Detection of circulating Asian H5N1 viruses by a newly established monoclonal antibody

Monoclonal antibodies (MAbs) against the recently emerged Asian H5N1 virus (A/crow/Kyoto/53/2004) were generated. From five anti-hemagglutinin (HA) MAbs, four antibodies (3C11, 4C12, 3H12, and 3H4) broadly in vitro recognized and neutralized H5 subtypes, including H5N1. By contrast, the 4G6 MAb specifically reacted with H5N1-HA and not with H5N2- or H5N3-HAs from previous epidemics. The 4G6 MAb was useful for immunofluorescence assays but not for immunoblotting, suggesting that this antibody recognizes a conformational epitope of the H5N1-HA protein. An intensive epitope-mapping analysis demonstrated that the 4G6 MAb recognizes Asp59, which is highly conserved among currently circulating H5N1 lineages. Further, a 4G6-based antigen capture enzyme-linked immunosorbent assay detected H5N1 even that derived from clade 2.2 (A/chicken/Egypt/CL-61/2007) from infected chicken lung before virus isolation. Taken together, these results suggest that the established MAbs, especially 4G6, are useful for rapid and specific detection of Asian H5N1 viruses.     

Identification of novel ribonucleo-protein complexes from the brain-specific snoRNA MBII-52

Small nucleolar RNAs (snoRNAs) guide nucleotide modifications within ribosomal RNAs or spliceosomal RNAs by base-pairing to complementary regions within their RNA targets. The brain-specific snoRNA MBII-52 lacks such a complementarity to rRNAs or snRNAs, but instead has been reported to target the serotonin receptor 2C pre-mRNA, thereby regulating pre-mRNA editing and/or alternative splicing. To understand how the MBII-52 snoRNA might be involved in these regulatory processes, we isolated the MBII-52 snoRNP from total mouse brain by an antisense RNA affinity purification approach. Surprisingly, by mass spectrometry we identified 17 novel candidates for MBII-52 snoRNA binding proteins, which previously had not been reported to be associated with canonical snoRNAs. Among these, Nucleolin and ELAVL1 proteins were confirmed to independently and directly interact with the MBII-52 snoRNA by coimmunoprecipitation. Our findings suggest that the MBII-52 snoRNA assembles into novel RNA-protein complexes, distinct from canonical snoRNPs.     

Localization of a flavonoid biosynthetic polyphenol oxidase in vacuoles

Aureusidin synthase, a polyphenol oxidase (PPO), specifically catalyzes the oxidative formation of aurones from chalcones, which are plant flavonoids, and is responsible for the yellow coloration of snapdragon (Antirrhinum majus) flowers. All known PPOs have been found to be localized in plastids, whereas flavonoid biosynthesis is thought to take place in the cytoplasm [or on the cytoplasmic surface of the endoplasmic reticulum (ER)]. However, the primary structural characteristics of aureusidin synthase and some of its molecular properties argue against localization of the enzyme in plastids and the cytoplasm. In this study, the subcellular localization of the enzyme in petal cells of the yellow snapdragon was investigated. Sucrose-density gradient and differential centrifugation analyses suggested that the enzyme (the 39-kDa mature form) is not located in plastids or on the ER. Transient assays using a green fluorescent protein (GFP) chimera fused with the putative propeptide of the PPO precursor suggested that the enzyme was localized within the vacuole lumen. We also found that the necessary information for vacuolar targeting of the PPO was encoded within the 53-residue N-terminal sequence (NTPP), but not in the C-terminal sequence of the precursor. NTPP-mediated ER-to-Golgi trafficking to vacuoles was confirmed by means of the co-expression of an NTPP-GFP chimera with a dominant negative mutant of the Arabidopsis GTPase Sar1 or with a monomeric red fluorescent protein (mRFP)-fused Golgi marker (an H+-translocating inorganic pyrophosphatase of Arabidopsis). We identified a sequence-specific vacuolar sorting determinant in the NTPP of the precursor. We have demonstrated the biosynthesis of a flavonoid skeleton in vacuoles. The findings of this metabolic compartmentation may provide a strategy for overcoming the biochemical instability of the precursor chalcones in the cytoplasm, thus leading to the efficient accumulation of aurones in the flower.     

Production of D-lactic acid from defatted rice bran by simultaneous saccharification and fermentation

Production of d-lactic acid from rice bran, one of the most abundant agricultural by-products in Japan, is studied. Lactobacillus delbrueckii subsp. delbrueckii IFO 3202 and defatted rice bran powder after squeezing rice oil were used for the production. Since the rice bran contains polysaccharides as starch and cellulose, we coupled saccharification with amylase and cellulase to lactic acid fermentation. The indigenous bacteria in the rice bran produced racemic lactic acid in the saccharification at pH 6.0-6.8. Thus the pH was controlled at 5.0 to suppress the growth of the indigenous bacteria. L. delbrueckii IFO 3202 produced 28 kgm(-3) lactic acid from 100 kgm(-3) rice bran after 36 h at 37 degrees C. The yield based on the amount of sugars soluble after 36-h hydrolysis of the bran by amylase and cellulase (36 kgm(-3) from 100 kgm(-3) of the bran) was 78%. The optical purity of produced d-lactic acid was 95% e.e.     

Newly discovered orally active pure antiestrogens

In order to develop orally active pure antiestrogens, we incorporated the carboxy-containing side chains into the 7alpha-position of the steroid scaffold and found that 17-keto derivative CH4893237 (12b) functioned as a pure antiestrogen with its oral activity much superior to clinically used pure antiestrogen, ICI182,780. Results from the pharmacokinetic evaluation indicated that the potent antiestrogen activity at oral dosing in mice attributed to both improved absorption from the intestinal wall and metabolic stability in liver.     

Pyrinadines B-G, new bis-pyridine alkaloids with an azoxy moiety from sponge Cribrochalina sp

Six new cytotoxic bis-3-alkylpyridine alkaloids with an azoxy moiety, pyrinadines B–G (1–6), have been isolated from an Okinawan marine sponge Cribrochalina sp., and the structures were elucidated by spectroscopic data and chemical means.     

An easy preparation of 'monolithic type' hydrophilic solid phase: capability for affinity resin to isolate target proteins

An easy preparation method of monolithic type hydrophilic solid phase was discussed. Newly invented functional monomer with a hydrophilic cross-linking agent was co-polymerized to realize well-controlled monolithic co-continuous structure by use of diethylene glycol as porogenic solvent. We were able to control the content of the functional monomer up to 40 vol% without loss of monolithic structure. Those prepared were utilized as affinity resins after immobilization of FK506, an immunosuppressive drug as a ligand. It was found that the affinity resins prepared were hydrophilic enough to eliminate non-specific adsorption of proteins, while two of the target proteins of FK506 tested were successfully captured.     

Preparation of antibodies against a novel immunosuppressant, FTY720, and development of an enzyme immunoassay for FTY720

FTY720 (1) is a novel immunosuppressant (immunomodulator), derived from ISP-I (2: myriocin and thermozymocidin). To clarify the pharmacokinetic properties of 1, antibodies against 1 were prepared and a competitive enzyme immunoassay (EIA) was developed. Two kinds of haptens, 3 and 4, for 1 were synthesized and coupled to ovalbumin (OVA). Rabbits were immunized with 3-OVA or 4-OVA, and corresponding antibodies were obtained. Both antibodies recognized the 2-amino-2-(2-phenylethyl)propane-1,3-diol moiety in 1. Using the anti-3-OVA antibody, a competitive EIA for 1 was developed and evaluated. The range of quantification by the EIA was 0.06-10 ng/mL. The application of the EIA has enabled us to measure the FTY720 concentration in serum after oral administration of 1 (1mg/kg) to rats.     

Vesicular storage and secretion of L-glutamate from glucagon-like peptide 1-secreting clonal intestinal L cells

Vesicular glutamate transporter (VGLUT) is responsible for the vesicular storage of l-glutamate, and plays an essential role in glutamate-mediated intercellular signal transmission in the CNS and in some neuroendocrine cells. Intestinal L cells are the glucose-responsive neuroendocrine cells responsible for the secretion of glucagon-like peptide 1 (GLP-1). We have shown that intestinal L cells express VGLUT2, a VGLUT isoform, which suggests that L cells secrete L-glutamate. In the present study, we investigated this possibility using GLUTag mouse clonal L cells. RT-PCR and northern blot analyses revealed expression of the VGLUT1 and VGLUT2 genes, but not of the VGLUT3 gene. Western blot analysis revealed immunological counterparts for VGLUT2, whereas an immunological counterpart of VGLUT1 was not detected. Indirect immunofluorescence microscopy revealed a punctate distribution of VGLUT2 immunoreactivity throughout the cells, which co-localized with GLP-1. Double-labeling immunoelectronmicroscopy confirmed the association of VGLUT2 with GLP-1-containing secretory granules. The membrane fraction exhibited ATP-dependent L-glutamate uptake, which was sensitive to bafilomycin A1 (a vacuolar proton ATPase inhibitor) and Evans blue (a VGLUT inhibitor) but insensitive to D,L-aspartate. Upon depolarization with KCl, GLUTag cells secreted appreciable amounts of L-glutamate and GLP-1. D-Glucose and methyl-alpha-D-glucopyranoside, stimulators of exocytosis of GLP-1, also triggered the secretion of L-glutamate. The L-glutamate secretion was partially dependent on Ca2+ and sensitive to bafilomycin A1. These results demonstrated that GLUTag cells stored L-glutamate in secretory granules and secreted it with GLP-1 by exocytosis. As GLUTag cells and intestinal L cells express kainate receptors and plasma membrane glutamate transporters, these results support the concept of L-glutamate-mediated intercellular signaling in the vicinity of intestinal L cells.     

Comprehensive trapping of polyubiquitinated proteins using the UIM peptide of ASB2a

An alternative splicing variant of E3 ubiquitin ligase ASB2, termed ASB2a, has a distinct N-terminal sequence containing a ubiquitin-interacting motif (UIM) consensus sequence. Examination of the minimal essential region for binding to polyubiquitinated proteins indicated that the UIM consensus sequence (residues 26–41) alone is not enough, and that amino acids 12–41 from the N-terminus of ASB2a is essential for binding. ASB2a(12–41) peptide was chemically synthesized and coupled to Sepharose 4B via disulfide bonds. This ASB2a(12–41) peptide-coupled affinity resin bound both K48- and K63-linked polyubiquitinated proteins in cell lysates and comprehensively captured polyubiquitinated proteins, including polyubiquitinated β-catenin, I-κB, and EGF receptor, which were eluted with 2-mercaptoethanol under non-denaturing conditions. These results indicate that this UIM affinity purification (designated as ubiquitin-trapping) is a useful method to discover polyubiquitinated proteins and their associated proteins.