Arf6 Guanine Nucleotide Exchange Factor Cytohesin-2 Binds to CCDC120 and Is Transported Along Neurites to Mediate Neurite Growth

The mechanism of neurite growth is complicated, involving continuous cytoskeletal rearrangement and vesicular trafficking. Cytohesin-2 is a guanine nucleotide exchange factor for Arf6, an Arf family molecular switch protein, controlling cell morphological changes such as neuritogenesis. Here, we show that cytohesin-2 binds to a protein with a previously unknown function, CCDC120, which contains three coiled-coil domains, and is transported along neurites in differentiating N1E-115 cells. Transfection of the small interfering RNA (siRNA) specific for CCDC120 into cells inhibits neurite growth and Arf6 activation. When neurites start to extend, vesicles containing CCDC120 and cytohesin-2 are transported in an anterograde manner rather than a retrograde one. As neurites continue extension, anterograde vesicle transport decreases. CCDC120 knockdown inhibits cytohesin-2 localization into vesicles containing CCDC120 and diffuses cytohesin-2 in cytoplasmic regions, illustrating that CCDC120 determines cytohesin-2 localization in growing neurites. Reintroduction of the wild type CCDC120 construct into cells transfected with CCDC120 siRNA reverses blunted neurite growth and Arf6 activity, whereas the cytohesin-2-binding CC1 region-deficient CCDC120 construct does not. Thus, cytohesin-2 is transported along neurites by vesicles containing CCDC120, and it mediates neurite growth. These results suggest a mechanism by which guanine nucleotide exchange factor for Arf6 is transported to mediate neurite growth.     

Fern gametophytes of Angiopteris lygodiifolia and Osmunda japonica harbor diverse Mucoromycotina fungi

Journal of Plant Research - Mycorrhizal symbiosis between plants and fungi is ubiquitous, and has been played key roles in plant terrestrialization and diversification. Although arbuscular...     

Formation of unidentified nitrogen in plants: an implication for a novel nitrogen metabolism

Plants take up inorganic nitrogen and store it unchanged or convert it to organic forms. The nitrogen in such organic compounds is stoichiometrically recoverable by the Kjeldahl method. The sum of inorganic nitrogen and Kjeldahl nitrogen has long been known to equal the total nitrogen in plants. However, in our attempt to study the mechanism of nitrogen dioxide (NO₂) metabolism, we unexpectedly discovered that about one-third of the total nitrogen derived from ¹⁵N-labeled NO₂ taken up by Arabidopsis thaliana (L.) Heynh. plants was converted to neither inorganic nor Kjeldahl nitrogen, but instead to an as yet unknown nitrogen compound(s). We here refer to this nitrogen as unidentified nitrogen (UN). The generality of the formation of UN across species, nitrogen sources and cultivation environments for plants has been shown as follows. Firstly, all of the other 11 plant species studied were found to form the UN in response to fumigation with ¹⁵NO₂. Secondly, tobacco (Nicotiana tabacum L.) plants fed with ¹⁵N-nitrate appeared to form the UN. And lastly, the leaves of naturally fed vegetables, grass and roadside trees were found to possess the UN. In addition, the UN appeared to comprise a substantial proportion of total nitrogen in these plant species. Collectively, all of our present findings imply that there is a novel nitrogen mechanism for the formation of UN in plants. Based on the analyses of the exhaust gas and residue fractions of the Kjeldahl digestion of a plant sample containing the UN, probable candidates for compounds that bear the UN were deduced to be those containing the heat-labile nitrogen–oxygen functions and those recalcitrant to Kjeldahl digestion, including organic nitro and nitroso compounds. We propose UN-bearing compounds may provide a chemical basis for the mechanism of the reactive nitrogen species (RNS), and thus that cross-talk may occur between UN and RNS metabolisms in plants. A mechanism for the formation of UN-bearing compounds, in which RNS are involved as intermediates, is proposed. The important broad impact of this novel nitrogen metabolism, not only on the general physiology of plants, but also on plant substances as human and animal food, and on plants as an integral part of the global environment, is discussed.Abbreviations NO Nitric oxide - NO₂ Nitrogen dioxide - RNS Reactive nitrogen species - UN Unidentified nitrogen - TNNAT, RNNAT, INNAT and UNNAT Total, Kjeldahl, inorganic and unidentified nitrogen in naturally fed plants, respectively - TNNIT, RNNIT, INNIT and UNNIT Total, Kjeldahl, inorganic and unidentified nitrogen derived from nitrate, respectively - TNNO2, RNNO2, INNO2 and UNNO2 Total, Kjeldahl, inorganic and unidentified nitrogen derived from NO₂, respectively     

Enzymatic and nonenzymatic formation of reactive oxygen species from 6-anilino-5,8-quinolinequinone

The nonenzymatic and enzymatic formation of reactive oxygen species (ROS) from LY83583 (6-anilino-5,8-quinolinequinone) was investigated by electron paramagnetic resonance (EPR) spectroscopy. In the presence of thiol compounds such as glutathione and L-cysteine, LY83583 underwent a one-electron reduction due to low redox potential (-0.3+/-0.01 V vs. SCE), followed by formation of LY83583 semiquinone anion radical. This species was characterized by EPR spectroscopy under an argon atmosphere at neutral pH. Under an aerobic condition, this species interacts with molecular oxygen to form a superoxide anion radical. GSH-conjugated LY83583 was also identified by NMR and FAB-MS. When LY83583 was applied to PC12 cells, ROS formation was completely inhibited by both the flavoenzyme inhibitor DPI and the DT-diaphorase inhibitor dicumarol. On the other hand, ROS generation occurred independent of intracellular GSH level. These results indicate that LY83583 can generate ROS both enzymatically and nonenzymatically, although the enzymatic formation is dominant over the nonenzymatic system in PC12 cells.     

Small RNA class transition from siRNA/piRNA to miRNA during pre-implantation mouse development

Recent studies showed that small interfering RNAs (siRNAs) and Piwi-interacting RNA (piRNA) in mammalian germ cells play important roles in retrotransposon silencing and gametogenesis. However, subsequent contribution of those small RNAs to early mammalian development remains poorly understood. We investigated the expression profiles of small RNAs in mouse metaphase II oocytes, 8–16-cell stage embryos, blastocysts and the pluripotent inner cell mass (ICM) using high-throughput pyrosequencing. Here, we show that during pre-implantation development a major small RNA class changes from retrotransposon-derived small RNAs containing siRNAs and piRNAs to zygotically synthesized microRNAs (miRNAs). Some siRNAs and piRNAs are transiently upregulated and directed against specific retrotransposon classes. We also identified miRNAs expression profiles characteristic of the ICM and trophectoderm (TE) cells. Taken together, our current study reveals a major reprogramming of functional small RNAs during early mouse development from oocyte to blastocyst.     

Modulation of rat cecal microbiota by administration of raffinose and encapsulated Bifidobacterium breve

To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192T cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.     

The human megakaryocytic cell line UT‐7/TPO expresses functional platelet agonist signals mediated through GPVI and thromboxane receptor

We have demonstrated that a unique megakaryocytic cell line UT‐7/TPO could respond to one of the primary platelet signals through GP (glycoprotein) VI and a secondary signal of the AA (arachidonic acid) cascade. Unlike other megakaryocytic cell lines, UT‐7/TPO was found to express GPVI and its associate signal molecule of FcRγ (Fc receptor γ chain). When UT‐7/TPO was stimulated with the GPVI agonist convulxin, the [Ca²⁺]i (intracellular Ca²⁺) was elevated in a convulxin concentration‐dependent manner, and [Ca²⁺]i elevation was blocked by pretreatment with the Src family kinase inhibitor PP2 and the phospholipase inhibitor U73122. These results strongly indicate that endogenously expressed GPVI signal molecules are functional in UT‐7/TPO. Concerning the AA cascade, the expression of COX (cyclooxygenase)‐1 and TX (thromboxane) synthase was observed, and this cell line was able to produce TX by exogenous AA, followed by [Ca²⁺]i elevation mediated through the TX receptor. It is worth noting that convulxin stimulation did not cause TX generation, even through the GPVI pathway and the AA cascade are functional in this cell line. As there are many reports that convulxin‐stimulated platelets failed to produce TX, it is suggested that UT‐7/TPO has the same property as the platelets in regards to convulxin stimulation. Thus, UT‐7/TPO is useful for the observation of both the GPVI pathway and AA cascade without requiring either the induction of differentiation or GPVI transfection. Furthermore, this cell line provides a new tool for research on platelet activation signals.     

In Situ Microspatial Imaging Using Two-Photon and Confocal Laser Scanning Microscopy of Bacteria and Extracellular Polymeric Secretions (EPS) Within Marine Stromatolites

The combination of a hydrophilic embedding resin, Nanoplast, with fluorescent probes, and subsequent imaging using two-photon and confocal laser scanning microscopy (2P-LSM and CLSM) has allowed in imaging of the in situ microspatial arrangements of microbial cells and their extracellular polymeric secretion (EPS) within marine stromatolites. Optical sectioning by 2P-LSM and CLSM allowed imaging of endolithic cyanobacteria cells, Solentia sp., seen within carbonate sand grains. 2P-LSM allowed very clear imaging with a high resolution of bacteria using DAPI, which normally require UV excitation and reduced photo-bleaching of fluorescent probes.