Sumoylation of CCAAT/enhancer-binding protein alpha and its functional roles in hepatocyte differentiation

The sumoylation of CCAAT/enhancer-binding proteins (C/EBPs) by small ubiquitin-related modifier-1 (SUMO-1) has been reported recently. In this study, we investigated the functional role of the sumoylation of C/EBPalpha in the differentiation of hepatocytes. The amount of sumoylated C/EBPalpha gradually decreased during the differentiation, which suggests that the sumoylation is important for the control of growth/differentiation especially in the fetal liver. To analyze the function of the sumoylation of C/EBPalpha in liver-specific gene expression, we studied its effects on the expression of the albumin gene. The C/EBPalpha-mediated transactivation of the albumin gene was reduced by sumoylation of C/EBPalpha in primary fetal hepatocytes. The enhancement of C/EBPalpha-mediated transactivation by BRG1, a core subunit of the SWI/SNF chromatin remodeling complex, was hampered by sumoylation in a luciferase reporter assay. In addition, we discovered that sumoylation of C/EBPalpha blocked its inhibitory effect on cell proliferation by leading to the disruption of a proliferation-inhibitory complex because of a failure of the sumoylated C/EBPalpha to interact with BRG1. BRG1 was recruited to the dihydrofolate reductase promoter in nonproliferating C33a cells but was not detected in proliferating cells where C/EBPalpha, BRG1, and SUMO-1 were overexpressed. This result suggests that BRG1 down-regulates the expression of the dihydrofolate reductase gene. These findings provide the insight that SUMO acts as a space regulator, which affects protein-protein interactions.     

Effects of disturbances on the carbon balance of tropical peat swamp forests

Tropical peatlands have accumulated huge soil carbon over millennia. However, the carbon pool is presently disturbed on a large scale by land development and management, and consequently has become vulnerable. Peat degradation occurs most rapidly and massively in Indonesia, because of fires, drainage, and deforestation of swamp forests coexisting with tropical peat. Peat burning releases carbon dioxide (CO₂) intensively but occasionally, whereas drainage increases CO₂ emission steadily through the acceleration of aerobic peat decomposition. Therefore, tropical peatlands present the threat of switching from a carbon sink to a carbon source to the atmosphere. However, the ecosystem‐scale carbon exchange is still not known in tropical peatlands. A long‐term field experiment in Central Kalimantan, Indonesia showed that tropical peat ecosystems, including a relatively intact peat swamp forest with little drainage (UF), a drained swamp forest (DF), and a drained burnt swamp forest (DB), functioned as net carbon sources. Mean annual net ecosystem CO₂ exchange (NEE) (± a standard deviation) for 4 years from July 2004 to July 2008 was 174 ± 203, 328 ± 204 and 499 ± 72 gC m^?2 yr^?1, respectively, for the UF, DF, and DB sites. The carbon emissions increased according to disturbance degrees. We found that the carbon balance of each ecosystem was chiefly controlled by groundwater level (GWL). The NEE showed a linear relationship with GWL on an annual basis. The relationships suggest that annual CO₂ emissions increase by 79–238 gC m^?2 every 0.1 m of GWL lowering probably because of the enhancement of oxidative peat decomposition. In addition, CO₂ uptake by vegetation photosynthesis was reduced by shading due to dense smoke from peat fires ignited accidentally or for agricultural practices. Our results may indicate that tropical peatland ecosystems are no longer a carbon sink under the pressure of human activities.     

Cerebellar globular cells receive monoaminergic excitation and monosynaptic inhibition from Purkinje cells

Inhibitory interneurons in the cerebellar granular layer are more heterogeneous than traditionally depicted. In contrast to Golgi cells, which are ubiquitously distributed in the granular layer, small fusiform Lugaro cells and globular cells are located underneath the Purkinje cell layer and small in number. Globular cells have not been characterized physiologically. Here, using cerebellar slices obtained from a strain of gene-manipulated mice expressing GFP specifically in GABAergic neurons, we morphologically identified globular cells, and compared their synaptic activity and monoaminergic influence of their electrical activity with those of small Golgi cells and small fusiform Lugaro cells. Globular cells were characterized by prominent IPSCs together with monosynaptic inputs from the axon collaterals of Purkinje cells, whereas small Golgi cells or small fusiform Lugaro cells displayed fewer and smaller spontaneous IPSCs. Globular cells were silent at rest and fired spike discharges in response to application of either serotonin (5-HT) or noradrenaline. The two monoamines also facilitated small Golgi cell firing, but only 5-HT elicited firing in small fusiform Lugaro cells. Furthermore, globular cells likely received excitatory monosynaptic inputs through mossy fibers. Because globular cells project their axons long in the transversal direction, the neuronal circuit that includes interplay between Purkinje cells and globular cells could regulate Purkinje cell activity in different microzones under the influence of monoamines and mossy fiber inputs, suggesting that globular cells likely play a unique modulatory role in cerebellar motor control.     

Extended linkage disequilibrium in noncoding regions in a conifer, Cryptomeria japonica

We measured linkage disequilibrium in mostly noncoding regions of Cryptomeria japonica, a conifer belonging to Cupressaceae. Linkage disequilibrium was extensive and did not decay even at a distance of 100 kb. The average estimate of the population recombination rate per base pair was 1.55 × 10(-5) and was <1/70 of that in the coding regions. We discuss the impact of low recombination rates in a large part of the genome on association studies.     

Serine 58 of 14-3-3ζ Is a Molecular Switch Regulating ASK1 and Oxidant Stress-Induced Cell Death

Oxidant stress is a ubiquitous stressor with negative impacts on multiple cell types. ASK1 is a central mediator of oxidant injury, but while mechanisms of its inhibition, such as sequestration by 14-3-3 proteins and thioredoxin, have been identified, mechanisms of activation have remained obscure and the signaling pathways regulating this are not clear. Here, we report that phosphorylation of 14-3-3ζ at serine 58 (S58) is dynamically regulated in the cell and that the phosphorylation status of S58 is a critical factor regulating oxidant stress-induced cell death. Phosphorylation of S58 releases ASK1 from 14-3-3ζ, and ASK1 then activates stress-activated protein kinases, leading to cell death. While several members of the mammalian sterile 20 (Mst) family of kinases can phosphorylate S58 when overexpressed, we identify Ste20/oxidant stress response kinase 1 (SOK-1), an Mst family member known to be activated by oxidant stress, as a central endogenous regulator of S58 phosphorylation and thereby of ASK1-mediated cell death. Our findings identify a novel pathway that regulates ASK1 activation and oxidant stress-induced cell death.Oxidant stress plays a central role in a wide variety of pathologies, and a critical mediator of oxidant injury is the protein kinase ASK1 (30). Indeed, ASK1 is required for several types of oxidant stress-induced cell death (32). Its activity is restrained by a large number of complementary mechanisms, a fact that attests to the importance of ASK1 being maintained in an inactive state in the cell. For example, reduced thioredoxin binds to the N-terminal region of ASK1, thereby inhibiting its activity (27). Following oxidant stress and oxidation of thioredoxin, ASK1 is released, allowing its activation. Multiple phosphorylation events, including phosphorylation of ASK1 at S83 by Akt and at S1033 by an unknown mechanism, also negatively regulate ASK1 (6, 41; reviewed in reference 30). Critical to the negative regulation of ASK1 is phosphorylation of S966, which drives the association of ASK1 with 14-3-3 proteins, thereby inhibiting ASK1-mediated activation of downstream signaling and cell death (8, 43). The kinases responsible for S966 phosphorylation are not known, but the protein phosphatase calcineurin has been shown to dephosphorylate S966, leading to dissociation of ASK1 from 14-3-3 (13). Thus, other than calcineurin-mediated dephosphorylation of ASK1, signaling mechanisms positively regulating the release of ASK1 from 14-3-3 proteins are not known, despite intense interest in this kinase as a potential target in cardiovascular and neurologic diseases (30). Therefore, we undertook studies to attempt to identify such a mechanism.14-3-3 proteins play protective roles in the cell by sequestering proapoptotic factors in a phosphorylation-dependent manner (1, 15, 23). These proapoptotic proteins that are sequestered by 14-3-3 proteins are typically phosphorylated on one or more 14-3-3 binding motifs (18, 39). For example, in addition to ASK1 phosphorylation at S966, (8, 30), Bad is phosphorylated by Akt and ribosomal S6 kinases at several residues, inhibiting its proapoptotic functions (4, 14, 42, 45). Acting in opposition to this is the well-characterized c-Jun N-terminal kinase (JNK)-mediated phosphorylation of serine 184 of 14-3-3 proteins, leading to release of the proapoptotic factors Bax, Bad, FOXO3a, and Abl (29, 33, 40). In addition to S184, the phosphorylation statuses of other 14-3-3 residues can regulate 14-3-3/client interactions, such as T233, which is phosphorylated by CKI, disrupting the 14-3-3/Raf-1 interaction (5).Although most of the attention to phosphorylation of 14-3-3 has been focused on S184 and T233 (1), S58 has been known to be phosphorylated in situ for some time, and several kinases have been implicated, including protein kinases A and D, Akt, mitogen-activated protein kinase-activated kinase 2 (MK2), and sphingosine-dependent protein kinase 1 (later identified as a cleavage fragment of protein kinase C δ) (9, 16, 17, 24, 25, 44). However, it is not clear which specific kinases mediate phosphorylation under specific circumstances, nor are the biological consequences clear. This is underscored by the fact that both pro- and antiapoptotic kinases have been reported to phosphorylate this residue (23). It does seem clear, however, that S58 phosphorylation disrupts 14-3-3 dimerization and that this reduces the binding of some proteins (e.g., Raf-1) (28, 34), though probably not all, since Woodcock et al. reported that 14-3-3ζ monomers phosphorylated at S58 remained competent to bind phosphopeptides (37).Thorson et al. and Wang et al. created 14-3-3 mutants that were deficient in binding phosphopeptides, and Xing et al. employed one of these, 14-3-3ζ(R56A/R60A), to show that it led to enhanced activation of the stress-activated protein kinases, JNKs and p38, and enhanced cell death in response to UVC irradiation, a model of oxidant stress (31, 36, 38). However, since S58 of 14-3-3ζ is in the center of the R56-R60 region, we hypothesized that phosphorylation of S58 might disrupt binding of 14-3-3ζ to ASK1, which is upstream from the JNKs and p38 in the response to oxidant stress. In addition, Preisinger et al. reported that Ste20/oxidant stress response kinase 1 (SOK-1)/STK25, a mammalian sterile 20 (Mst) family member also known as yeast sterile 20 kinase 1 (YSK1), phosphorylated 14-3-3ζ on S58, leading to reorganization of the Golgi structure and cell motility (26). However, we and others had initially identified SOK-1 as a kinase activated by oxidant stress (20-22), and we have also shown that SOK-1 exits the Golgi apparatus following cellular stress (19), potentially giving SOK-1 greater access to the ASK1/14-3-3 complex (which is primarily cytosolic). Thus, we hypothesized that oxidant stress-induced activation of SOK-1 might lead to S58 phosphorylation, release of ASK1 from 14-3-3ζ, activation of JNKs and p38, and cell death. Here, we confirm these hypotheses and identify S58 as a molecular switch regulating ASK1-mediated oxidant stress-induced cell death.     

Lbx2 regulates formation of myofibrils

Background  

Skeletal muscle differentiation requires assembly of contractile proteins into organized myofibrils. The Drosophila ladybird homeobox gene (lad) functions in founder cells of the segmental border muscle to promote myoblast fusion and muscle shaping. Tetrapods have two homologous genes (Lbx). Lbx1 functions in migration and/or proliferation of hypaxial myoblasts, whereas the function of Lbx2 is poorly understood.     

Vesnarinone Represses the Fibrotic Changes in Murine Lung Injury Induced by Bleomycin

We investigated the potential usefulness of vesnarinone, a novel cytokine inhibitor, for the treatment of lung fibrosis using a murine model of bleomycin (BLM)-induced pulmonary fibrosis. Mice were fed a control diet (n=42), or a diet containing low (n=42) or high (n=42) dose of vesnarinone. Dietary intake of vesnarinone minimized the BLM toxicity as reflected by significant decreases in numbers of inflammatory cells, KC, and soluble TNF receptors in the bronchoalveolar lavage fluid. A quantitative evaluation of histology demonstrated significantly mild lung parenchymal lesions in BLM-treated mice fed with diet containing high dose of vesnarinone than in the control diet group. Consistent with the histopathology, hydroxyproline levels in lung tissue from BLM-treated mice fed with diet containing vesnarinone were significantly lower than that from mice fed with control diet. We concluded that vesnarinone inhibits BLM-induced pulmonary fibrosis, at least in part, by the inhibition of acute lung injuries in the early phase.