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).     

Analysis of conformational motions and related key residue interactions responsible for a specific function of proteins with elastic network model

Protein collective motions play a critical role in many biochemical processes. How to predict the functional motions and the related key residue interactions in proteins is important for our understanding in the mechanism of the biochemical processes. Normal mode analysis (NMA) of the elastic network model (ENM) is one of the effective approaches to investigate the structure-encoded motions in proteins. However, the motion modes revealed by the conventional NMA approach do not necessarily correspond to a specific function of protein. In the present work, a new analysis method was proposed to identify the motion modes responsible for a specific function of proteins and then predict the key residue interactions involved in the functional motions by using a perturbation approach. In our method, an internal coordinate that accounts for the specific function was introduced, and the Cartesian coordinate space was transformed into the internal/Cartesian space by using linear approximation, where the introduced internal coordinate serves as one of the axes of the coordinate space. NMA of ENM in this internal/Cartesian space was performed and the function-relevant motion modes were identified according to their contributions to the specific function of proteins. Then the key residue interactions important for the functional motions of the protein were predicted as the interactions whose perturbation largely influences the fluctuation along the internal coordinate. Using our proposed methods, the maltose transporter (MalFGK₂) from E. Coli was studied. The functional motions and the key residue interactions that are related to the channel-gating function of this protein were successfully identified.     

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.     

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.     

Merkel cell tumor of the back detected during pregnancy

Merkel cell tumor is an unusual, aggressive malignancy of skin that has been considered to be derived from cutaneous Merkel cells. We are reporting a case of Merkel cell tumor overlying the left scapula with metastases to the thoracic spine and pleura. The tumor was found incidentally in a 23-year-old pregnant black woman. The tumor recurred locally 5 months after initial wide excision. Subsequently, a second wide excision of the recurrent tumor with ipsilateral axillary dissection was performed. The course of the disease was complicated by local recurrence and formation of distant metastases to pleura and spine. At the end-stage of the disease, the patient was found to have a cardiac murmur, and echocardiography revealed a mass in the anterior wall of the right ventricle that was suspicious for a metastatic lesion. The patient expired from extensive distant metastases 23 months after diagnosis.