Phencyclidine animal models of schizophrenia: approaches from abnormality of glutamatergic neurotransmission and neurodevelopment

In humans, phencyclidine (PCP), a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, reproduces a schizophrenia-like psychosis including positive symptoms, negative symptoms and cognitive dysfunction. Thus, the glutamatergic neuronal dysfunction hypothesis is one of the main explanatory hypotheses and PCP-treated animals have been utilized as an animal model of schizophrenia. The adult rodents treated with PCP repeatedly exhibit hyperlocomotion as an index of positive symptoms, a social behavioral deficit in a social interaction test and enhanced immobility in a forced swimming test as indices of negative symptoms. They also show a sensorimotor gating deficits and cognitive dysfunctions in several learning and memory tests. Some of these behavioral changes endure after withdrawal from repeated PCP treatment. Furthermore, repeated PCP treatment induces some neurochemical and neuroanatomical changes. On the other hand, the exposure to viral or environmental insult in the second trimester of pregnancy increases the probability of subsequently developing schizophrenia as an adult. NMDA receptor has been implicated in controlling the structure and plasticity of developing brain circuitry. Based on neurodevelopment hypothesis of schizophrenia, schizophrenia model rats treated with PCP at the perinatal stage is developed. Perinatal PCP treatment impairs neuronal development and induces long-lasting schizophrenia-like behaviors in adult period. Many findings suggest that these PCP animal models would be useful for evaluating novel therapeutic candidates and for confirming pathological mechanisms of schizophrenia.     

Involvement of signaling of VEGF and TGF-β in differentiation of sinusoidal endothelial cells during culture of fetal rat liver cells

Embryonic development of the liver is closely associated with vascular organization. However, little is known about the mechanisms of vascular differentiation during liver development. Our previous study showed that the maturation of sinusoidal endothelial cells (SECs) occurred during embryonic day 13.0 (E13.0) to E15.0. To improve our understanding of SEC differentiation, we examined here the expression of maturation markers, SE-1 and stabilin-2, in fetal livers and also attempted to establish an in vitro SEC differentiation system by culturing E13.5 fetal liver cells. Immunohistochemical examination of SE-1 and stabilin-2 expression during fetal rat liver development revealed that these differentiation markers were co-expressed in SECs in the late stage of liver development, although stabilin-2 was expressed in almost all vascular endothelial cells in the early stage. Liver cells from the E13.5 rat fetus were cultured in EBM-2 medium containing vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1) and VEGF plus SB-431542 (an inhibitor of the TGF-β1 receptor, activin receptor-like kinase 5 [ALK-5]). In vitro SEC differentiation, as indicated by the appearance of cells co-expressing SE-1 and stabilin-2 and of cells with cytoplasmic fenestrae in endothelial sheets, was induced by the addition of both VEGF and SB-431542, an inhibitor of the phosphorylation of Smad2/3 but not that of Smad1/5/8 in the cultured cells. These results indicate for the first time that both VEGF signaling and the blocking of the ALK-5-Smad2/3 signal pathway are important for the fetal differentiation of SECs.     

Transforming activity of purinergic receptor P2Y, G-protein coupled, 2 revealed by retroviral expression screening

Colorectal cancer (CRC) is one of the leading causes of cancer death in humans. In order to identify novel cancer-promoting genes in CRC, we here constructed a retroviral cDNA expression library from a CRC cell line RKO, and used it for a focus formation assay with mouse 3T3 fibroblasts, leading to the identification of 42 independent cDNAs. One of such cDNAs turned out to encode purinergic receptor P2Y, G-protein coupled, 2 (P2RY2). The oncogenic potential of P2RY2 was confirmed in vitro with the focus formation assay as well as soft agar-growth assay, and also in vivo with a tumorigenicity assay in nude mice. While our P2RY2 cDNA encodes a protein with two amino-acid substitutions compared to the reported one, we have confirmed that the wild-type P2RY2 has a strong transforming potential as well. These results indicate an unexpected role of P2RY2 in the carcinogenesis of human cancers.     

Identification of critical structural determinants responsible for octopamine binding to the alpha-adrenergic-like Bombyx mori octopamine receptor

Octopamine (OA) is a biogenic amine with a widespread distribution in the insect nervous system. OA modulates and/or regulates various behavioral patterns of insects as a neurotransmitter, neuromodulator, and neurohormone. OA receptors (OARs) belong to one of the families of G protein-coupled receptors (GPCRs). The binding of OA to OARs is coupled to the activation of the specific G proteins, which induces the release of intracellular second messengers such as cAMP and/or calcium. We previously reported the isolation of an OAR (BmOAR1) from Bombyx mori. In the study presented here, five mutated BmOAR1s were constructed with a point mutation in the putative binding crevice and expressed in HEK-293 cells. The S202A mutant receptor was found to retain the cAMP response to OA as does the wild-type receptor, but such function was impaired in the other four mutants (D103A, S198A, Y412F, and S198A/S202A). Furthermore, competition binding assays using [3H]OA and calcium mobilization assays gave results that were approximately consistent with those of the cAMP assays. Taken together, the results indicate that D103 and S198 are involved in the binding and activation of BmOAR1 with OA through electrostatic or hydrogen bond interactions, but S202 does not appear to participate in this process. Y412 seems to be involved in one of the active forms of BmOAR1. These findings should prove helpful in designing new pest control chemicals.     

Rad50 is involved in MMS-induced recombination between homologous chromosomes in mitotic cells

The structural maintenance of chromosomes (SMC) family proteins (Smc1-Smc6) typically consist of two coiled-coil domains, an amino-terminal head domain, and a carboxyl-terminal tail domain. Rad50, a component of the Mre11/Rad50/Xrs2 (MRX) complex, has a similar domain structure to the SMC proteins. In Saccharomyces cerevisiae, the MRX complex appears to be essential for recombination between homologous chromosomes in meiotic cells, but not in cells undergoing vegetative growth. Here we provide for the first time evidence that Rad50, like Smc6, is required for the induction of recombination between homologous chromosomes in cells in the vegetative growth state upon exposure to methyl methanesulfonate. However, UV-induced recombination between homologous chromosomes is intact in both rad50 and smc6-56 mutant cells.     

Abundance of Collembola (Insecta) inhabiting the hyphal mat of an ectomycorrhizal fungus,Sarcodon scabrosus,in a Pinus densiflora forest

The total number of collembolans collected from the hyphal mat of the ectomycorrhizal fungus Sarcodon scabrosus was less than half of that collected from the adjacent non-mat soil. The same was true of all families of collembolans except one, although not all differences were significant. The exception was the Hypogastruridae, with more individuals in the hyphal mat on the sampling day in the fruiting season; these were also the most abundant collembolans on the fruiting bodies. These results indicate that most collembolans avoid the hyphal mat of S. scabrosus in a Japanese red pine forest.     

Design and synthesis of 3-pyridylacetamide derivatives as dipeptidyl peptidase IV (DPP-4) inhibitors targeting a bidentate interaction with Arg125

We have previously discovered nicotinic acid derivative 1 as a structurally novel dipeptidyl peptidase IV (DPP-4) inhibitor. In this study, we obtained the X-ray co-crystal structure between nicotinic acid derivative 1 and DPP-4. From these X-ray co-crystallography results, to achieve more potent inhibitory activity, we targeted Arg125 as a potential amino acid residue because it was located near the pyridine core, and some known DPP-4 inhibitors were reported to interact with this residue. We hypothesized that the guanidino group of Arg125 could interact with two hydrogen-bond acceptors in a bidentate manner. Therefore, we designed a series of 3-pyridylacetamide derivatives possessing an additional hydrogen-bond acceptor that could have the desired bidentate interaction with Arg125. We discovered the dihydrochloride of 1-{[5-(aminomethyl)-2-methyl-4-(4-methylphenyl)-6-(2-methylpropyl)pyridin-3-yl]acetyl}-l-prolinamide (13j) to be a potent and selective DPP-4 inhibitor that could interact with the guanidino group of Arg125 in a unique bidentate manner.     

High sensitivity of RBL-2H3 cells to cadmium and manganese: an implication of the role of ZIP8

Cellular incorporation of Cd involves multiple transport systems for other metals such as Fe, Zn, Mn, and Ca. Metal transporters including divalent metal transporter 1, Zrt/Irt-related protein (ZIP) 8, and ZIP14, and certain types of voltage-dependent Ca channels have been shown to be involved in cellular Cd uptake. However, tissue- or cell-specific roles of these metal transporters in the accumulation and toxicity of Cd remains unclear. In the present study, we compared the sensitivity to and accumulation of Cd, Mn, and Zn among four types of rat cell lines. Rat basophilic leukemia RBL-2H3 cells showed the highest sensitivity to Cd and Mn due to the highest accumulation of Cd and Mn among the four cell lines. The high accumulation of Cd and Mn was caused by high uptake rates of Cd and Mn. Since relatively high expression of ZIP8 and ZIP14 was found in RBL-2H3 cells, siRNAs of ZIP8 and ZIP14 were transfected into RBL-2H3 cells. The knockdown of ZIP8, but not of ZIP14, significantly reduced the uptake rates of Cd and Mn in RBL-2H3 cells, especially in the presence of bicarbonate. These results suggest that the high expression of ZIP8, which is known to have affinities for both Cd and Mn, resulted in high accumulation of Cd and Mn, leading to high sensitivity to these metals in RBL-2H3 cells. Thus, RBL-2H3 cells may serve as a good model for clarifying the mechanisms of Cd and Mn transport via ZIP8.     

Enzymatic properties of pierisin-1 and its N-terminal domain, a guanine-specific ADP-ribosyltransferase from the cabbage butterfly

The cabbage butterfly, Pieris rapae, produces an ADP-ribosylating cytotoxic protein, pierisin-1. Unlike other ADP-ribosylating toxins, the acceptor site for ADP-ribosylation by pierisin-1 is the N-2 position of guanine bases in DNA. The present study was designed to characterize this novel guanine-specific ADP-ribosyltransferase, pierisin-1. The N-terminal polypeptide from Met-1 to Arg-233, but not the C-terminal Ser-234-Met-850 polypeptide, was found to exhibit guanine ADP-ribosyltransferase activity. Trypsin-treated pierisin-1, which is considered to be a "nicked" full-length form composed of associated N- and C-terminal fragments, also demonstrated such activity. Optimum conditions for the N-terminal polypeptide of pierisin-1 were pH 8-10, 37-40 degrees C, in the presence of 100-200 mM NaCl or KCl. Other metal ions such as Ca(2+) or Mg(2+) were not required. Kinetic studies demonstrated potent ADP-ribosyltransferase activity with a K(M) value for NAD of 0.17 mM and k(cat) of 55 per second. Under these optimum conditions, the specific activity of trypsin-treated pierisin-1 was about half (k(cat) = 25 per second). When the conditions were changed to pH 5-7 or 10-20 degrees C, some activity (6-55% or 5-20%, respectively, of that under optimal conditions) of the N-terminal polypeptide was still evident; however, almost all of the trypsin-treated enzyme activity disappeared. This implies the inhibition of the N-terminal enzyme domain by the associated C-terminal fragment. Long-term reactions indicated that a single molecule of pierisin-1 has the capacity to generate more than 10(6) ADP-ribosylated DNA adducts, which could cause the death of a mammalian cell.