Sprouty proteins are targeted to membrane ruffles upon growth factor receptor tyrosine kinase activation. Identification of a novel translocation domain

Sprouty (Spry) was first identified in a genetic screen in Drosophila to be an antagonist of fibroblast growth factor and epidermal growth factor (EGF) signaling, seemingly by inhibiting the Ras/MAP kinase pathway. Data base searches lead to the identification and cloning of, to date, four mammalian sprouty genes. The primary sequences of the mammalian sprouty gene products share a well conserved cysteine-rich C-terminal domain with the Drosophila protein. The N-terminal regions, however, do not exhibit significant homology. This study aimed at determining the disposition of Spry proteins in intact cells before and after stimulation of the EGF receptor tyrosine kinase. Full-length or deletion mutants of Spry, tagged at the N termini with the FLAG-epitope, were expressed in COS-1 cells by transient transfection and analyzed by immunofluorescence microscopy before and after EGF stimulation of the cells. In unstimulated cells, the Spry proteins were distributed throughout the cytosol except for human Sprouty2 (hSpry2), which, although generally located in the cytosol, co-localized with microtubules. In all cases, the Spry proteins underwent rapid translocation to membrane ruffles following EGF stimulation. The optimal translocation domain was identified by deletion and immunofluorescence analysis to be a highly conserved 105-amino acid domain in the C-terminal half of the hSpry2 protein. The translocation of this conserved domain, based on hSpry2 data, was independent of the activation of phosphatidylinositol-3 kinase.     

A highly sensitive biosensor with (Con A/HRP)n multilayer films based on layer-by-layer technique for the detection of reduced thiols

The bilayer of Con A/HRP through the biospecific affinity of concanavalin A (Con A) and glycoprotein horseradish peroxidase (HRP) was prepared on the surface of an Au electrode modified by the precursor film consisted of poly(allylamine hydrochloride) poly(sodium-p-styrene-sulfonate). Atomic force microscopy and electrochemical impedance spectroscopy were adopted to monitor the uniform layer-by-layer assembly of the Con A/HRP bilayers. The amperometric measurement was based on the inhibition of reduced thiols and performed in the presence of the electron mediator hydroquinone in 0.2 M phosphate buffer of pH 6.5 at an applied potential of −0.15 V versus Ag/AgCl. Under the optimal conditions, the biosensor presented a linear response for cysteine from 0.1 to 23.5 μM, with a detection limit of 0.02 μM. The biosensor demonstrated high stability and repeatability. A series of reduced thiols were detected by this inhibition biosensor and oxidized thiols showed no effect on the current response of the biosensor.     

Structural hybridization of pyrrolidine-based T-type calcium channel inhibitors and exploration of their analgesic effects in a neuropathic pain model

Highly effective and safe drugs for the treatment of neuropathic pain are urgently required and it was shown that blocking T-type calcium channels can be a promising strategy for drug development for neuropathic pain. We have developed pyrrolidine-based T-type calcium channel inhibitors by structural hybridization and subsequent assessment of in vitro activities against Ca_v3.1 and Ca_v3.2 channels. Profiling of in vitro ADME properties of compounds was also carried out. The representative compound 17h showed comparable in vivo efficacy to gabapentin in the SNL model, which indicates T-type calcium channel inhibitors can be developed as effective therapeutics for neuropathic pain.     

七姊妹山常绿落叶阔叶混交林物种多样性格局及其环境解释

依托七姊妹山自然保护区6 hm²森林动态监测样地研究平台,基于样地和物种基本信息数据,采用多元回归树和冗余分析研究方法,探讨地形因子对生境的塑造作用及物种分布特征,分析不同群丛类型下物种多样性的变化规律。结果表明：(1)依据“1 SE”规则,4次分割依次以海拔(1 453 m)、坡度(23.13°)、海拔(1 398 m)、凹凸度(4.094)为分界点可将150个样地分为5个群丛。(2)冗余分析表明地形因子对物种分布解释量为0.077 6,解释率为16.36%,各环境因子对物种分布的解释力度依次为：海拔>坡度>凹凸度;坡向与物种的分布无显著相关性。(3)5个群丛中立木密度与胸高截面积最高的均为群丛5(527.4株/400 m²;3.495 cm²/株),立木密度与平均胸高截面积最低为群丛4(225.4株/400 m²;3.057 cm²/株)。(4)5个群丛中Shannon Winener丰富度指数与Simpson优势度指数最高的均为群丛2,最低的为群丛5,物种多样性尺度效应明显;Pielou均匀度指数最高为群丛4,最低为群丛5。(5)两两群丛间Jaccard相似性系数最低为群丛1 群丛2(0.331),最高的为群丛4 群丛5(0.645),海拔对β多样性格局影响较大。研究认为,七姊妹山自然保护区6 hm²样地地形因子对该区域生境的塑造具有一定作用,海拔、坡度、凹凸度组成的“环境筛”影响了该区域的物种分布及多样性格局。     

The Soluble Periplasmic Domains of Escherichia coli Cell Division Proteins FtsQ/FtsB/FtsL Form a Trimeric Complex with Submicromolar Affinity

Cell division in Escherichia coli involves a set of essential proteins that assembles at midcell to form the so-called divisome. The divisome regulates the invagination of the inner membrane, cell wall synthesis, and inward growth of the outer membrane. One of the divisome proteins, FtsQ, plays a central but enigmatic role in cell division. This protein associates with FtsB and FtsL, which, like FtsQ, are bitopic inner membrane proteins with a large periplasmic domain (denoted FtsQ_p, FtsB_p, and FtsL_p) that is indispensable for the function of each protein. Considering the vital nature and accessible location of the FtsQBL complex, it is an attractive target for protein-protein interaction inhibitors intended to block bacterial cell division. In this study, we expressed FtsQ_p, FtsB_p, and FtsL_p individually and in combination. Upon co-expression, FtsQ_p was co-purified with FtsB_p and FtsL_p from E. coli extracts as a stable trimeric complex. FtsB_p was also shown to interact with FtsQ_p in the absence of FtsL_p albeit with lower affinity. Interactions were mapped at the C terminus of the respective domains by site-specific cross-linking. The binding affinity and 1:1:1 stoichiometry of the FtsQ_pB_pL_p complex and the FtsQ_pB_p subcomplex were determined in complementary surface plasmon resonance, analytical ultracentrifugation, and native mass spectrometry experiments.     

Effect of pre-treatment with St John's Wort on nephrotoxicity of cisplatin in rats

An herbal health care supplement, St John's Wort (SJW, Hypericum perforatum) has become widely used in the treatment of depression, and is known to interact with therapeutic drugs. Here we report a preventive effect of SJW on cisplatin nephrotoxicity in rats. Rats were given SJW (400 mg/kg/day, p.o.) for 10 consecutive days, and were injected with cisplatin (5 mg/kg, i.v.) on the day after the final SJW treatment. Cisplatin treatment increased the serum creatinine level, which is an index of nephrotoxicity, to 1.51+/-0.22 mg/dl (mean+/-SE) from 0.28+/-0.05 mg/dl (control) on day 5 after the cisplatin injection. This increase fell significantly to 0.86+/-0.13 mg/dl by pre-treatment with SJW. Cisplatin-induced histological abnormality of the kidney was blocked by pre-treatment with SJW. When SJW was administered for 10 days, the amounts of renal metallothionein (MT) and hepatic multidrug resistance protein 2 (Mrp2) were increased to 164.8+/-13.0% and 220.8+/-39.3% (mean+/-SE) of controls, respectively. GSH levels in the kidney and liver were not changed. Total and free cisplatin concentration in serum was not influenced by SJW treatment. In conclusion, the results suggest that pre-treatment with SJW may diminish cisplatin nephrotoxicity.     

The myocardial response to adrenomedullin involves increased cAMP generation as well as augmented Akt phosphorylation

In this work we aimed to observe (1) the changes in adrenomedullin (AM) and its receptor system - calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) - in myocardial ischemic injury and (2) the response of injuried myocardia to AM and the phosphorylation of Akt to illustrate the protective mechanism of AM in ischemic myocardia. Male SD rats were subcutaneously injected with isoproterenol (ISO) to induce myocardial ischemia. The mRNA levels of AM, CRLR, RAMP1, RAMP2 and RAMP3 were determined by RT-PCR. Protein levels of Akt, phosphor-Akt, CRLR, RAMP1, RAMP2 and RAMP3 were assayed by Western blot. Results showed that, compared with that of the controls, ISO-treated rats showed lower cardiac function and myocardial injury. The mRNA relative amount of AM, CRLR, RAMP1, RAMP2 and RAMP3 in the myocardia of ISO-treated rats was increased. The elevated mRNA levels of CRLR, RAMP1, RAMP2 and RAMP3 were positively correlated with AM content in injured myocardia. The protein levels of CRLR, RAMP1, RAMP2 and RAMP3 in injured myocardia were increased compared with that of control myocardia. AM-stimulated cAMP generation in myocardia was elevated in the ISO group, and was antagonized by AM(22-52) and CGRP(8-37). Western blot analyses revealed that AM significantly enhanced Akt phosphorylation in injured myocardia, which was blocked by pretreatment with AM(22-52) or CGRP(8-37). Ischemia-injured myocardia hyper-expressed AM and its receptors - CRLR, RAMP1, RAMP2 and RAMP3 - and the response of ischemic myocardia to AM was potentiated, and the level of Akt phosphorylation was also increased, which suggests that changes in cardiac AM/AM receptor might play an important role in the pathogenesis of myocardial ischemic injury.     

Design,synthesis and biological evaluation of hypolipidemic compounds based on BRD4 inhibitor RVX-208

Bromodomain-containing protein 4 (BRD4) is a new therapeutic target for the treatment of diseases including cardiovascular diseases, cancer, inflammation and central nervous system (CNS) disorders. In this study, we introduced the pharmacophore of fibrates to a BRD4 inhibitor, RVX-208, to design dual-active hypolipidemic compounds, and found that some of new analogues showed favorable hypolipidemic activities. Synthetic accessibility towards this class of compounds optimized RVX-208 as well as would supply more thoughts on hypolipidemic drugs.