Structural basis for the dual RNA-recognition modes of human Tra2-β RRM

Human Transformer2-β (hTra2-β) is an important member of the serine/arginine-rich protein family, and contains one RNA recognition motif (RRM). It controls the alternative splicing of several pre-mRNAs, including those of the calcitonin/calcitonin gene-related peptide (CGRP), the survival motor neuron 1 (SMN1) protein and the tau protein. Accordingly, the RRM of hTra2-β specifically binds to two types of RNA sequences [the CAA and (GAA)(2) sequences]. We determined the solution structure of the hTra2-β RRM (spanning residues Asn110-Thr201), which not only has a canonical RRM fold, but also an unusual alignment of the aromatic amino acids on the β-sheet surface. We then solved the complex structure of the hTra2-β RRM with the (GAA)(2) sequence, and found that the AGAA tetra-nucleotide was specifically recognized through hydrogen-bond formation with several amino acids on the N- and C-terminal extensions, as well as stacking interactions mediated by the unusually aligned aromatic rings on the β-sheet surface. Further NMR experiments revealed that the hTra2-β RRM recognizes the CAA sequence when it is integrated in the stem-loop structure. This study indicates that the hTra2-β RRM recognizes two types of RNA sequences in different RNA binding modes.     

Use of high-performance liquid chromatography for quantitative determination of the native form of the third component C3 of human complement

A high-performance liquid chromatography method is reported for the quantitative determination of the intact C3, the third component of complement, in solutions containing inactivated C3. This method is simple and far more sensitive than a conventional method using 5,5'-dithiobis(2-nitrobenzoic acid).     

Hypoosmotic Shock Induces Increases in Cytosolic Ca2+ in Tobacco Suspension-Culture Cells

Hypoosmotic shock treatment increased cytosolic Ca2+ ion concentration ([Ca2+]cyt) in tobacco (Nicotiana tabacum) suspension-culture cells. [Ca2+]cyt measurements were made by genetically transforming these cells to express apoaequorin and by reconstituting the Ca2+-dependent photoprotein, aequorin, in the cytosol by incubation with chemically synthesized coelenterazine. Measurement of Ca2+-dependent luminescence output thus allowed the direct monitoring of [Ca2+]cyt changes. When cells were added to a hypoosmotic medium, a biphasic increase in [Ca2+]cyt was observed; an immediate small elevation (phase 1) was observed first, followed by a rapid, large elevation (phase 2). Phase 1 [Ca2+]cyt was stimulated by the V-type ATPase inhibitor bafilomycin A1. Phase 2 was inhibited by the protein kinase inhibitor K-252a and required the continued presence of the hypoosmotic stimulus to maintain it. Although Ca2+ in the medium was needed to produce phase 2, it was not needed to render the cells competent to the hypoosmotic stimulus. If cells were subject to hypoosmotic shock in Ca2+- depleted medium, increases in luminescence could be induced up to 20 min after the shock by adding Ca2+ to the medium. These data suggest that hypoosmotic shock-induced [Ca2+]cyt elevation results from the activity of a Ca2+ channel in the plasma membrane or associated hypoosmotic sensing components that require Ca2+- independent phosphorylation and a continued stimulus to maintain full activity.     

Effect on the epigallocatechin gallate/epigallocatechin ratio in a green tea (Camellia sinensis L.) extract of different extraction temperatures and its effect on IgA production in mice

We found that the epigallocatechin gallate (EGCG)/epigallocatechin (EGC) ratio in a green tea (Camellia sinensis L.) extract was affected by the extraction temperature. The EGCG/EGC ratio in the 4 °C extract was around 1:3-4, whereas in the 100 °C extract, it was around 1:0.7. Oral administration of the mixture with a high EGC ratio (1:2-3 = EGCG/EGC) resulted in greater IgA production by murine Peyer's patch cells.     

Specific degradation of H. pylori urease by a catalytic antibody light chain

Catalytic antibodies capable of digesting crucial proteins of pathogenic bacteria have long been sought for potential therapeutic use. Helicobacter pylori urease plays a crucial role for the survival of this bacterium in the highly acidic conditions of human stomach. The HpU-9 monoclonal antibody (mAb) raised against H. pylori urease recognized the alpha-subunit of the urease, but only slightly recognized the beta-subunit. However, when isolated both the light and the heavy chains of this antibody were mostly bound to the beta-subunit. The cleavage reaction catalyzed by HpU-9 light chain (HpU-9-L) followed the Michaelis-Menten equation with a K(m) of 1.6 x 10(-5) m and a k(cat) of 0.11 min(-1), suggesting that the cleavage reaction was enzymatic. In a cleavage test using H. pylori urease, HpU-9-L efficiently cleaved the beta-subunit but not the alpha-subunit, indicating that the degradation by HpU-9-L had a specificity. The cleaved peptide bonds in the beta-subunit were L121-A122, E124-G125, S229-A230, Y241-D242, and M262-A263. BSA was hardly cleaved by HpU-9-L, again indicating the digestion by HpU-9-L was specific. In summary, we succeeded in the preparation of a catalytic antibody light chain capable of specifically digesting the beta-subunit of H. pylori urease.     

Atelocollagen-mediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo

Silencing gene expression by siRNAs is rapidly becoming a powerful tool for the genetic analysis of mammalian cells. However, the rapid degradation of siRNA and the limited duration of its action call for an efficient delivery technology. Accordingly, we describe here that Atelocollagen complexed with siRNA is resistant to nucleases and is efficiently transduced into cells, thereby allowing long-term gene silencing. Site-specific in vivo administration of an anti-luciferase siRNA/Atelocollagen complex reduced luciferase expression in a xenografted tumor. Furthermore, Atelocollagen-mediated transfer of siRNA in vivo showed efficient inhibition of tumor growth in an orthotopic xenograft model of a human non-seminomatous germ cell tumor. Thus, for clinical applications of siRNA, an Atelocollagen-based non-viral delivery method could be a reliable approach to achieve maximal function of siRNA in vivo.