Chemoenzymatic synthesis and application of glycopolymers containing multivalent sialyloligosaccharides with a poly(L-glutamic acid) backbone for inhibition of infection by influenza viruses

Highly water-soluble glycopolymers with poly(alpha-L-glutamic acid) (PGA) backbones carrying multivalent sialyl oligosaccharides units were chemoenzymatically synthesized as polymeric inhibitors of infection by human influenza viruses. p-Aminophenyl disaccharide glycosides were coupled with gamma-carboxyl groups of PGA side chains and enzymatically converted to Neu5Acalpha2-3Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-6Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-3Galbeta1-3GalNAcalpha-, and Neu5Acalpha2-3Galbeta1-3GalNAcbeta- units, respectively, by alpha2,3- or alpha2,6-sialytransferases. The glycopolymers synthesized were used for neutralization of human influenza A and B virus infection as assessed by measurement of the degree of cytopathic inhibitory effect in virus-infected MDCK cells. Among the glycopolymers tested, alpha2,6-sialo-PGA with a high molecular weight (260 kDa) most significantly inhibited infection by an influenza A virus, strain A/Memphis/1/71 (H3N2), which predominantly binds to alpha2-6 Neu5Ac residue. The alpha2,6-sialo-PGA also inhibited infection by an influenza B virus, B/Lee/40. The binding preference of viruses to terminal sialic acids was affected by core determinants of the sugar chain, Galbeta1-4GlcNAcbeta- or Galbeta1-3GalNAcalpha/beta- units. Inhibition of infection by viruses was remarkably enhanced by increasing the molecular weight and sialic acid content of glycopolymers.     

Role of nociceptin systems in learning and memory

This article summarizes our recent finding that the nociceptin system is involved in the regulation of learning and memory. The nociceptin-knockout mice show greater learning ability in the water maze task, an enhanced latent learning in the water finding task, better memory in the passive avoidance task, and further, larger long-term potentiation in the hippocampal CA1 region than wild-type mice. Nociceptin itself induces an impairment of passive avoidance task in wild-type mice, which is reversed by naloxone benzoylhydrazone (NalBzoH). Thus, the nociceptin system seems to play negative roles in learning and memory, and NalBzoH may act as a potent antagonist for the nociceptin receptor.     

Metabolism of amyloid precursor protein in COS cells transfected with a β-secretase candidate

Thimet oligopeptidase (TOP) is a thiol- andmetallo-dependent peptidase and has been shown to beone of the -secretase candidates. TOPexpressed in COS cells cleaved amyloid precursorprotein (APP) at the -secretase site, and wefound a proteolytic product of APP called secretedform of APP by -secretase (sAPP) in theconditioned media. Here we demonstrate thatsAPP was increased in conditioned media whenTOP was coexpressed in COS cells with APP and treatedwith an ADAM inhibitor SI-27. In addition, althoughTOP expressed in COS cell was localized at nuclei orGolgi apparatus, it exclusively colocalized at Golgiapparatus when APP was coexpressed with TOP.     

Inhibition of angiotensin-converting enzyme protects endothelial cell against hypoxia/reoxygenation injury

Cardiovascular tissue injury in ischemia/reperfusion has been shown to be prevented by angiotensin-converting enzyme (ACE) inhibitors. However, the mechanism on endothelial cells has not been assessed in detail. Cultured human aortic endothelial cells (HAEC) were exposed to hypoxia with or without reoxygenation. Hypoxia enhanced apoptosis along with the activation of caspase-3. Reoxygenation increased lactate dehydrogenase release time-dependently, along with an increase of intracellular oxygen radicals. ACE inhibitor quinaprilat and bradykinin significantly lessened apoptosis and lactate dehydrogenase release with these effects being diminished by a kinin B2 receptor antagonist and a nitric oxide synthase inhibitor. In conclusion, hypoxia activated the suicide pathway leading to apoptosis of HAEC by enhancing caspase-3 activity, while subsequent reoxygenation induced necrosis by enhancing oxygen radical production. Quinaprilat could ameliorate both apoptosis and necrosis through the upregulation of constitutive endothelial nitric oxide synthase via an increase of bradykinin, with the resulting increase of nitric oxide.     

Autocrine/Paracrine secretion of IL-6 family cytokines causes angiotensin II-induced delayed STAT3 activation

We recently reported that angiotensin II (AngII) biphasically activates the JAK/STAT pathway and induces delayed phosphorylation of STAT3 in the late stage (120 min) in cardiomyocytes. This study was designed to determine the mechanism of delayed phosphorylation of STAT3. Conditioned medium prepared from AngII-stimulated cardiomyocytes could reproduce the tyrosine phosphorylation of STAT3 at 5 min. This delayed phosphorylation was almost completely inhibited by anti-gp130 blocking antibody RX435, but not by TAK044 (ET-A/B-R antagonist), prazosin, or propranolol. AngII induced phosphorylation of gp130 in the late stage, which was temporally in parallel with the delayed phosphorylation of STAT3. AngII augmented IL-6, CT-1, and LIF mRNA expression at 30-60 min, but not CNTF expression. AngII increased IL-6 protein levels by 3-fold in the conditioned media at 2 h compared with the control. These findings indicated that AngII-induced delayed activation of STAT3 is caused by autocrine/paracrine secreted IL-6 family cytokines.     

Cellular internalization of arginine‐rich peptides into tobacco suspension cells: a structure–activity relationship study

Translocation of several fluorescently labeled arginine‐rich peptides into intact plant cells was quantitatively examined in order to investigate the structural factors required for efficient cellular internalization, and thereby, to evaluate the potential of arginine‐rich peptides as intracellular delivery vectors in plants. Cell‐penetrating peptides (CPPs) such as arginine‐rich peptides permit the direct introduction of biologically active macromolecules into plant cytoplasm to manipulate various intracellular processes. While a significant level of adsorption of applied arginine‐rich peptides was observed in the cell walls rich in negative charges, removal of adsorbed peptides by trypsin treatment allowed determination of the amount of internalized peptides in a quantitative manner using spectrofluorometric analysis. The internalization of arginine‐rich peptides depended on the number of arginine residues, and the peptide containing eight arginine residues showed most effective internalization. Besides, the position of small cargoes attached to the arginine‐rich peptides markedly affected the internalization efficiency. The results obtained in this study provide useful information for the development of efficient intracellular delivery tools in plant science. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Selection of Orthologous Genes for Construction of a Highly Resolved Phylogenetic Tree and Clarification of the Phylogeny of Trichosporonales Species

The order Trichosporonales (Tremellomycotina, Basidiomycota) includes various species that have clinical, agricultural and biotechnological value. Thus, understanding why and how evolutionary diversification occurred within this order is extremely important. This study clarified the phylogenetic relationships among Tricosporonales species. To select genes suitable for phylogenetic analysis, we determined the draft genomes of 17 Trichosporonales species and extracted 30 protein-coding DNA sequences (CDSs) from genomic data. The CDS regions of Trichosporon asahii and T. faecale were identified by referring to mRNA sequence data since the intron positions of the respective genes differed from those of Cryptococcus neoformans (outgroup) and are not conserved within this order. A multiple alignment of the respective gene was first constructed using the CDSs of T. asahii, T. faecale and C. neoformans, and those of other species were added and aligned based on codons. The phylogenetic trees were constructed based on each gene and a concatenated alignment. Resolution of the maximum-likelihood trees estimated from the concatenated dataset based on both nucleotide (72,531) and amino acid (24,173) sequences were greater than in previous reports. In addition, we found that several genes, such as phosphatidylinositol 3-kinase TOR1 and glutamate synthase (NADH), had good resolution in this group (even when used alone). Our study proposes a set of genes suitable for constructing a phylogenetic tree with high resolution to examine evolutionary diversification in Trichosporonales. These can also be used for epidemiological and biogeographical studies, and may also serve as the basis for a comprehensive reclassification of pleomorphic fungi.