Synthesis, gene-silencing activity and nuclease resistance of 3'-3'-linked double short hairpin RNA

To improve the nuclease resistance of siRNA while reducing its induction of an innate immune response and maintaining its biological activity for possible therapeutic application, we designed and synthesized a series of double short hairpin RNAs (dshRNAs). Each dshRNA consisted of two identical short hairpin RNAs (shRNAs) linked at their 3' ends by glycerol. The dshRNAs were synthesized on a glycerol-derivatized solid support from amidites with 2-cyanoethoxymethyl (CEM) as the 2'-hydroxyl protecting group. Synthesis was carried out in a single run on a DNA/RNA synthesizer, without the need for enzymatic ligation. The dshRNAs showed structure-dependent gene-silencing activity at the protein level, and dshRNAs in which the 3' end of the two sense regions were linked showed especially high activity. Inclusion of 2'-O-methyluridine residues in the loop region was associated with 1.6- to 2.4-fold lower induction of interferon-α than was siRNA, without loss of gene-silencing activity. dshRNA also showed higher exonuclease resistance than siRNA or canonical shRNA. Our studies provide a new approach to gene silencing based on the concept of linking the 3' end of the sense regions of two shRNA molecules to form a double shRNA.     

A CD36-related Transmembrane Protein Is Coordinated with an Intracellular Lipid-binding Protein in Selective Carotenoid Transport for Cocoon Coloration

The transport pathway of specific dietary carotenoids from the midgut lumen to the silk gland in the silkworm, Bombyx mori, is a model system for selective carotenoid transport because several genetic mutants with defects in parts of this pathway have been identified that manifest altered cocoon pigmentation. In the wild-type silkworm, which has both genes, Yellow blood (Y) and Yellow cocoon (C), lutein is transferred selectively from the hemolymph lipoprotein to the silk gland cells where it is accumulated into the cocoon. The Y gene encodes an intracellular carotenoid-binding protein (CBP) containing a lipid-binding domain known as the steroidogenic acute regulatory protein-related lipid transfer domain. Positional cloning and transgenic rescue experiments revealed that the C gene encodes Cameo2, a transmembrane protein gene belonging to the CD36 family genes, some of which, such as the mammalian SR-BI and the fruit fly ninaD, are reported as lipoprotein receptors or implicated in carotenoid transport for visual system. In C mutant larvae, Cameo2 expression was strongly repressed in the silk gland in a specific manner, resulting in colorless silk glands and white cocoons. The developmental profile of Cameo2 expression, CBP expression, and lutein pigmentation in the silk gland of the yellow cocoon strain were correlated. We hypothesize that selective delivery of lutein to specific tissue requires the combination of two components: 1) CBP as a carotenoid transporter in cytosol and 2) Cameo2 as a transmembrane receptor on the surface of the cells.     

Isolation of porphyran-degrading marine microorganisms from the surface of red alga, Porphyra yezoensis

Marine microorganisms degrading porphyran (POR) were found on the surface of thalli of Porphyra yezoensis. Fifteen crude microorganism groups softened and liquefied the surface of agar-rich plate medium. Among these, 11 microorganism groups degraded porphyran that consisted of sulfated polysaccharide in Porphyra yezoensis. Following isolation, 7 POR-degradable microorganisms were isolated from the 11 POR-degradable microorganism groups.     

The crystal structure of the apoenzyme of the iron-sulphur cluster-free hydrogenase

The iron-sulphur cluster-free hydrogenase (Hmd, EC 1.12.98.2) from methanogenic archaea is a novel type of hydrogenase that tightly binds an iron-containing cofactor. The iron is coordinated by two CO molecules, one sulphur and a pyridone derivative, which is linked via a phosphodiester bond to a guanosine base. We report here on the crystal structure of the Hmd apoenzyme from Methanocaldococcus jannaschii at 1.75 A and from Methanopyrus kandleri at 2.4 A resolution. Homodimeric Hmd reveals a unique architecture composed of one central and two identical peripheral globular units. The central unit is composed of the intertwined C-terminal segments of both subunits, forming a novel intersubunit fold. The two peripheral units consist of the N-terminal domain of each subunit. The Rossmann fold-like structure of the N-terminal domain contains a mononucleotide-binding site, which could harbour the GMP moiety of the cofactor. Another binding site for the iron-containing cofactor is most probably Cys176, which is located at the bottom of a deep intersubunit cleft and which has been shown to be essential for enzyme activity. Adjacent to the iron of the cofactor modelled as a ligand to Cys176, an extended U-shaped extra electron density, interpreted as a polyethyleneglycol fragment, suggests a binding site for the substrate methenyltetrahydromethanopterin.     

Enhanced accumulation of adipocytes in bone marrow stromal cells in the presence of increased extracellular and intracellular [Ca²⁺

The bone marrow stroma contains osteoblasts and adipocytes that have a common precursor: the pluripotent mesenchymal stem cell found in bone marrow stromal cells (BMSCs). Local bone marrow Ca(2+) levels can reach high concentrations due to bone resorption, which is one of the notable features of the bone marrow stroma. Here, we describe the effects of high [Ca(2+)](o) on the accumulation of adipocytes in the bone marrow stroma. Using primary mouse BMSCs, we evaluated the level of adipocyte accumulation by measuring Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity. High [Ca(2+)](o) enhanced the accumulation of adipocytes following treatment with both insulin and dexamethasone together but not in the absence of this treatment. This enhanced accumulation was the result of both the accelerated proliferation of BMSCs and their differentiation into adipocytes. Using the fura-2 method, we also showed that high [Ca(2+)](o) induces an increase in [Ca(2+)](i). An intracellular Ca(2+) chelator suppressed the enhancement in adipocyte accumulation due to increased [Ca(2+)](o) in BMSCs. These data suggest a new role for extracellular Ca(2+) in the bone marrow stroma: increased [Ca(2+)](o) induces an increase in [Ca(2+)](i) levels, which in turn enhances the accumulation of adipocytes under certain conditions.     

Protection of bioreactor from microbial contamination by high dissolved oxygen tension

Summary Repeated batch hydrolysis of casein using immobilized protease were carried out at various do tensions and pH. At any of the pH tested, microbial contamination could be satisfactorily suppressed by maintaining high do. In continuous reaction, contamination could be suppressed throughout 40 days under the do of 190 ppm, whereas contamination was observed after 24 hours with the do of 2.5 ppm.     

Activation Process of Dipteran-Specific Insecticidal Protein Produced by Bacillus thuringiensis subsp. israelensis

Dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 130 kDa in Bacillus thuringiensis subsp. israelensis. Here we performed the in vitro processing of Cry4A and showed that the 130-kDa protoxin of Cry4A was processed into the two protease-resistant fragments of 20 and 45 kDa through the intramolecular cleavage of a 60-kDa intermediate. The processing into these two fragments was also observed in vivo. To investigate functional properties of the two fragments, GST (glutathione S-transferase) fusion proteins of the 60-kDa intermediate and the 20- and 45-kDa fragments were constructed. Neither the GST–20-kDa fusion protein (GST-20) nor the GST–45-kDa fusion protein (GST-45) was actively toxic against mosquito larvae of Culex pipiens, whereas the GST–60-kDa intermediate fusion protein (GST-60) exhibited significant toxicity. However, when the two fusion proteins GST-20 and GST-45 coexisted, significant toxicity was observed. The coprecipitation experiment demonstrated that the two fragments associated with each other. Therefore, it is strongly suggested that the two fragments formed an active complex of apparently 60 kDa. A mutant of the 60-kDa protein which was apparently resistant to the intramolecular cleavage with the midgut extract of C. pipiens larvae had toxicity slightly lower than that of GST-60.