Rab5-mediated endosome formation is regulated at the trans-Golgi network

Early endosomes, also called sorting endosomes, are known to mature into late endosomesvia the Rab5-mediated endolysosomal trafficking pathway. Thus, early endosome existence isthought to be maintained by the continual fusion of transport vesicles from the plasmamembrane and thetrans-Golgi network (TGN). Here we show instead that endocytosis isdispensable and post-Golgi vesicle transport is crucial for the formation of endosomes andthe subsequent endolysosomal traffic regulated by yeast Rab5 Vps21p. Fittingly, all threeproteins required for endosomal nucleotide exchange on Vps21p arefirst recruited to theTGN before transport to the endosome, namely the GEF Vps9p and the epsin-relatedadaptors Ent3/5p. The TGN recruitment of these components is distinctly controlled, withVps9p appearing to require the Arf1p GTPase, and the Rab11s, Ypt31p/32p. These resultsprovide a different view of endosome formation and identify the TGN as a critical location forregulating progress through the endolysosomal trafficking pathway

Structure and expression of 12-oxophytodienoate reductase (OPR) subgroup I gene in pea and oxidoreductase activity of their recombinant proteins

Recently, we observed that expression of a pea gene (S64) encoding an oxophytodienoic acid reductase (OPR) was induced by a suppressor of pea defense responses, secreted by the pea pathogen Mycosphaerella pinodes. Because it is known that OPRs are usually encoded by families of homologous genes, we screened for genomic and cDNA clones encoding members of this putative OPR family in pea. We isolated five members of the OPR gene family from a pea genomic DNA library, and amplified six cDNA clones, including S64, by RT-PCR (reverse transcriptase-PCR). Sequencing analysis revealed that S64 corresponds to PsOPR2, and the amino acid sequences of the predicted products of the six OPR-like genes shared more than 80% identity with each other. Based on their sequence similarity, all these OPR-like genes code for OPRs of subgroup I, i.e., enzymes which are not required for jasmonic acid biosynthesis. However, the genes varied in their exon/intron organization and in their promoter sequences. To investigate the expression of each individual OPR-like gene, RT-PCR was performed using gene-specific primers. The results indicated that the OPR-like gene most strongly induced by the inoculation of pea plants with a compatible pathogen and by treatment with the suppressor from M. pinodes was PsOPR2. Furthermore, the ability of the six recombinant OPR-like proteins to reduce a model substrate, 2-cyclohexen-1-one (2-CyHE), was investigated. The results indicated that PsOPR1, 4 and 6 display robust activity, and PsOPR2 has a most remarkable ability to reduce 2-CyHE, whereas PsOPR3 has little and PsOPR5 does not reduce this compound. Thus, the six OPR-like proteins can be classified into four types. Interestingly, the gene structures, expression profiles, and enzymatic activities used to classify each member of the pea OPR-like gene family are clearly correlated, indicating that each member of this OPR-like family has a distinct function.</p

Cofilin Phosphorylation by Protein Kinase Testicular Protein Kinase 1 and Its Role in Integrin-mediated Actin Reorganization and Focal Adhesion Formation

科研費報告書収録論文(課題番号:11480213・基盤研究(B)(2)・H11～H12/研究代表者:水野, 健作/LIMキナーゼによるアクチン細胞骨格の制御機構の解明

Controlling the XUV Transparency of Helium Using Two-Pathway Quantum Interference

Atoms irradiated with combined femtosecond laser and extreme ultraviolet (XUV) fields ionize through multiphoton processes, even when the energy of the XUV photon is below the ionization potential. However, in the presence of two different XUV photons and an intense laser field, it is possible to induce full electromagnetic transparency. Taking helium as an example, the laser field modifies its electronic structure, while the presence of two different XUV photons and the laser field leads to two distinct ionization pathways that can interfere destructively. This work demonstrates a new approach for coherent control in a regime of highly excited states and strong optical fields

A Self-Consistent Model for Positronium Formation from Helium Atoms

The differential and total cross sections for electron capture by positrons from helium atoms are calculated using a first-order distorted wave theory satisfying the Coulomb boundary conditions. In this formalism a parametric potential is used to describe the electron screening in a consistent and realistic manner. The present procedure is self consistent because (i) it satisfies the correct boundary conditions and post-prior symmetry, and (ii) the potential and the electron binding energies appearing in the transition amplitude are consistent with the wave functions describing the collision system. The results are compared with the other theories and with the available experimental measurements. At the considered range of collision energies, the results agree reasonably well with recent experiments and theories. [Note: This paper will be published on volume 42 of the Brazilian Journal of Physics

Direct Visualization of Laser-Driven Electron Multiple Scattering and Tunneling Distance in Strong-Field Ionization

Using a simple model of strong-field ionization of atoms that generalizes the well-known 3-step model from 1D to 3D, we show that the experimental photoelectron angular distributions resulting from laser ionization of xenon and argon display prominent structures that correspond to electrons that pass by their parent ion more than once before strongly scattering. The shape of these structures can be associated with the specific number of times the electron is driven past its parent ion in the laser field before scattering. Furthermore, a careful analysis of the cutoff energy of the structures allows us to experimentally measure the distance between the electron and ion at the moment of tunnel ionization. This work provides new physical insight into how atoms ionize in strong laser fields and has implications for further efforts to extract atomic and molecular dynamics from strong-field physics

Characterization of recombinant β-fructofuranosidase from Bifidobacterium adolescentis G1

<p>Abstract</p> <p>Background</p> <p>We have previously reported on purification and characterization of β-fructofuranosidase (β-FFase) from <it>Bifidobacterium adolescentis </it>G1. This enzyme showed high activity of hydrolysis on fructo-oligosaccharides with a low degree of polymerization. Recently, genome sequences of <it>B. longum </it>NCC2705 and <it>B. adolescentis </it>ATCC 15703 were determined, and <it>cscA </it>gene in the both genome sequences encoding β-FFase was predicted. Here, cloning of <it>cscA </it>gene encoding putative β-FFase from <it>B. adolescentis </it>G1, its expression in <it>E. coli </it>and properties of the recombinant protein are described.</p> <p>Results</p> <p>Using the information of <it>cscA </it>gene from <it>Bifidobacterium adolescentis </it>ATCC 15703, <it>cscA </it>gene from <it>B. adolescentis </it>G1 was cloned and sequenced. The N-terminal amino acid sequence of purified β-FFase from <it>B. adolescentis </it>G1 was identical to the deduced amino acid sequences of <it>cscA </it>gene from <it>B. adolescentis </it>G1. To confirm the translated product of the <it>cscA </it>gene, the recombinant protein was expressed in <it>Escherichia coli</it>. Molecular mass of the purified recombinant enzyme was estimated to be about 66,000 by SDS-PAGE and 60,300 by MALDI TOF-MS. The optimum pH of the enzyme was 5.7 and the enzyme was stable at pH 5.0-8.6. The thermostability of the enzyme was up to 50°C. The <it>K</it>_m(mM), <it>V</it>_max(μmol/mg of protein/min), <it>k</it>₀(sec^-1) and <it>k</it>₀/<it>K</it>_m(mM^-1sec^-1) for 1-kestose, neokestose, nystose, fructosylnystose, sucrose and inulin were 1.7, 107, 107.5, 63.2, and 1.7, 142, 142.7, 83.9, and 3.9, 152, 152.8, 39.2, and 2.2, 75, 75.4, 34.3, and 38, 79, 79.4, 2.1, and 25.9, 77, 77.4, 3.0, respectively. The hydrolytic activity was strongly inhibited by AgNO₃, SDS, and HgCl₂.</p> <p>Conclusion</p> <p>The recombinant enzyme had similar specificity to the native enzyme, high affinity for 1-kestose, and low affinity for sucrose and inulin, although properties of the recombinant enzyme showed slight difference from those of the native one previously described.</p