basis of hormone binding and receptor activation should provide insights helpful in the development of such drugs. We previously reported that Cys residues and the perimembranous residues in the extracellular loops and amino-terminal tail of the motilin receptor are critical for peptide ligand, motilin, binding and biological activity. In the current work, we focused on the predicted extracellular domains of the human GHS receptor 1a, and identified functionally important residues by using sequential deletions ranging from one to twelve amino acid residues and site-directed replacement mutagenesis approach. Each construct was transiently expressed in COS cells, and characterized for ghrelin- and growth hormone releasing peptide (GHRP)-6-stimulated intracellular calcium responses and ghrelin radioligand binding. Cys residues in positions 116 and 198 in the first and second extracellular loops and the perimembranous Glu¹⁸⁷ residue in the second extracellular loop were critical for ghrelin and GHRP-6 biological activity. These results suggest that Cys residues in the extracellular domains in this family of Class A G protein-coupled receptor is likely involved in the highly conserved and functionally important disulfide bond, and that the perimembranous residues contribute peptide ligand binding and signaling.     

Detection of antibodies to Neospora caninum in cattle by enzyme-linked immunosorbent assay with truncated NcSRS2 expressed in Escherichia coli

The surface antigen 1-related sequence 2 of Neospora caninum (NcSRS2) is considered as an immunodominant antigen. In this study, the gene encoding truncated NcSRS2 (NcSRS2t) lacking an N-terminal signal peptide and C-terminal hydrophobic regions was expressed in Escherichia coli, and its diagnostic potential in an enzyme-linked immunosorbent assay (ELISA) was evaluated. ELISA could discriminate clearly between known N. caninum-positive and -negative sera from cattle. Field serum samples collected from cattle in Brazil were examined for the diagnosis of N. caninum infection using ELISA. Of the 197 samples analyzed, 64 (32.5%) samples were positive for antibodies to N. caninum. Of the 64 ELISA-positive samples, 58 (90.6%) were confirmed as positive by Western blot analysis with whole-parasite antigens. These results suggest that ELISA with recombinant NcSRS2t is an effective method for diagnosis of N. caninum infection in cattle.     

Degradation of DNA into 5'-monodeoxyribonucleotides in the presence of Mn(2+) ions

DNA is known to be aggregated by metal ions including Mn(2+) ions, but analysis of the aggregation process from a chemical viewpoint, which means identification of the product yielded during the process, has not been performed yet. On examination of the kinds of degraded materials that were in the supernatant obtained on centrifugation of a DNA mixture aggregated under conditions of 10 mM Mn(2+) ions ([Mn]/[P] = 46.3) at 70 degrees C for 1 h, the degradation products were found to be dAMP, dCMP, dGMP, and TMP. These dNMPs were purified by HPLC on TSKgel ODS-80Ts and identified by LC-TOF/MS. The degradation activity was lost on pretreatment of the DNA with a phenol-chloroform mixture, and the activity was recovered by pretreatment with a mixture of DMSO and a buffer containing surfactants. Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), and Cd(2+), as transition element metal ions, were effective as to the degradation into dNMP. Mg(2+), Ca(2+), Sr(2+), and Ba(2+), as alkali earth element metal ions, were not effective as to the degradation. Monovalent anions such as Cl(-), CH(3)OO(-), and NO(3)(-) were found to increase the degradation rate. Sixty mug of the 120 mug of the starting DNA in 450 mul was degraded into dNMP on reaction for 1 h in the presence of 100 mM NaCl and 10 mM Mn(2+) ions. In this process, aggregation did not occur, and thus was not considered to be necessary for degradation. The degradation was found not to occur at pH 7.0, and to be very sensitive to pH. The OH(-) ion should have a critical role in cleavage of the phosphodiester linkages in this case. The dNMP obtained in the degradation process was found to be only 5'-NMP, based on the H(1)NMR spectra. This prosess should prove to be a new process for the production of 5'-dNMP in addtion to the exonuclease.     

The HA and NS Genes of Human H5N1 Influenza A Virus Contribute to High Virulence in Ferrets

Highly pathogenic H5N1 influenza A viruses have spread across Asia, Europe, and Africa. More than 500 cases of H5N1 virus infection in humans, with a high lethality rate, have been reported. To understand the molecular basis for the high virulence of H5N1 viruses in mammals, we tested the virulence in ferrets of several H5N1 viruses isolated from humans and found A/Vietnam/UT3062/04 (UT3062) to be the most virulent and A/Vietnam/UT3028/03 (UT3028) to be avirulent in this animal model. We then generated a series of reassortant viruses between the two viruses and assessed their virulence in ferrets. All of the viruses that possessed both the UT3062 hemagglutinin (HA) and nonstructural protein (NS) genes were highly virulent. By contrast, all those possessing the UT3028 HA or NS genes were attenuated in ferrets. These results demonstrate that the HA and NS genes are responsible for the difference in virulence in ferrets between the two viruses. Amino acid differences were identified at position 134 of HA, at positions 200 and 205 of NS1, and at positions 47 and 51 of NS2. We found that the residue at position 134 of HA alters the receptor-binding property of the virus, as measured by viral elution from erythrocytes. Further, both of the residues at positions 200 and 205 of NS1 contributed to enhanced type I interferon (IFN) antagonistic activity. These findings further our understanding of the determinants of pathogenicity of H5N1 viruses in mammals.     

Prevention of experimental autoimmune encephalomyelitis by transfer of embryonic stem cell-derived dendritic cells expressing myelin oligodendrocyte glycoprotein peptide along with TRAIL or programmed death-1 ligand

Experimental autoimmune encephalomyelitis (EAE) is caused by activation of myelin Ag-reactive CD4+ T cells. In the current study, we tested a strategy to prevent EAE by pretreatment of mice with genetically modified dendritic cells (DC) presenting myelin oligodendrocyte glycoprotein (MOG) peptide in the context of MHC class II molecules and simultaneously expressing TRAIL or Programmed Death-1 ligand (PD-L1). For genetic modification of DC, we used a recently established method to generate DC from mouse embryonic stem cells (ES cells) in vitro (ES-DC). ES cells were sequentially transfected with an expression vector for TRAIL or PD-L1 and an MHC class II-associated invariant chain-based MOG epitope-presenting vector. Subsequently, double-transfectant ES cell clones were induced to differentiate to ES-DC, which expressed the products of introduced genes. Treatment of mice with either of the double-transfectant ES-DC significantly reduced T cell response to MOG, cell infiltration into spinal cord, and the severity of MOG peptide-induced EAE. In contrast, treatment with ES-DC expressing MOG alone, irrelevant Ag (OVA) plus TRAIL, or OVA plus PD-L1, or coinjection with ES-DC expressing MOG plus ES-DC-expressing TRAIL or PD-L1 had no effect in reducing the disease severity. In contrast, immune response to irrelevant exogenous Ag (keyhole limpet hemocyanin) was not impaired by treatment with any of the genetically modified ES-DC. The double-transfectant ES-DC presenting Ag and simultaneously expressing immune-suppressive molecules may well prove to be an effective therapy for autoimmune diseases without inhibition of the immune response to irrelevant Ag.     

Three new species of bipolar budding yeasts of the genus Hanseniaspora and its anamorph Kloeckera isolated in Thailand

In the course of a survey of yeast biodiversity in the natural substrates in Thailand, eight strains were found to represent three hitherto undescribed species of Hanseniaspora/Kloeckera . They were isolated from insect frass, flower, lichen, rotted fruit and rotted wood. Based on the morphological and physiological characteristics, and sequences of D1/D2 domain, six strains represent a single species of the genus Hanseniaspora , described as Hanseniaspora thailandica sp. nov. (type BCC 14938^T=NBRC 104216^T=CBS 10841^T), and another strain as Hanseniaspora singularis sp. nov. (type BCC 15001^T=NBRC 104214^T=CBS 10840^T). A further strain, which belongs to Kloeckera and does not produce ascospores, is described as Kloeckera hatyaiensis sp. nov. (type BCC 14939^T=NBRC 104215^T=CBS 10842^T). Strains belonging to H. thailandica sp. nov. differed by 17–19 nucleotide substitutions from Hanseniaspora meyeri , the closest species. DNA reassociation between the two taxa showed 30–48% relatedness. Kloeckera hatyaiensis sp. nov. and H. singularis sp. nov. differed by eight and 16 nucleotide substitutions with one gap from the nearest species, Hanseniaspora clermontiae and Hanseniaspora valbyensis , respectively.     

A Mass Conserved Reaction–Diffusion System Captures Properties of Cell Polarity

Cell polarity is a general cellular process that can be seen in various cell types such as migrating neutrophils and Dictyostelium cells. The Rho small GTP(guanosine 5′-tri phosphate)ases have been shown to regulate cell polarity; however, its mechanism of emergence has yet to be clarified. We first developed a reaction–diffusion model of the Rho GTPases, which exhibits switch-like reversible response to a gradient of extracellular signals, exclusive accumulation of Cdc42 and Rac, or RhoA at the maximal or minimal intensity of the signal, respectively, and tracking of changes of a signal gradient by the polarized peak. The previous cell polarity models proposed by Subramanian and Narang show similar behaviors to our Rho GTPase model, despite the difference in molecular networks. This led us to compare these models, and we found that these models commonly share instability and a mass conservation of components. Based on these common properties, we developed conceptual models of a mass conserved reaction–diffusion system with diffusion–driven instability. These conceptual models retained similar behaviors of cell polarity in the Rho GTPase model. Using these models, we numerically and analytically found that multiple polarized peaks are unstable, resulting in a single stable peak (uniqueness of axis), and that sensitivity toward changes of a signal gradient is specifically restricted at the polarized peak (localized sensitivity). Although molecular networks may differ from one cell type to another, the behaviors of cell polarity in migrating cells seem similar, suggesting that there should be a fundamental principle. Thus, we propose that a mass conserved reaction–diffusion system with diffusion-driven instability is one of such principles of cell polarity.