Chemical synthesis of a very long oligoribonucleotide with 2-cyanoethoxymethyl (CEM) as the 2'-O-protecting group: structural identification and biological activity of a synthetic 110mer precursor-microRNA candidate

A long RNA oligomer, a 110mer with the sequence of a precursor-microRNA candidate, has been chemically synthesized in a single synthesizer run by means of standard automated phosphoramidite chemistry. The synthetic method involved the use of 2-cyanoethoxymethyl (CEM), a 2′-hydroxyl protecting group recently developed in our laboratory. We improved the methodology, introducing better coupling and capping conditions. The overall isolated yield of highly pure 110mer was 5.5%. Such a yield on a 1-μmol scale corresponds to 1mg of product and emphasizes the practicality of the CEM method for synthesizing oligomers of more than 100nt in sufficient quantity for biological research. We confirmed the identity of the 110mer by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, as well as HPLC, electrophoretic methods, and RNase-digestion experiments. The 110mer also showed sense-selective specific gene-silencing activity. As far as we know, this is the longest chemically synthesized RNA oligomer reported to date. Furthermore, the identity of the 110mer was confirmed by both physicochemical and biological methods.     

START-GAP3/DLC3 is a GAP for RhoA and Cdc42 and is localized in focal adhesions regulating cell morphology

In the human genome there are three genes encoding RhoGAPs that contain the START (steroidogenic acute regulatory protein (StAR)-related lipid transfer)—domain. START-GAP3/DLC3 is a tumor suppressor gene similar to two other human START-GAPs known as DLC1 or DLC2. Although expression of START-GAP3/DLC3 inhibits the proliferation of cancer cells, its molecular function is not well understood. In this study we carried out biochemical characterization of START-GAP3/DLC3, and explored the effects of its expression on cell morphology and intracellular localization. We found that START-GAP3/DLC3 serves as a stimulator of PLCδ1 and as a GAP for both RhoA and Cdc42 in vitro. Moreover, we found that the GAP activity is responsible for morphological changes. The intracellular localization of endogenous START-GAP3/DLC3 was explored by immunocytochemistry and was revealed in focal adhesions. These results indicate that START-GAP3/DLC3 has characteristics similar to other START-GAPs and the START-GAP family seems to share common characteristics.     

Transition of ovalbumin to thermostable structure entails conformational changes involving the reactive center loop

Ovalbumin is a serpin without inhibitory activity against proteases. During embryonic development, ovalbumin in the native (N) form undergoes changes and takes a heat-stable form, which was previously named HS-ovalbumin. It has been known that N-ovalbumin is artificially converted to another thermostable form called S-ovalbumin by heating at an alkaline pH. Here, we characterized further the three ovalbumin forms, N, HS, and S. The epitope of the monoclonal antibody 2B3/2H11, which recognizes N- and HS-ovalbumin but not S-ovalbumin, was found to reside in the region Glu-Val-Val-Gly-Ala-Ser-Glu-Ala-Gly-Val-Asp-Ala-Ala-Ser-Val-Ser-Glu-Glu-Phe-Arg, which corresponds to 340-359 of amino acid residues and is contained in the reactive center loop (RCL). Removal of RCL by elastase or subtilisin mitigated binding of the antibody. Dephosphorylation experiments indicated that the phosphorylated Ser-344 residue located on RCL is crucial for the epitope recognition. We suggest that the shift to the heat-stable form of ovalbumin accompanies a movement of RCL.     

Production of bioactive lysophosphatidic acid by lysophospholipase D in hen egg white

Lysophosphatidic acid (LPA), a lysophospholipid mediator, is produced extracellularly by lysophospholipase D (lysoPLD) secreted in several animal body fluids including blood plasma. Previously, we reported that hen egg white contains polyunsaturated fatty acid-rich LPA. In this study, we examined whether lysoPLD is involved in the production of LPA in hen egg white. LysoPLD activity was measured by determining LPA and choline by mass spectrometric and enzyme-linked fluorometric analyses, respectively. LysoPLD increased with increased dilution of egg white, indicating that one or more components of egg white strongly inhibit its lysoPLD activity. This dilution-dependent increase in the lysoPLD activity was masked by co-incubation of the egg white with lysozyme, a major protein in hen egg white. Furthermore, addition of Zn(2+), Mn(2+), Ni(2+), or Co(2+) to diluted egg white altered preference patterns of lysoPLD toward choline-containing substrates. In particular, the egg white lysoPLD activity was greatly increased when Co(2+) was added. The cation-requirement of lysoPLD activity in hen egg white resembled that of plasma autotaxin (ATX)/lysoPLD. Western blot analysis revealed that egg white contained a protein that was immunostained with anti-ATX antibody. These results suggested that LPA in hen egg white is produced from lysophospholipids, especially LPC, by the action of ATX/lysoPLD, possibly originating from hen oviduct fluid.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the dwarf pomegranate (<Emphasis Type="Italic">Punica granatum</Emphasis> L. var. <Emphasis Type="Italic">nana</Emphasis>)

The dwarf pomegranate (Punica granatum L. var. nana) is a dwarf ornamental plant that has the potential to be the model plant of perennial fruit trees because it bears fruits within 1 year of seedling. We established an Agrobacterium-mediated transformation system for the dwarf pomegranate. Adventitious shoots regenerated from leaf segments were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBin19-sgfp, which contains neomycin phosphotransferase (npt II) and green fluorescent protein (gfp) gene as a selectable and visual marker, respectively. After co-cultivation, the inoculated adventitious shoots were cut into small pieces to induce regeneration, and then selected on MS medium supplemented with 0.5 μM α-naphthaleneacetic acid (NAA), 5 μM N⁶-benzyladenine (BA), 0.3% gellan gum, 50 mg/l kanamycin, and 10 mg/l meropenem. Putative transformed shoots were regenerated after 6–8 months of selection. PCR and PCR-Southern blot analysis revealed the integration of the transgene into the plant genome. Transformants bloomed and bore fruits within 3 months of being potted, and the inheritance of the transgene was confirmed in T₁ generations. The advantage of the transformation of dwarf pomegranate was shown to be the high transformation rate. The establishment of this transformation system is invaluable for investigating fruit-tree-specific phenomena.     

Direct electrical power generation from urine, wastes and biomass with simultaneous photodecomposition and cleaning

Electric power was for the first time generated directly from urine, wastes, and biomass with simultaneous photodecomposition and cleaning by using a biophotofuel cell (BPFC) composed of a nanoporous TiO2 film semiconductor photoanode and an O2-reducing cathode. Human urine exhibited a PFC characteristics with J(sc) 0.086 mA cm(-2), Voc 0.56 V, and fill factor (FF) 0.50 under irradiation by a solar simulator with AM 1.5 G and 100 mW cm(-2) incident light intensity. Both the soluble and residual parts of waste paper partially solubilized by a H3PO4 aqueous solution were also photodecomposed with simultaneous electrical power generation. As trials of various biomass materials, Coca-Cola (to test colored sample), Japanese rice wine (to test alcohol aqueous solution), and grated radish (to test slurry state sample) also generated effectively electrical power during photodecomposition by a solar simulator.     

On-fiber display of a functional peptide at sites distant from the cell surface using a long bacterionanofiber of a trimeric autotransporter adhesin

In the cell surface display system, the distance of a surface-displayed molecule from the cell surface should influence its functionality due to the interference by other surface structures. For the purpose of developing this distance-variable surface display system, we utilized a long fibrous adhesin, Acinetobacter trimeric autotransporter adhesin (AtaA) of the strain Tol 5. We constructed His-tagged full-length and shorter AtaA fibers designed by N-terminal deletion and expressed them in the ΔataA mutant. Immunoelectron microscopy clearly showed that they formed fibers on the cell surface and the His-tag was displayed on the fiber tip located at fixed distances from the cell surface. N-terminal deletion of AtaA shortened the distance between the His-tag and the cell surface, as designed. Time-course analyses of the cell-to-Ni-Sepharose beads binding revealed that cells producing the longer fibers bound more rapidly to the beads. The His-tagged AtaA derivatives were also displayed on Escherichia coli cells, and a similar tendency was shown; the His-tag on the longer fiber was more functional than that on the shorter one. Thus, we developed an on-fiber display system of a functional peptide using a long trimeric autotransporter adhesin (TAA) fiber, which can vary the distance between the displayed molecule and the cell surface.     

Quantitative analysis of redox-sensitive proteome with DIGE and ICAT

Oxidative modifications of protein thiols are important mechanisms for regulating protein functions. The present study aimed to compare the relative effectiveness of two thiol-specific quantitative proteomic techniques, difference gel electrophoresis (DIGE) and isotope coded affinity tag (ICAT), for the discovery of redox-sensitive proteins in heart tissues. We found that these two methods were largely complementary; each could be used to reveal a set of unique redox-sensitive proteins. Some of these proteins are low-abundant signaling proteins and membrane proteins. From DIGE analysis, we found that both NF-kappaB-repressing protein and epoxide hydrolase were sensitive to H 2O 2 oxidation. In ICAT analysis, we found that specific cysteines within sacroplasmic endoplamic reticulum calcium ATPase 2 and voltage-dependent anion-selective channel protein 1 were sensitive to H 2O 2 oxidation. From these analyses, we conclude that both methods should be employed for proteome-wide studies, to maximize the possibility of identifying proteins containing redox-sensitive cysteinyl thiols in complex biological systems.     

Methyl jasmonate stimulates the formation and the accumulation of anthocyanin in <Emphasis Type="Italic">Kalanchoe blossfeldiana</Emphasis>

Methyl jasmonate (JA-Me) at concentrations of 0.1, 0.5 and 1.0 % (w/w) greatly stimulated anthocyanins accumulation in shoots of young plants of Kalanchoe blossfeldiana when it was applied around the stem as a lanolin paste. Stimulatory effect of JA-Me was evidently observed as early as two days after treatment. Anthocyanins were formed in the main and lateral shoots, including petioles, both below and above portions of the treatment. When leaves were removed from the plant, almost no anthocyanin formation was observed. It should be mentioned that leaves are necessary for the anthocyanin accumulation in stems induced by JA-Me.