A novel Netrin-1–sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching

Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1–sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.     

Halocynthiibacter namhaensis gen. nov., sp. nov., a novel alphaproteobacterium isolated from sea squirt Halocynthia roretzi

A Gram-negative, non-motile and rod-shaped bacterial strain, designated RA2-3^T, was isolated from a sea squirt (Halocynthia roretzi) collected from the South Sea, Korea, and its taxonomic position was investigated by using a polyphasic approach. Strain RA2-3^T was observed to grow optimally at 25 °C, at pH 7.0–7.5 and in the presence of 2 % (w/v) NaCl. Strain RA2-3^T exhibited the highest 16S rRNA gene sequence similarity values to the type strains of Litoreibacter meonggei (95.7 %), Planktotalea frisia (95.6 %), Thalassobius gelatinovorus (95.5 %) and Pelagicola litoralis (95.4 %). A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain RA2-3^T clustered with the type strains of Planktotalea frisia, Pelagicola litoralis, Pacificibacter maritimus and Roseovarius marinus. Strain RA2-3^T was found to contain Q-10 as the predominant ubiquinone and C_18:1 ω7c as the major fatty acid. The major polar lipids detected in strain RA2-3^T were phosphatidylcholine, phosphatidylglycerol, one unidentified aminolipid and one unidentified lipid. The DNA G+C content of strain RA2-3^T was 52.9 mol%. On the basis of the phylogenetic, chemotaxonomic and phenotypic properties, strain RA2-3^T is considered to represent a new genus and species within the family Rhodobacteraceae, for which the name Halocynthiibacter namhaensis gen. nov., sp. nov. is proposed. The type strain of H. namhaensis is RA2-3^T (=KCTC 32362^T=NBRC 109999^T).     

Distinctive roles of receptor‐interacting protein kinases 1 and 3 in caspase‐independent cell death of L929

Upon tumour necrosis factor alpha (TNFα) stimulation, cells respond actively by way of cell survival, apoptosis or programmed necrosis. The receptor‐interacting proteins 1 (RIP1) and 3 (RIP3) are responsible for TNFα‐mediated programmed necrosis. To delineate the differential contributions of RIP3 and RIP1 to programmed necrosis, L929 cells were stimulated with TNFα, carbobenzoxy‐valyl‐alanyl‐aspartyl‐[O‐methyl]‐fluoromethylketone (zVAD) or zVAD along with TNFα following RNA interference against RIP1 and RIP3, respectively. RIP1 silencing did not protect cells from TNFα‐mediated cell death, while RIP3 down‐regulation made them refractory to TNFα. The heat shock protein 90 inhibitor geldanamycin (GA) down‐regulated both RIP1 and RIP3 expression, which rendered cells resistant to zVAD/TNFα‐mediated cell death but not to TNFα‐mediated cell death alone. Therefore, the protective effect of GA on zVAD/TNFα‐stimulated necrosis might be attributed to RIP3, not RIP1, down‐regulation. Pretreatment of L929 cells with rapamycin mitigated zVAD‐mediated cell death, while the autophagy inhibitor chloroquine did not affect necrotic cell death. Meanwhile, necrotic cell death by zVAD and TNFα was caused by reactive oxygen species generation and effectively diminished by lipid‐soluble butylated hydroxyanisole. Taken together, the results indicate that RIP1 and RIP3 can independently mediate death signals being transduced by two different death stimuli, zVAD and TNFα. Copyright © 2013 John Wiley & Sons, Ltd.     

2-(Trimethylammonium) Ethyl (R)-3-Methoxy-3-oxo-2-Stearamidopropyl Phosphate Suppresses Osteoclast Maturation and Bone Resorption by Targeting Macrophage-Colony Stimulating Factor Signaling

2-(Trimethylammonium) ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate [(R)-TEMOSPho], a derivative of an organic chemical identified from a natural product library, promotes highly efficient megakaryopoiesis. Here, we show that (R)-TEMOSPho blocks osteoclast maturation from progenitor cells of hematopoietic origin, as well as blocking the resorptive function of mature osteoclasts. The inhibitory effect of (R)-TEMOSPho on osteoclasts was due to a disruption of the actin cytoskeleton, resulting from impaired downstream signaling of c-Fms, a receptor for macrophage-colony stimulating factor linked to c-Cbl, phosphoinositol-3-kinase (PI3K), Vav3, and Rac1. In addition, (R)-TEMOSPho blocked inflammation-induced bone destruction by reducing the numbers of osteoclasts produced in mice. Thus, (R)-TEMOSPho may represent a promising new class of antiresorptive drugs for the treatment of bone loss associated with increased osteoclast maturation and activity.     

Dynamic changes of microtubule and actin structures in CV-1 cells during electrofusion

To study the involvement of the cytoskeletal system in the fusion of animal cells, we examined the dynamic changes of cytoskeletal proteins during the various stages of cell fusion. CV-1 cells were fused by applying a radio-frequency electrical pulse. Structural changes of microtubules (MTs) and F-actin were monitored simultaneously by double-label fluorescence microscopy. It was observed that in a few minutes after the initiation of cell fusion, MT bundles began to extend into the cytoplasmic bridges which were formed by fusing the membranes of neighboring cells. Later, a network of parallel MT bundles appeared between the adjacent nuclei of the fusing cells; such MT bundles may provide the mechanical links that are responsible for nuclear aggregation. The structural changes of F-actin during cell fusion were more complicated. We observed many different patterns of actin distribution in the fusing cells, including some giant, ring-shaped structures. Reorganization of actin is unlikely to be involved in the nuclear aggregation process. Instead, actin bundles condensed at the cell edges may help to widen the cytoplasmic bridges to allow merging of cellular contents between the fusing cells.     

Effects of hyperthermia and photodynamic therapy on BALB/c mice bearing subcutaneous tumors

The therapeutic effects of photodynamic therapy and hyperthermia on mice bearing subcutaneous tumors were investigated. Ehrlich ascites tumor cells (1 x 10(7)) were implanted subcutaneously into the femoral area of BALB/c mice. A total of 134 tumor-bearing mice were treated with photodynamic therapy, i.e., administration of laser irradiation (514.5 nm, 112.5 mW/cm2 for 11.12 min with a total energy 75 J/cm2) after injection (i.p.) of hematoporphyrin derivative (HPD, 7.5 and 10.0 mg/kg body weight) and/or hyperthermia (by electric heating needles to 44 and 45 degrees C for 30 min) once a day for three successive days. The results revealed that the therapeutic effects of the combination of photodynamic therapy and hyperthermia were improved when compared with photodynamic therapy or hyperthermia alone. A combination of photodynamic therapy (10.0 mg HPD/kg body weight and 75 J/cm2 of total laser irradiation energy) and hyperthermia (44 degrees C for 30 min) had the best therapeutic effect, indicating that the mortality rate within 120 days (MR120) was 12.5% and the mean survival time (MST120) was 113.8 days.     

Post-embryonic development of the Furongian (late Cambrian) trilobite Tsinania canens: implications for life mode and phylogeny

SUMMARY The current concept of the order Asaphida was proposed to accommodate some Cambrian and Ordovician trilobite clades that are characterized by the possession of a ventral median suture. The family Tsinaniidae was recently suggested to be a member of the order Asaphida on the basis of its close morphological similarity to Asaphidae. Postembryonic development of the tsinaniid trilobite, Tsinania canens , from the Furongian (late Cambrian) Hwajeol Formation of Korea, reveals that this trilobite had an adult-like protaspis. Notable morphological changes with growth comprise the effacement of dorsal furrows, sudden degeneration of pygidial spines, regression of genal spines, and loss of a triangular rostral plate to form a ventral median suture. Programmed cell death may be responsible for degenerating the pygidial and genal spines during ontogeny. Morphological changes with growth, such as the loss of pygidial spines, modification of pleural tips, and effacement of dorsal furrows, suggest that T. canens changed its life mode during ontogeny from benthic crawling to infaunal. The protaspid morphology and the immature morphology of T. canens retaining genal and pygidial spines suggest that tsinaniids bear a close affinity to leiostegioids of the order Corynexochida. Accordingly, development of a ventral median suture in T. canens demonstrates that the ventral median suture could have evolved polyphyletically, and thus the current concept of the order Asaphida needs to be revised.