No abstract

Proliferation of vascular smooth muscle cells (VSMC) is triggered by two types of growth factors. One activates tyrosine kinase-type receptors and the other activates G-protein-coupled receptors. We found that a conditioned medium of rat VSMC contained a growth-potentiating activity for the latter type of growth factor, and we purified a 70-kDa growth-potentiating factor (GPF) from the conditioned medium. Analyses of GPF and its cDNA revealed GPF to be a gamma-carboxyglutamic acid-containing protein encoded by a growth arrest-specific gene, gas6, which related to protein S. GPF specifically potentiated cell proliferation mediated by Ca(2+)-mobilizing receptors. The presence of a specific binding site suggests that the effect of GPF is mediated by a receptor. Thus, GPF may be a new type of extracellular factor regulating VSMC proliferation.

Traumatic brain injury (TBI) is currently a major cause of morbidity and poor quality of life in Western society, with an estimate of 2.5 million people affected per year in Europe, indicating the need for advances in TBI treatment. Within the first 24 h after TBI, several inflammatory response factors become upregulated, including the lectin galectin-3. In this study, using a controlled cortical impact (CCI) model of head injury, we show a large increase in the expression of galectin-3 in microglia and also an increase in the released form of galectin-3 in the cerebrospinal fluid (CSF) 24 h after head injury. We report that galectin-3 can bind to TLR-4, and that administration of a neutralizing antibody against galectin-3 decreases the expression of IL-1β, IL-6, TNFα and NOS2 and promotes neuroprotection in the cortical and hippocampal cell populations after head injury. Long-term analysis demonstrated a significant neuroprotection in the cortical region in the galectin-3 knockout animals in response to TBI. These results suggest that following head trauma, released galectin-3 may act as an alarmin, binding, among other proteins, to TLR-4 and promoting inflammation and neuronal loss. Taking all together, galectin-3 emerges as a clinically relevant target for TBI therapy.

Gas6 (encoded by growth-arrest-specific gene 6) is a gamma-carboxyglutamic acid (Gla)-containing protein which is released from growth-arrested vascular smooth muscle cells (VSMCs) and potentiates VSMC proliferation induced by Ca2+-mobilizing growth factors, but not that induced by receptor tyrosine kinases. In this study we examined the importance of Gla residues for the biological activities of Gas6 and tried to assess the importance of endogenous Gas6 in VSMC proliferation. We demonstrated that Gla-deficient Gas6 lacked receptor-binding and growth-potentiating activities. Therefore the vitamin K-dependent modification of Gas6 appeared to be essential for its biological activities. Next we used warfarin, an inhibitor of vitamin K-dependent gamma-carboxylation, to estimate the contribution of endogenous Gas6 to VSMC proliferation. Warfarin markedly inhibited the thrombin-induced proliferation of VSMC without affecting the mRNA or protein expression of Gas6. Therefore the inhibition seems to be due to prevention of the vitamin K-dependent modification of Gas6. However, warfarin did not affect epidermal growth factor-induced proliferation. A neutralizing antibody against Gas6 gave a similar result, i.e. it inhibited thrombin-induced VSMC proliferation but not that induced by epidermal growth factor. These results indicate that endogenously produced Gas6 is very important for VSMC proliferation induced by Ca2+-mobilizing growth factors.

No abstract

Activated microglia can phagocytose dying, stressed, or excess neurons and synapses via the phagocytic receptor Mer tyrosine kinase (MerTK). Galectin-3 (Gal-3) can cross-link surface glycoproteins by binding galactose residues that are normally hidden below terminal sialic acid residues. Gal-3 was recently reported to opsonize cells via activating MerTK. We found that LPS-activated BV-2 microglia rapidly released Gal-3, which was blocked by calcineurin inhibitors. Gal-3 bound to MerTK on microglia and to stressed PC12 (neuron-like) cells, and it increased microglial phagocytosis of PC12 cells or primary neurons, which was blocked by inhibition of MerTK. LPS-activated microglia exhibited a sialidase activity that desialylated PC12 cells and could be inhibited by Tamiflu, a neuraminidase (sialidase) inhibitor. Sialidase treatment of PC12 cells enabled Gal-3 to bind and opsonize the live cells for phagocytosis by microglia. LPS-induced microglial phagocytosis of PC12 was prevented by small interfering RNA knockdown of Gal-3 in microglia, lactose inhibition of Gal-3 binding, inhibition of neuraminidase with Tamiflu, or inhibition of MerTK by UNC569. LPS-induced phagocytosis of primary neurons by primary microglia was also blocked by inhibition of MerTK. We conclude that activated microglia release Gal-3 and a neuraminidase that desialylates microglial and PC12 surfaces, enabling Gal-3 binding to PC12 cells and their phagocytosis via MerTK. Thus, Gal-3 acts as an opsonin of desialylated surfaces, and inflammatory loss of neurons or synapses may potentially be blocked by inhibiting neuraminidases, Gal-3, or MerTK.

A system was established in which single cells differentiated to embryos at a high frequency. Small spherical single cells from a carrot (Daucus carota L. cv "Kurodagosun") cell suspension culture were obtained by fractionation through sieving, using nylon screens and then density gradient centrifugation in Percoll solutions. Eighty-five to 90% of these small single cells differentiated to embryos when they were cultured in a medium containing 2,4-dichlorophenoxyacetic acid (5 x 10' molar), zeatin (10-' molar), and mannitol (0.2 molar) for 7 days, followed by transfer to a medium containing zeatin (10-' molar) but no auxin. This indicates that there are at least two phases in the differentiation of embryos from single cells. The progression of the first phase required exogenous auxin, whereas that of the second phase was inhibited by the same growth regulator. The relationship between the morphology and potency for embryogenesis from single cells was discussed. The system established here is a useful one for investigation of differentiation process from a single cell to a whole plant via embryogenesis, especially in its early stage.Somatic embryogenesis in cultured cells is a useful system for research on differentiation to a whole plant from a single cell. For biochemical investigation of somatic embryogenesis, however, a system in which embryogenesis occurs synchronously at high frequency is required. Fujimura and Komamine (3) established such a synchronous system of somatic embryogenesis in a carrot suspension culture. The method comprised fractionation of cell clusters (composed of 3-10 cells), by sieving and density gradient centrifugation, and transfer of the fractionated cell clusters to a medium containing zeatin but no auxin. In this system, more than 90% of the cell clusters synchronously differentiated to embryos, and physiological and biochemical studies of the process of embryogenesis from the cell clusters were performed using this system (4-6). However, since the initial materials used in the system were cell cultures composed of 3 to 10 cells, which differentiated to embryos in an auxin-free medium, the possibility remained that differentiation to embryos had already been determined in the initial cell clusters.In this present work, we report on the establishment of a system in which single cells selected give rise to small cell clusters which can be induced to undergo embryogenesis at high frequency in a carrot cell suspension culture. University, Sendai, 980, Japan carota L. cv "Kurodagosun") seedling. The cells were subcultured every 7 d for 6 to 12 months in a modified Lin and Staba medium (9) containing 5 x I0-M 2,4-D. The stock cell suspension culture was grown at 27C in 500-ml conical flasks on a reciprocal shaker (70 strokes/min and 5 cm amplitude) in the dark. MATERIALS AND METHODS PlantThe method of induction of embryo formation was as according to Fujimura and Komamine (3). Embryo formation was induced by transfer of the cell clusters to a medium containing 10-7 M zeatin but n...

. (2017) Structural analysis of MDM2 RING separates degradation from regulation of p53 transcription activity. Nature Structural and Molecular Biology, 24(7), pp. 578-587. (doi:10.1038/nsmb.3414) This is the author's final accepted version.There may be differences between this version and the published version.