Two motifs essential for nuclear import of the hnRNP A1 nucleocytoplasmic shuttling sequence M9 core

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 regulates mRNA genesis. It shuttles between the nucleus and cytoplasm. Its shuttling signal is a 38-residue sequence M9. We studied the nuclear import and export of M9 by mutational analysis. Heterokaryon assay indicated that the 19-residue sequence SNFGPMKGGNFGGRSSGPY (M9 core) is necessary and sufficient for shuttling. Moreover, M9 core mutation revealed that in addition to the hitherto characterized N-terminal motif SNFGPMK, the C-terminal motif PY is crucial for nuclear import as well as for binding to transportin. Key residues of the motifs are conserved in the shuttling signals of hnRNP D and JKTBP.     

Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 degrees C, but stopped growing at the non-permissive temperature of 39 degrees C. In the presence of receptor activator of NFkappaB ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 degrees C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.     

Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression

CD82, also known as KAI1, was recently identified as a prostate cancer metastasis suppressor gene on human chromosome 11p1.2 (ref. 1). The product of CD82 is KAI1, a 40- to 75-kDa tetraspanin cell-surface protein also known as the leukocyte cell-surface marker CD82 (refs. 1,2). Downregulation of KAI1 has been found to be clinically associated with metastatic progression in a variety of cancers, whereas overexpression of CD82 specifically suppresses tumor metastasis in various animal models. To define the mechanism of action of KAI1, we used a yeast two-hybrid screen and identified an endothelial cell-surface protein, DARC (also known as gp-Fy), as an interacting partner of KAI1. Our results indicate that the cancer cells expressing KAI1 attach to vascular endothelial cells through direct interaction between KAI1 and DARC, and that this interaction leads to inhibition of tumor cell proliferation and induction of senescence by modulating the expression of TBX2 and p21. Furthermore, the metastasis-suppression activity of KAI1 was significantly compromised in DARC knockout mice, whereas KAI1 completely abrogated pulmonary metastasis in wild-type and heterozygous littermates. These results provide direct evidence that DARC is essential for the function of CD82 as a suppressor of metastasis.     

Mitochondrial superoxide production contributes to vancomycin-induced renal tubular cell apoptosis

Vancomycin chloride (VCM), a glycopeptide antibiotic, is widely used for the therapy of infections caused by methicillin-resistant Staphylococcus aureus. However, nephrotoxicity is a major adverse effect in VCM therapy. In this study, we investigated the cellular mechanisms underlying VCM-induced renal tubular cell injury in cultured LLC-PK1 cells. VCM induced a concentration- and time-dependent cell injury in LLC-PK1 cells. VCM caused increases in the numbers of annexin V-positive/PI-negative cells and TUNEL-positive cells, indicating the involvement of apoptotic cell death in VCM-induced renal cell injury. The VCM-induced apoptosis was accompanied by the activation of caspase-9 and caspase-3/7 and reversed by inhibitors of these caspases. Moreover, VCM caused an increase in intracellular reactive oxygen species production and mitochondrial membrane depolarization, which were reversed by vitamin E. In addition, mitochondrial complex I activity was inhibited by VCM as well as by the complex I inhibitor rotenone, and rotenone mimicked the VCM-induced LLC-PK1 cell injury. These findings suggest that VCM causes apoptotic cell death in LLC-PK1 cells by enhancing mitochondrial superoxide production leading to mitochondrial membrane depolarization followed by the caspase activities. Moreover, mitochondrial complex I may play an important role in superoxide production and renal tubular cell apoptosis induced by VCM.     

FIA functions as an early signal component of abscisic acid signal cascade in Vicia faba guard cells

An abscisic acid (ABA)-insensitive Vicia faba mutant, fia (fava bean impaired in ABA-induced stomatal closure) had previously been isolated. In this study, it was investigated how FIA functions in ABA signalling in guard cells of Vicia faba. Unlike ABA, methyl jasmonate (MeJA), H(2)O(2), and nitric oxide (NO) induced stomatal closure in the fia mutant. ABA did not induce production of either reactive oxygen species or NO in the mutant. Moreover, ABA did not suppress inward-rectifying K(+) (K(in)) currents or activate ABA-activated protein kinase (AAPK) in mutant guard cells. These results suggest that FIA functions as an early signal component upstream of AAPK activation in ABA signalling but does not function in MeJA signalling in guard cells of Vicia faba.     

The effect of surface charge property on Escherichia coli initial adhesion and subsequent biofilm formation

Polyethylene (PE) sheets were modified by radiation-induced graft polymerization (RIGP) of an epoxy-group containing monomer glycidyl methacrylate (GMA). The epoxy group of GMA was opened by introducing sodium sulfite (SS) and diethylamine (DEA) as representatives of negatively and positively charged functional groups, respectively. These modified surfaces by RIGP, termed GMA, SS, and DEA sheets, were investigated to elucidate their effects on initial adhesion and subsequent biofilm formation of Escherichia coli. Initial adhesion test revealed that E. coli density and viability were governed by sheet surface electrostatic property: E. coli cell density on the DEA sheet was 23 times higher than that on the SS sheet after 8 h incubation. The viability of E. coli cells dramatically decreased after contact with the DEA sheet, but remained high on the SS sheet. E. coli biofilm structure on the DEA sheet was dense, homogeneous, and uniform, with biomass higher than that of the GMA and SS sheets by factors of 14.0 and 37.5, respectively. On the contrary, biofilm structure on the SS sheet was sparse, heterogeneous, and mushroom-shaped. More than 40% of E. coli biofilm on the DEA sheet was retained under a high liquid shear force condition (5,000 s(-1)), whereas 97% and 100% of biofilms on the GMA and SS sheets were sloughed, indicating that E. coli biofilm robustness depends on surface charge property of the substratum. This suggests that substratum surface fabrication by RIGP may enhance or suppress biofilm formation, a finding with potentially important practical implications.     

Gain of function by phosphorylation in Presenilin 1-mediated regulation of insulin signaling

During pregnancy, activation of the maternal immune system results in inflammation in the foetal nervous system. The causative agents are pro-inflammatory cytokines like interleukin-1β (IL-1β), produced by the foetus. In this study, we examine the effect of IL-1β on the proliferation and differentiation of neural progenitor cells (NPCs) to better understand its potential effects on the developing brain. We find that the IL-1β receptor (IL-1R1) is expressed in the ventral mesencephalon of the developing brain. Furthermore, IL-1R1 is expressed on Nestin-positive, Sox-2-positive NPCs. IL-1β treatment reduced the numbers of proliferating NPCs, an effect prevented by the IL-1R1 receptor antagonist. LDH and MTT assays, and western blot analysis for cleaved caspase 3 and poly(ADP-ribose) polymerase, confirmed that this was not due to an increase in cell death but rather an induction of differentiation. To further study the effects of IL-1β on cell fate determination, we differentiated NPCs in the presence and absence of IL-1β. Il-1β promoted gliogenesis and inhibited neurogenesis, an effect that required p38-MAPK kinase signalling. In summary, these data show that exposure of NPCs to IL-1β affects their development. This necessitates an examination of the consequences that maternal immune system activation during pregnancy has on the cellular architecture of the developing brain.     

A novel anti-inflammatory role for spleen-derived interleukin-10 in obesity-induced hypothalamic inflammation

Obesity can be associated with systemic low-grade inflammation that contributes to obesity-related metabolic disorders. Recent studies raise the possibility that hypothalamic inflammation contributes to the pathogenesis of diet-induced obesity (DIO), while another study reported that obesity decreases the expression of pro-inflammatory cytokines in spleen. The following study examines the hypothesis that obesity suppresses the splenic synthesis of the anti-inflammatory cytokine, interleukin (IL)-10, thereby resulting in chronic hypothalamic inflammation. The results showed that due to oxidative stress or apoptosis, the synthesis of splenic IL-10 was decreased in DIO when compared with non-obesity rats. Splenectomy (SPX) accelerated DIO-induced inflammatory responses in the hypothalamus. Interestingly, SPX suppressed the DIO-induced increases in food intake and body weight and led to a hypothalamic pro-inflammatory state that was similar to that produced by DIO, indicating that hypothalamic inflammation exerts a dual effect on energy metabolism. These SPX-induced changes were inhibited by the systemic administration of IL-10. Moreover, SPX had no effect on hypothalamic inflammatory responses in IL-10-deficient mice. These data suggest that spleen-derived IL-10 plays an important role in the prevention of hypothalamic inflammation and may be a therapeutic target for the treatment of obesity and hypothalamic inflammation.     

Structural insight into receptor-selectivity for lurasidone

Lurasidone is a novel antipsychotic agent with high affinity for dopamine D₂, 5-hydroxyltryptamine 5-HT_2A, and 5-HT₇ receptors. Lurasidone has negligible affinity for histamine H₁ and muscarinic M₁ receptors, which are thought to contribute to side effects such as weight gain, sedation, and worsening of cognitive deficits. Our interests focus on why lurasidone has such high selectivity for only a part of these aminergic G-protein coupled receptors (GPCRs) and the different binding profile from ziprasidone, which has the same benzisothiazolylpiperazine moiety as lurasidone. In order to address these issues, we constructed structural models of lurasidone–GPCR complexes by homology modeling of receptors, exhaustive docking of ligand, and molecular dynamics simulation-based refinement of complexes. This computational study gave reliable structural models for D₂, 5-HT_2A, and 5-HT₇, which had overall structural complementarities with a salt bridge anchor at the center of the lurasidone molecule, but not for H₁ and M₁ owing to steric hindrance between the norbornane-2,3-dicarboximide and/or cyclohexane part of lurasidone and both receptors. By comparison with the structural models of olanzapine–GPCRs and ziprasidone–GPCRs constructed using the same computational protocols, it was suggested that the bulkiness of the norbornane-2,3-dicarboximide part and the rigidity and the bulkiness of the cyclohexyl linker gave lurasidone high selectivity for the desired aminergic GPCRs. Finally, this structural insight was validated by a binding experiment of the novel benzisothiazolylpiperazine derivatives. This knowledge on the structural mechanism behind the receptor selectivity should help to design new antipsychotic agents with preferable binding profiles, and the established computational protocols realize virtual screening and structure-based drug design for other central nervous system drugs with desired selectivity for multiple targets.