Potent and selective disruption of protein kinase D functionality by a benzoxoloazepinolone

Protein kinase D (PKD) is a novel family of serine/threonine kinases targeted by the second messenger diacylglycerol. It has been implicated in many important cellular processes and pathological conditions. However, further analysis of PKD in these processes is severely hampered by the lack of a PKD-specific inhibitor that can be readily applied to cells and in animal models. We now report the discovery of the first potent and selective cell-active small molecule inhibitor for PKD, benzoxoloazepinolone (CID755673). This inhibitor was identified from the National Institutes of Health small molecule repository library of 196,173 compounds using a human PKD1 (PKCmu)-based fluorescence polarization high throughput screening assay. CID755673 suppressed half of the PKD1 enzyme activity at 182 nm and exhibited selective PKD1 inhibition when compared with AKT, polo-like kinase 1 (PLK1), CDK activating kinase (CAK), CAMKIIalpha, and three different PKC isoforms. Moreover, it was not competitive with ATP for enzyme inhibition. In cell-based assays, CID755673 blocked phorbol ester-induced endogenous PKD1 activation in LNCaP cells in a concentration-dependent manner. Functionally, CID755673 inhibited the known biological actions of PKD1 including phorbol ester-induced class IIa histone deacetylase 5 nuclear exclusion, vesicular stomatitis virus glycoprotein transport from the Golgi to the plasma membrane, and the ilimaquinone-induced Golgi fragmentation. Moreover, CID755673 inhibited prostate cancer cell proliferation, cell migration, and invasion. In summary, our findings indicate that CID755673 is a potent and selective PKD1 inhibitor with valuable pharmacological and cell biological potential.     

Natural occurrence of microbial sulphur oxidation by long-range electron transport in the seafloor

Recently, a novel mode of sulphur oxidation was described in marine sediments, in which sulphide oxidation in deeper anoxic layers was electrically coupled to oxygen reduction at the sediment surface. Subsequent experimental evidence identified that long filamentous bacteria belonging to the family Desulfobulbaceae likely mediated the electron transport across the centimetre-scale distances. Such long-range electron transfer challenges some long-held views in microbial ecology and could have profound implications for sulphur cycling in marine sediments. But, so far, this process of electrogenic sulphur oxidation has been documented only in laboratory experiments and so its imprint on the seafloor remains unknown. Here we show that the geochemical signature of electrogenic sulphur oxidation occurs in a variety of coastal sediment environments, including a salt marsh, a seasonally hypoxic basin, and a subtidal coastal mud plain. In all cases, electrogenic sulphur oxidation was detected together with an abundance of Desulfobulbaceae filaments. Complementary laboratory experiments in intertidal sands demonstrated that mechanical disturbance by bioturbating fauna destroys the electrogenic sulphur oxidation signal. A survey of published geochemical data and 16S rRNA gene sequences identified that electrogenic sulphide oxidation is likely present in a variety of marine sediments with high sulphide generation and restricted bioturbation, such as mangrove swamps, aquaculture areas, seasonally hypoxic basins, cold sulphide seeps and possibly hydrothermal vent environments. This study shows for the first time that electrogenic sulphur oxidation occurs in a wide range of marine sediments and that bioturbation may exert a dominant control on its natural distribution.     

Prolonged membrane potential depolarization in cingulate pyramidal cells after digit amputation in adult rats

The anterior cingulate cortex (ACC) plays an important role in higher brain functions including learning, memory, and persistent pain. Long-term potentiation of excitatory synaptic transmission has been observed in the ACC after digit amputation, which might contribute to plastic changes associated with the phantom pain. Here we report a long-lasting membrane potential depolarization in ACC neurons of adult rats after digit amputation in vivo. Shortly after digit amputation of the hind paw, the membrane potential of intracellularly recorded ACC neurons quickly depolarized from ~-70 mV to ~-15 mV and then slowly repolarized. The duration of this amputation-induced depolarization was about 40 min. Intracellular staining revealed that these neurons were pyramidal neurons in the ACC. The depolarization is activity-dependent, since peripheral application of lidocaine significantly reduced it. Furthermore, the depolarization was significantly reduced by a NMDA receptor antagonist MK-801. Our results provide direct in vivo electrophysiological evidence that ACC pyramidal cells undergo rapid and prolonged depolarization after digit amputation, and the amputation-induced depolarization in ACC neurons might be associated with the synaptic mechanisms for phantom pain.     

细胞周期与细胞凋亡

从海洋生物胚胎细胞到哺乳动物的细胞周期,主要是在其细胞周期基因产物周期素及Ｐ３４的调控下启动,运行和脱出周期的;某些原癌基因或抑癌基因的产物如ｐ５３,ｐＲＢ也直接调控着细胞周期。     

The effect of genistein supplementation on performance and antioxidant status of Japanese Quail under heat stress

Genistein, a phytoestrogen found in soybeans, is a powerful antioxidant. We evaluated the effects of genistein supplementation on performance, carcass characteristics, levels of malondialdehyde (MDA), homocysteine, vitamins C, E, A in Japanese quail (Coturnix coturnix japonica) exposed to high ambient temperature of 34°C. Two hundred and forty Japanese quails (10 d old) were randomly assigned to eight treatment groups consisting of 10 replicates of three birds. The birds were kept in an environmental controlled room either for 24 h/d at 22°C with (thermoneutral, TN groups) or for 16 h/d at 22°C and for 8 h/d (09.00 am to 05.00 pm) at 34°C (heat stress, HS groups). Birds were fed either a basal (control) diet (TN and HS) or the basal diet supplemented with 200, 400 or 800 mg of genistein per kg of diet. Heat exposure decreased birds' performance when basal diet was fed. Increase in feed intake and body weight, and improvement of feed efficiency and carcass traits were found in genistein-supplemented quails reared under heat stress conditions. Growth rate and feed efficiency improved in quails reared under thermo-neutral conditions as well. Concentration of serum vitamins C, E, and A increased in supplemented birds reared at high temperature, while non-significant changes occurred in TN groups. With genistein supplementation homocysteine levels in serum and MDA levels in serum and liver decreased in all birds of both TN and HS groups. Effects of genistein were relatively greater in heat-stressed quails than in quails kept under thermo-neutral conditions. Results of the present study suggest that supplementation with genistein can be considered to be protective by reducing the negative effects of oxidative stress induced by heat stress in quail.     

Identifying actives from HTS data sets: practical approaches for the selection of an appropriate HTS data-processing method and quality control review

High-throughput screening (HTS) has achieved a dominant role in drug discovery over the past 2 decades. The goal of HTS is to identify active compounds (hits) by screening large numbers of diverse chemical compounds against selected targets and/or cellular phenotypes. The HTS process consists of multiple automated steps involving compound handling, liquid transfers, and assay signal capture, all of which unavoidably contribute to systematic variation in the screening data. The challenge is to distinguish biologically active compounds from assay variability. Traditional plate controls-based and non-controls-based statistical methods have been widely used for HTS data processing and active identification by both the pharmaceutical industry and academic sectors. More recently, improved robust statistical methods have been introduced, reducing the impact of systematic row/column effects in HTS data. To apply such robust methods effectively and properly, we need to understand their necessity and functionality. Data from 6 HTS case histories are presented to illustrate that robust statistical methods may sometimes be misleading and can result in more, rather than less, false positives or false negatives. In practice, no single method is the best hit detection method for every HTS data set. However, to aid the selection of the most appropriate HTS data-processing and active identification methods, the authors developed a 3-step statistical decision methodology. Step 1 is to determine the most appropriate HTS data-processing method and establish criteria for quality control review and active identification from 3-day assay signal window and DMSO validation tests. Step 2 is to perform a multilevel statistical and graphical review of the screening data to exclude data that fall outside the quality control criteria. Step 3 is to apply the established active criterion to the quality-assured data to identify the active compounds.     

Identification of druggable targets for radiation mitigation using a small interfering RNA screening assay

Currently, there is a serious absence of pharmaceutically attractive small molecules that mitigate the lethal effects of an accidental or intentional public exposure to toxic doses of ionizing radiation. Moreover, cellular systems that emulate the radiobiologically relevant cell populations and that are suitable for high-throughput screening have not been established. Therefore, we examined two human pluripotent embryonal carcinoma cell lines for use in an unbiased phenotypic small interfering RNA (siRNA) assay to identify proteins with the potential of being drug targets for the protection of human cell populations against clinically relevant ionizing radiation doses that cause acute radiation syndrome. Of the two human cell lines tested, NCCIT cells had optimal growth characteristics in a 384 well format, exhibited radiation sensitivity (D(0) = 1.3 ± 0.1 Gy and ? = 2.0 ± 0.6) comparable to the radiosensitivity of stem cell populations associated with human death within 30 days after total-body irradiation. Moreover, they internalized siRNA after 4 Gy irradiation enabling siRNA library screening. Therefore, we used the human NCCIT cell line for the radiation mitigation study with a siRNA library that silenced 5,520 genes known or hypothesized to be potential therapeutic targets. Exploiting computational methodologies, we identified 113 siRNAs with potential radiomitigative properties, which were further refined to 29 siRNAs with phosphoinositide-3-kinase regulatory subunit 1 (p85α) being among the highest confidence candidate gene products. Colony formation assays revealed radiation mitigation when the phosphoinositide-3-kinase inhibitor LY294002 was given after irradiation of 32D cl 3 cells (D(0) = 1.3 ± 0.1 Gy and ? = 2.3 ± 0.3 for the vehicle control treated cells compared to D(0) = 1.2 ± 0.1 Gy and ? = 6.0 ± 0.8 for the LY294002 treated cells, P = 0.0004). LY294002 and two other PI3K inhibitors, PI 828 and GSK 1059615, also mitigated radiation-induced apoptosis in NCCIT cells. Treatment of mice with a single intraperitoneal LY294002 dose of 30 mg/kg at 10 min, 4, or 24 h after LD(50/30) whole-body dose of irradiation (9.25 Gy) enhanced survival. This study documents that an unbiased siRNA assay can identify new genes, signaling pathways, and chemotypes as radiation mitigators and implicate the PI3K pathway in the human radiation response.