Design,synthesis, and anti-tumor activities of novel triphenylethylene–coumarin hybrids,and their interactions with Ct-DNA

Novel triphenylethylene–coumarin hybrid derivatives containing different amounts of amino side chains were designed and synthesized in good yields under microwave radiation. The derivatives 5b–d which possessed two amino side chains (except morpholinyl) showed a broad-spectrum and good anti-proliferative activity against five tumor cells and low cytotoxicity in osteoblast. UV–vis, fluorescence, and circular dichroism (CD) spectroscopies and thermal denaturation exhibited that compounds 10c, 5c, and 13c bearing amino side chain (except morpholinyl) on 4-phenyl had significant interactions with Ct-DNA by the intercalative mode of binding. Structure–activity relationships (SARs) analysis suggested that the amino alkyl chain would play an important role both in the compounds against tumor cells proliferation and their interactions with DNA.     

Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative,nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes

In the present study, one has determined the relative role of plasma membrane equilibrative (Na⁺-independent) ENT nucleoside transport proteins (particularly ENT2) in the uptake of antiviral nucleoside analogues for comparison with the previously reported drug transport properties of concentrative (Na⁺-dependent) CNT nucleoside transport proteins. The human and rat nucleoside transport proteins hENT1, rENT1, hENT2 and rENT2 were produced in Xenopus oocytes and investigated for their ability to transport three 3'-deoxy-nucleoside analogues, ddC (2' 3'-dideoxycytidine), AZT (3'-azido-3'-deoxythymidine)and ddI (2' 3'-dideoxyinosine), used in human immunodeficiency virus (HIV) therapy. The results show, for the first time, that the ENT2 transporter isoform represents a mechanism for cellular uptake of these clinically important nucleoside drugs. Recombinant h/rENT2 transported ddC, ddI and AZT, whilst h/rENT1 transported only ddC and ddI. Relative to uridine, h/rENT2 mediated substantially larger fluxes of ddC and ddI than h/rENT1. Transplanting the amino-terminal half of rENT2 into rENT1 rendered rENT1 transport-positive for AZT and enhanced the uptake of both ddC and ddI, identifying this region as a major site of 3'-deoxy-nucleoside drug interaction.     

Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP

Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB_313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF₃ support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF₃ through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.     

Regulation of a Dynamic Interaction between Two Microtubule-binding Proteins,EB1 and TIP150, by the Mitotic p300/CBP-associated Factor (PCAF) Orchestrates Kinetochore Microtubule Plasticity and Chromosome Stability during Mitosis

The microtubule cytoskeleton network orchestrates cellular dynamics and chromosome stability in mitosis. Although tubulin acetylation is essential for cellular plasticity, it has remained elusive how kinetochore microtubule plus-end dynamics are regulated by p300/CBP-associated factor (PCAF) acetylation in mitosis. Here, we demonstrate that the plus-end tracking protein, TIP150, regulates dynamic kinetochore-microtubule attachments by promoting the stability of spindle microtubule plus-ends. Suppression of TIP150 by siRNA results in metaphase alignment delays and perturbations in chromosome biorientation. TIP150 is a tetramer that binds an end-binding protein (EB1) dimer through the C-terminal domains, and overexpression of the C-terminal TIP150 or disruption of the TIP150-EB1 interface by a membrane-permeable peptide perturbs chromosome segregation. Acetylation of EB1-PCAF regulates the TIP150 interaction, and persistent acetylation perturbs EB1-TIP150 interaction and accurate metaphase alignment, resulting in spindle checkpoint activation. Suppression of the mitotic checkpoint serine/threonine protein kinase, BubR1, overrides mitotic arrest induced by impaired EB1-TIP150 interaction, but cells exhibit whole chromosome aneuploidy. Thus, the results identify a mechanism by which the TIP150-EB1 interaction governs kinetochore microtubule plus-end plasticity and establish that the temporal control of the TIP150-EB1 interaction by PCAF acetylation ensures chromosome stability in mitosis.     

Soluble interleukin‐1 receptor,a potential negative regulator of orange‐spotted grouper Epinephelus coioides interleukin‐1 system

In this study, the cDNA sequence encoding interleukin‐1 (Il‐1) receptor‐like protein of orange‐spotted grouper Epinephelus coioides was obtained. The newly identified sequence was named soluble type I Il‐1 receptor (sIl‐1rI) owing to its structural composition, which had two Ig‐like domains, lack of transmembrane region and the Toll/interleukin‐1 receptor (TIR) domain, similar to the brown rat Rattus norvegicus soluble Il‐1rI. In addition, sequence comparison and phylogenetic analysis indicated that E. coioides sequence had a closer relationship with Il‐1rI than Il‐1rII. Real‐time PCR revealed that sil‐1rI mRNA expression presented a process of decrease, restoration and increase in Cryptocaryon irritans‐infected E. coioides. The negative correlation between Il‐1β and sil‐1rI mRNA in C. irritans‐infected head‐kidney implied the potential negative regulatory role of sil‐1rI in E. coioides Il‐1 system. The leucocytes incubated with lipopolysaccharide or polyriboinosinic polyribocytidylic acid exhibited different expression profiles of sil‐1rI. Recombinant Il‐1β (rIl‐1β) protein was capable of inducing sil‐1rI mRNA under the concentration of 100 ng ml^?1, suggesting that high dosage or excess Il‐1β would stimulate the expression of sil‐1rI to maintain the homoeostasis of E. coioides Il‐1 system. For the first time, the role of teleost Il‐1rI in parasite infection has been identified, and soluble Il‐1r was found in fish.     

Cortical GluK1 kainate receptors modulate scratching in adult mice

Recent investigations into the mechanisms mediating itch transmission have focused on spinal mechanisms, whereas few studies have investigated the role of the cerebral cortex in itch‐related behaviors. Human imaging studies show that several cortical regions are active in correspondence with itch, including the anterior cingulate cortex (ACC). We present here evidence of cortical modulation of pruritogen‐induced scratching behavior. We combine pharmacological, genetic, and electrophysiological approaches to show that cortical GluK1‐containing kainate (KA) receptors are involved in scratching induced by histamine and non‐histamine‐dependent itching stimuli. We further show that scratching corresponds with enhanced excitatory transmission in the ACC through KA receptor modulation of inhibitory circuitry. In addition, we found that inhibiting GluK1‐containing KA receptors in the ACC also reduced behavioral nociceptive responses induced by formalin. Our results reveal a new role of the cortex in pruritogen‐induced scratching.

     

Drought-induced H2O2 accumulation in subsidiary cells is involved in regulatory signaling of stomatal closure in maize leaves

Increasing H₂O₂ levels in guard cells in response to environmental stimuli are recently considered a general messenger involved in the signaling cascade for the induction of stomatal closure. But little is known as to whether subsidiary cells participate in the H₂O₂-mediated stomatal closure of grass plants. In the present study, 2-week-old seedlings of maize (Zea mays) were exposed to different degrees of soil water deficit for 3 weeks. The effects of soil water contents on leaf ABA and H₂O₂ levels and stomatal aperture were investigated using physiological, biochemical, and histochemical approaches. The results showed that even under well-watered conditions, significant amounts of H₂O₂ were observed in guard cells, whereas H₂O₂ concentrations in the subsidiary cells were negligible. Decreasing soil water contents led to a significant increase in leaf ABA levels associated with significantly enhanced O₂ ^? and H₂O₂ contents, consistent with reduced degrees of stomatal conductance and aperture. The significant increase in H₂O₂ appeared in both guard cells and subsidiary cells of the stomatal complex, and H₂O₂ levels increased with decreasing soil water contents. Drought-induced increase in the activity of antioxidative enzymes could not counteract the significant increase in H₂O₂ levels in guard cells and subsidiary cells. These results indicate that subsidiary cells participate in H₂O₂-mediated stomatal closure, and drought-induced H₂O₂ accumulation in subsidiary cells is involved in the signaling cascade regulating stomatal aperture of grass plants such as maize.