Hepatic zinc response via metallothionein induction after tumor transplantation

Based on previous findings that liver zinc and metallothionein (MT) levels increase after tumor transplantation, zinc metabolism in tumor-bearing mice was studied to clarify the role of zinc-MT in host defense systems. Zinc in the hepatic cytosolic MT fraction did not increase in tumor-bearing mice fed a zinc-deficient diet, suggesting that dietary zinc is necessary for apo-MT induction in the liver after tumor transplantation and is then incorporated into the apo-MT. When (65)ZnCl(2) was intravenously injected, liver (65)Zn levels in the tumor-bearing mice were higher than those in control mice for 72 h after the injection. Pancreatic and blood (65)Zn levels in tumor-bearing mice were lower than those in controls for 24 h (pancreas) and 6 h (blood) after the injection. These findings indicate that the hepatic zinc response via MT induction influences zinc metabolism in the body after tumor transplantation. Moreover, (65)Zn uptake in the liver of MT-deficient tumor-bearing mice was lower than that in control tumor-bearing mice 1 h after injection. (65)Zn uptake in the tumor and blood (65)Zn levels in the MT-deficient tumor-bearing mice were higher than those in the control tumor-bearing mice. Tumor weight increased more in MT-deficient mice than in control mice. The formation of zinc-MT in the liver of tumor-bearing mice might decrease blood zinc availability for tumors and other tissues, such as the pancreas.     

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.     

Alteration of a DNA-dependent ATPase activity in xeroderma pigmentosum complementation group C cells.

DNA-dependent ATPase activities in crude extracts prepared from HeLa cells were separated into five peaks by fast protein liquid chromatography Mono Q column chromatography. Similar elution profiles were observed with the extracts from human cells normal in repair and xeroderma pigmentosum cells belonging to complementation groups A through G except for group C. An alteration in elution of one of the five ATPases, designated DNA-dependent ATPase Q1, was observed with a cell line of complementation group C. This alteration was observed with all tested cell lines that belonged to group C. ATPase Q1 in HeLa cell extracts exhibited about 2-fold higher activity with ultraviolet light-irradiated DNA as compared to that with non-irradiated DNA, whereas little difference in the effects of two DNAs was observed with the ATPase activities in the extract from group C cells.     

Vertebrate WRNIP1 and BLM are required for efficient maintenance of genome stability

Bloom syndrome (BS) is rare autosomal recessive disorder associated with chromosomal instability. The gene responsible for BS, BLM, encodes a protein belonging to the RecQ helicase family. Disruptions of the SGS1 gene of Saccharomyces cerevisiae, which encodes the RecQ helicase homologue in the budding yeast, causes accelerated aging, and this phenotype is enhanced by the disruption of MGS1, the budding yeast homologue for WRNIP1. To examine the functional relationship between RecQ and WRNIP1 in vertebrate cells, we generated and characterized wrnip1/blm cells derived from the chicken B-lymphocyte line DT40. wrnip1/blm cells showed an additive elevation of sister chromatid exchange (SCE), suggesting that both genes independently contribute to the suppression of excess SCE formation. The double mutants were more sensitive to DNA damage from camptothecin (CPT), but not to damage from methyl methanesulfonate, than either single mutant. This result suggests that WRNIP1 and BLM function independently to repair DNA or induce tolerance to the lesions induced by CPT.     

Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin

The serine/threonine protein kinase Akt is involved in a variety of cellular processes including cell proliferation, survival, metabolism and gene expression. It is essential in vascular endothelial growth factor (VEGF)-mediated angiogenesis; however, it is not known how Akt regulates the migration of endothelial cells, a crucial process for vessel sprouting, branching and the formation of networks during angiogenesis. Here we report that Akt-mediated phosphorylation of Girdin, an actin-binding protein, promotes VEGF-dependent migration of endothelial cells and tube formation by these cells. We found that exogenously delivered adenovirus harbouring Girdin short interfering RNA in Matrigel embedded in mice, markedly inhibited VEGF-mediated angiogenesis. Targeted disruption of the Girdin gene in mice impaired vessel remodelling in the retina and angiogenesis from aortic rings, whereas Girdin was dispensable for embryonic vasculogenesis. These findings demonstrate that the Akt/Girdin signalling pathway is essential in VEGF-mediated postneonatal angiogenesis.     

Synthesis and biological evaluation of N-mercaptoacylproline and N-mercaptoacylthiazolidine-4-carboxylic acid derivatives as leukotriene A4 hydrolase inhibitors

We studied the synthetic modification of structurally similar N-mercaptoacyl-L-proline and (4R)-N-mercaptoacylthiazolidine-4-carboxylic acid to obtain potent leukotriene A(4) (LTA(4)) hydrolase inhibitors. An N-mercaptoacyl group, (2S)-3-mercapto-2-methylpropionyl group, was effective for both scaffolds. Additional introduction of a large substituent such as 4-isopropylbenzylthio (3f), 4-tert-butylbenzylthio (3l) or 4-cyclohexylbenzylthio group (3m) with (S)-configuration at the C(4) position of proline yielded much more potent LTA(4) hydrolase inhibitors (IC(50); 52, 31, and 34 nM, respectively) than captopril (IC(50); 630,000 nM).     

LTD windows of the STDP learning rule and synaptic connections having a large transmission delay enable robust sequence learning amid background noise

Spike-timing-dependent synaptic plasticity (STDP) is a simple and effective learning rule for sequence learning. However, synapses being subject to STDP rules are readily influenced in noisy circumstances because synaptic conductances are modified by pre- and postsynaptic spikes elicited within a few tens of milliseconds, regardless of whether those spikes convey information or not. Noisy firing existing everywhere in the brain may induce irrelevant enhancement of synaptic connections through STDP rules and would result in uncertain memory encoding and obscure memory patterns. We will here show that the LTD windows of the STDP rules enable robust sequence learning amid background noise in cooperation with a large signal transmission delay between neurons and a theta rhythm, using a network model of the entorhinal cortex layer II with entorhinal-hippocampal loop connections. The important element of the present model for robust sequence learning amid background noise is the symmetric STDP rule having LTD windows on both sides of the LTP window, in addition to the loop connections having a large signal transmission delay and the theta rhythm pacing activities of stellate cells. Above all, the LTD window in the range of positive spike-timing is important to prevent influences of noise with the progress of sequence learning.     

Accumulation of sumoylated Rad52 in checkpoint mutants perturbed in DNA replication

Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA-damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, chromatin immunoprecipitation (ChIP) on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double mutation of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD53 single mutation. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed.     

NMDA receptor regulates migration of newly generated neurons in the adult hippocampus via Disrupted-In-Schizophrenia 1 (DISC1)

In the mammalian brain, new neurons are continuously generated throughout life in the dentate gyrus (DG) of the hippocampus. Previous studies have established that newborn neurons migrate a short distance to be integrated into a pre-existing neuronal circuit in the hippocampus. How the migration of newborn neurons is governed by extracellular signals, however, has not been fully understood. Here, we report that NMDA receptor (NMDA-R)-mediated signaling is essential for the proper migration and positioning of newborn neurons in the DG. An intraperitoneal injection of the NMDA-R antagonists, memantine, or 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) into adult male mice caused the aberrant positioning of newborn neurons, resulting in the overextension of their migration in the DG. Interestingly, we revealed that the administration of NMDA-R antagonists leads to a decrease in the expression of Disrupted-In-Schizophrenia 1 (DISC1), a candidate susceptibility gene for major psychiatric disorders such as schizophrenia, which is also known as a critical regulator of neuronal migration in the DG. Furthermore, the overextended migration of newborn neurons induced by the NMDA-R antagonists was significantly rescued by exogenous expression of DISC1. Collectively, these results suggest that the NMDA-R signaling pathway governs the migration of newborn neurons via the regulation of DISC1 expression in the DG.