Gadd45a, the gene induced by the mood stabilizer valproic acid, regulates neurite outgrowth through JNK and the substrate paxillin in N1E-115 neuroblastoma cells

Valproic acid (VPA), a mood stabilizer and anticonvulsant, has a variety of neurotrophic functions; however, less is known about how VPA regulates neurite outgrowth. Here, using N1E-115 neuroblastoma cells as the model, we show that VPA upregulates Gadd45a to trigger activation of the downstream JNK cascade controlling neurite outgrowth. VPA induces the phosphorylation of c-Jun N-terminal kinase (JNK) and the substrate paxillin, while VPA induction of neurite outgrowth is inhibited by JNK inhibitors (SP600125 and the small JNK-binding peptide) or a paxillin construct harboring a Ser 178-to-Ala mutation at the JNK phosphorylation. Transfection of Gadd45a, acting through the effector MEKK4, leads to the phosphorylation of the JNK cascade. Conversely, knockdown of Gadd45a with siRNA reduces the effect of VPA. Taken together, these results suggest that upregulation of Gadd45a explains one of the mechanisms whereby VPA induces the neurotrophic effect, providing a new role of Gadd45a in neurite outgrowth.     

Incorporating experimental design and error into coalescent/mutation models of population history

Coalescent theory provides a powerful framework for estimating the evolutionary, demographic, and genetic parameters of a population from a small sample of individuals. Current coalescent models have largely focused on population genetic factors (e.g., mutation, population growth, and migration) rather than on the effects of experimental design and error. This study develops a new coalescent/mutation model that accounts for unobserved polymorphisms due to missing data, sequence errors, and multiple reads for diploid individuals. The importance of accommodating these effects of experimental design and error is illustrated with evolutionary simulations and a real data set from a population of the California sea hare. In particular, a failure to account for sequence errors can lead to overestimated mutation rates, inflated coalescent times, and inappropriate conclusions about the population. This current model can now serve as a starting point for the development of newer models with additional experimental and population genetic factors. It is currently implemented as a maximum-likelihood method, but this model may also serve as the basis for the development of Bayesian approaches that incorporate experimental design and error.     

Role of MAPK phosphatase-1 in the induction of monocyte chemoattractant protein-1 during the course of adipocyte hypertrophy

Monocyte chemoattractant protein-1 (MCP-1), an important chemokine whose expression is increased during the course of obesity, plays a role in macrophage infiltration into obese adipose tissue. This study was designed to elucidate the role of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the induction of MCP-1 during the course of adipocyte hypertrophy. We examined the time course of MKP-1 and MCP-1 mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation in the adipose tissue from mice rendered mildly obese by a short term high fat diet. We also studied the role of MKP-1 in the induction of MCP-1 in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. MCP-1 mRNA expression was increased, followed by ERK activation and down-regulation of MKP-1, an inducible dual specificity phosphatase to inactivate ERK, in the adipose tissue at the early stage of obesity induced by a short term high fat diet, when macrophages are not infiltrated. Down-regulation of MKP-1 preceded ERK activation and increased production of MCP-1 in 3T3-L1 adipocytes in vitro during the course of adipocyte hypertrophy. Adenovirus-mediated restoration of MKP-1 in hypertrophied 3T3-L1 adipocytes reduced the otherwise increased ERK phosphorylation, thereby leading to the significant reduction of MCP-1 mRNA expression. This study provides evidence that the down-regulation of MKP-1 is critical for increased production of MCP-1 during the course of adipocyte hypertrophy.     

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.     

Genome-wide association study of idiopathic hypersomnia in a Japanese population

Sleep and Biological Rhythms - Idiopathic hypersomnia (IH) is a rare sleep disorder characterized by excessive daytime sleepiness, great difficulty upon awakening, and prolonged sleep time. In...     

Hepatic neuroendocrine carcinoma in a Japanese macaque (Macaca fuscata)

Primary neuroendocrine neoplasm of the liver is extremely rare in both humans and non‐human primates. The present report describes the clinical and pathological findings of an aged Japanese macaque (Macaca fuscata) with hepatic neuroendocrine carcinoma. To our knowledge, this is the first report of hepatic neuroendocrine neoplasm in macaques.     

Involvement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase in alpha1B-adrenergic receptor/Galphaq-induced inhibition of cell proliferation

Certain G protein-coupled receptors (GPCRs) stimulate the activities of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), members of the MAPK family. We investigated the role of JNK and p38 MAPK activation induced by the alpha1B-adrenergic receptor in the proliferation of human embryonic kidney 293T cells. Activation of the alpha1B-adrenergic receptor resulted in inhibition of cell proliferation. This receptor-induced inhibition of proliferation was blocked by a kinase-deficient MKK4 and by the p38 MAPK inhibitor SB203580. Additionally, transfection of constitutively activated Galphaq into cells also led to inhibition of proliferation in a JNK- and p38 MAPK-dependent manner. These results demonstrate that the alpha1B-adrenergic receptor/Galphaq signaling inhibits cell proliferation through pathways involving JNK and p38 MAPK.     

A unique phosphatidylinositol bearing a novel branched-chain fatty acid from Rhodococcus equi binds to influenza virus hemagglutinin and inhibits the infection of cells

From the aquatic bacterium Rhodococcus equi strain S(420), we isolated a substance that strongly binds to influenza viruses. Structural analyses revealed that it is a unique type of phosphatidylinositol (PtdIns) bearing a branched-chain fatty acid (14-methyloctadecanoic acid). In a TLC/virus-binding immunostaining assay, this PtdIns bound to all subtypes of hemagglutinin (HA) of influenza A viruses tested, isolated from humans, ducks and swine, and also to human influenza B viruses. Furthermore, the PtdIns significantly prevented the infection of MDCK cells by influenza viruses, and also inhibited the virus-mediated hemagglutination and low pH-induced hemolysis of human erythrocytes, which represents the fusogenic activities of the viral HA. We also used purified hemagglutinin instead of virions to examine the interaction between viral HA and PtdIns, showing that the PtdIns binds to hemagglutinin. These findings indicate that the inhibitory mechanism of PtdIns on the influenza virus infection may be through its binding to viral HA spikes and host cell endosomal/lysosomal membranes, which are mediated by the function of viral HA.     

Activation of calcium/calmodulin-dependent protein kinase IV in long term potentiation in the rat hippocampal CA1 region

The importance of well characterized calcium/calmodulin-dependent protein kinase (CaMK) II in hippocampal long term potentiation (LTP) is widely well established; however, several CaMKs other than CaMKII are not yet clearly characterized and understood. Here we report the activation of CaMKIV, which is phosphorylated by CaMK kinase and localized predominantly in neuronal nuclei, and its functional role as a cyclic AMP-responsive element-binding protein (CREB) kinase in high frequency stimulation (HFS)-induced LTP in the rat hippocampal CA1 region. CaMKIV was transiently activated in neuronal nuclei after HFS, and the activation returned to the basal level within 30 min. Phosphorylation of CREB, which is a CaMKIV substrate, and expression of c-Fos protein, which is regulated by CREB, increased during LTP. This increase was inhibited mainly by CaMK inhibitors and also by an inhibitor for mitogen-activated protein kinase cascade, although to a lesser extent. Our results suggest that CaMKIV functions as a CREB kinase and controls CREB-regulated gene expression during HFS-induced LTP in the rat hippocampal CA1 region.     

Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines

System L is a major nutrient transport system responsible for the transport of large neutral amino acids including several essential amino acids. We previously identified a transporter (L-type amino acid transporter 1: LAT1) subserving system L in C6 rat glioma cells and demonstrated that LAT1 requires 4F2 heavy chain (4F2hc) for its functional expression. Since its oncofetal expression was suggested in the rat liver, it has been proposed that LAT1 plays a critical role in cell growth and proliferation. In the present study, we have examined the function of human LAT1 (hLAT1) and its expression in human tissues and tumor cell lines. When expressed in Xenopus oocytes with human 4F2hc (h4F2hc), hLAT1 transports large neutral amino acids with high affinity (K(m)= approximately 15- approximately 50 microM) and L-glutamine and L-asparagine with low affinity (K(m)= approximately 1.5- approximately 2 mM). hLAT1 also transports D-amino acids such as D-leucine and D-phenylalanine. In addition, we show that hLAT1 accepts an amino acid-related anti-cancer agent melphalan. When loaded intracellularly, L-leucine and L-glutamine but not L-alanine are effluxed by extracellular substrates, confirming that hLAT1 mediates an amino acid exchange. hLAT1 mRNA is highly expressed in the human fetal liver, bone marrow, placenta, testis and brain. We have found that, while all the tumor cell lines examined express hLAT1 messages, the expression of h4F2hc is varied particularly in leukemia cell lines. In Western blot analysis, hLAT1 and h4F2hc have been confirmed to be linked to each other via a disulfide bond in T24 human bladder carcinoma cells. Finally, in in vitro translation, we show that hLAT1 is not a glycosylated protein even though an N-glycosylation site has been predicted in its extracellular loop, consistent with the property of the classical 4F2 light chain. The properties of the hLAT1/h4F2hc complex would support the roles of this transporter in providing cells with essential amino acids for cell growth and cellular responses, and in distributing amino acid-related compounds.