A quantitative analysis of kinase inhibitor selectivity

Kinase inhibitors are a new class of therapeutics with a propensity to inhibit multiple targets. The biological consequences of multi-kinase activity are poorly defined, and an important step toward understanding the relationship between selectivity, efficacy and safety is the exploration of how inhibitors interact with the human kinome. We present interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome. The data constitute the most comprehensive study of kinase inhibitor selectivity to date and reveal a wide diversity of interaction patterns. To enable a global analysis of the results, we introduce the concept of a selectivity score as a general tool to quantify and differentiate the observed interaction patterns. We further investigate the impact of panel size and find that small assay panels do not provide a robust measure of selectivity.     

短毛柽柳不同部位中总黄酮含量及稳定性研究

目的：比较短毛柽柳中不同部位总黄酮的含量差异,研究黄酮化合物保存的条件,方法：采用超声波乙醇浸提法,在正交优先的最佳条件（料液比为1∶15,超声处理时间为40min,乙醇浓度为45%,超声温度50℃,提取3次）下提取不同部位总黄酮,并测定不同条件下其吸光度的变化。结果：柽柳花、叶、茎中总黄酮得率分别为24.585mg/g、25.623mg/g、41.726mg/g,柽柳中提取的黄酮溶液在光照、高温、高浓度蔗糖溶液和Na＋存在条件下不稳定;pH 4~7条件下较稳定;氧化剂对其稳定性的影响不大。结论：柽柳茎中总黄酮含量明显高于其他两个部位。柽柳黄酮保存时应避免接触碳水化合物和盐离子,在避光、常温、中性条件下保存。     

Spatial and temporal control of cofilin activity is required for directional sensing during chemotaxis

BACKGROUND: Previous work has led to the hypothesis that cofilin severing, as regulated by PLC, is involved in chemotactic sensing. We have tested this hypothesis by investigating whether activation of endogenous cofilin is spatially and temporally linked to sensing an EGF point source in carcinoma cells. RESULTS: We demonstrate that inhibition of endogenous cofilin activity with either siRNA or overexpression of LIMK suppresses directional sensing in carcinoma cells. LIMK siRNA knockdown, which suppresses cofilin phosphorylation, and microinjection of S3C cofilin, a cofilin mutant that is constitutively active and not phosphorylated by LIMK, also inhibits directional sensing and chemotaxis. These results indicate that phosphorylation of cofilin by LIMK, in addition to cofilin activity, is required for chemotaxis. Cofilin activity concentrates rapidly at the newly formed leading edge facing the gradient, whereas cofilin phosphorylation increases throughout the cell. Quantification of these results indicates that the amplification of asymmetric actin polymerization required for protrusion toward the EGF gradient occurs at the level of cofilin but not at the level of PLC activation by EGFR. CONCLUSIONS: These results indicate that local activation of cofilin by PLC and its global inactivation by LIMK phosphorylation combine to generate the local asymmetry of actin polymerization required for chemotaxis.     

Interaction of calmodulin with Sec61α limits Ca2+ leakage from the endoplasmic reticulum

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)-binding motif in the cytosolic N-terminus of mammalian Sec61α that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. In single-channel measurements, CaM potently mediated Sec61-channel closure in Ca2+-dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+-CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER.     

水分梯度下胡杨荒漠河岸林种内竞争及空间格局变化特征

为查明塔里木河下游不同水分梯度下胡杨种内竞争以及空间分布格局的变化规律,在塔里木河下游阿拉干断面选取3条样带,每条样带内依照水分梯度布设3个样方,采用Hegyi单木竞争模型与点格局等分析方法,探究了水分梯度下胡杨林种内竞争及空间格局变化特征。结果表明：1)塔里木河下游胡杨的最适竞争范围为10m; 2)胡杨竞争指数与胸径服从幂函数关系(P<0.001),胡杨竞争指数随对象木胸径的增加而逐渐减小,当对象木胸径达到20cm以上时,其竞争指数逐渐稳定,且维持在较低水平;3)随水分梯度的降低,胡杨林分竞争指数呈下降趋势;4)在塔里木河下游,胡杨空间格局主要为聚集分布,随空间尺度的增加,胡杨逐渐趋向于随机分布;5)随水分梯度的降低,胡杨空间格局由聚集分布逐渐转变为随机分布。水分是决定极端干旱区荒漠植被空间分布与结构的主要因子;在胡杨林分管理中,应充分考虑胡杨的种内竞争、空间格局及其与水分梯度之间关系,为塔里木河下游胡杨林的保护与更新提供科学依据。     

In Vivo Long-Term Monitoring of Circulating Tumor Cells Fluctuation during Medical Interventions

The goal of this research was to study the long-term impact of medical interventions on circulating tumor cell (CTC) dynamics. We have explored whether tumor compression, punch biopsy or tumor resection cause dissemination of CTCs into peripheral blood circulation using in vivo fluorescent flow cytometry and breast cancer-bearing mouse model inoculated with MDA-MB-231-Luc2-GFP cells in the mammary gland. Two weeks after tumor inoculation, three groups of mice were the subject of the following interventions: (1) tumor compression for 15 minutes using 400 g weight to approximate the pressure during mammography; (2) punch biopsy; or (3) surgery. The CTC dynamics were determined before, during and six weeks after these interventions. An additional group of tumor-bearing mice was used as control and did not receive an intervention. The CTC dynamics in all mice were monitored weekly for eight weeks after tumor inoculation. We determined that tumor compression did not significantly affect CTC dynamics, either during the procedure itself (P = 0.28), or during the 6-week follow-up. In the punch biopsy group, we observed a significant increase in CTC immediately after the biopsy (P = 0.02), and the rate stayed elevated up to six weeks after the procedure in comparison to the tumor control group. The CTCs in the group of mice that received a tumor resection disappeared immediately after the surgery (P = 0.03). However, CTC recurrence in small numbers was detected during six weeks after the surgery. In the future, to prevent these side effects of medical interventions, the defined dynamics of intervention-induced CTCs may be used as a basis for initiation of aggressive anti-CTC therapy at time-points of increasing CTC number.     

Mechanisms of Blood Brain Barrier Disruption by Different Types of Bacteria,and Bacterial–Host Interactions Facilitate the Bacterial Pathogen Invading the Brain

This review aims to elucidate the different mechanisms of blood brain barrier (BBB) disruption that may occur due to invasion by different types of bacteria, as well as to show the bacteria–host interactions that assist the bacterial pathogen in invading the brain. For example, platelet-activating factor receptor (PAFR) is responsible for brain invasion during the adhesion of pneumococci to brain endothelial cells, which might lead to brain invasion. Additionally, the major adhesin of the pneumococcal pilus-1, RrgA is able to bind the BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1), thus leading to invasion of the brain. Moreover, Streptococcus pneumoniae choline binding protein A (CbpA) targets the common carboxy-terminal domain of the laminin receptor (LR) establishing initial contact with brain endothelium that might result in BBB invasion. Furthermore, BBB disruption may occur by S. pneumoniae penetration through increasing in pro-inflammatory markers and endothelial permeability. In contrast, adhesion, invasion, and translocation through or between endothelial cells can be done by S. pneumoniae without any disruption to the vascular endothelium, upon BBB penetration. Internalins (InlA and InlB) of Listeria monocytogenes interact with its cellular receptors E-cadherin and mesenchymal-epithelial transition (MET) to facilitate invading the brain. L. monocytogenes species activate NF-κB in endothelial cells, encouraging the expression of P- and E-selectin, intercellular adhesion molecule 1 (ICAM-1), and Vascular cell adhesion protein 1 (VCAM-1), as well as IL-6 and IL-8 and monocyte chemoattractant protein-1 (MCP-1), all these markers assist in BBB disruption. Bacillus anthracis species interrupt both adherens junctions (AJs) and tight junctions (TJs), leading to BBB disruption. Brain microvascular endothelial cells (BMECs) permeability and BBB disruption are induced via interendothelial junction proteins reduction as well as up-regulation of IL-1α, IL-1β, IL-6, TNF-α, MCP-1, macrophage inflammatory proteins-1 alpha (MIP1α) markers in Staphylococcus aureus species. Streptococcus agalactiae or Group B Streptococcus toxins (GBS) enhance IL-8 and ICAM-1 as well as nitric oxide (NO) production from endothelial cells via the expression of inducible nitric oxide synthase (iNOS) enhancement, resulting in BBB disruption. While Gram-negative bacteria, Haemophilus influenza OmpP2 is able to target the common carboxy-terminal domain of LR to start initial interaction with brain endothelium, then invade the brain. H. influenza type b (HiB), can induce BBB permeability through TJ disruption. LR and PAFR binding sites have been recognized as common routes of CNS entrance by Neisseria meningitidis. N. meningitidis species also initiate binding to BMECs and induces AJs deformation, as well as inducing specific cleavage of the TJ component occludin through the release of host MMP-8. Escherichia coli bind to BMECs through LR, resulting in IL-6 and IL-8 release and iNOS production, as well as resulting in disassembly of TJs between endothelial cells, facilitating BBB disruption. Therefore, obtaining knowledge of BBB disruption by different types of bacterial species will provide a picture of how the bacteria enter the central nervous system (CNS) which might support the discovery of therapeutic strategies for each bacteria to control and manage infection.     

Accumulation of cadmium, lead and strontium, and a role of calcium oxalate in water hyacinth tolerance

Eichhornia crassipes plants brought from the River Nile were cultured in jars containing river water supplemented with various concentrations of Cd, Pb, and Sr (0 to 100 μg cm-3), added simultaneously. Treatment continued for 20 d during which each cultivation solution was being replaced with fresh one every 3 d. The growth of Eichhornia was drastically retarded at heavy metal concentrations higher than 15 μg cm-3. At concentrations 15 or 25 μg cm-3, the accumulation of Cd and Pb to levels several times higher than those in control plants was found. More than 50 % of the uptaken metals were retained by roots alone. Leaves and leaf petiols received around 30 and 20 % of the accumulated metals, respectively. X-ray microanalysis indicated the presence of the three heavy metals in Ca oxalate crystals. Content of metals in the crystals increased progressively over time of exposure in a way similar to those in whole plant tissues. These results suggest a possible role for Ca oxalate crystalization in toxic heavy metal deposition and thus tolerance by Eichhornia. This revised version was published online in August 2006 with corrections to the Cover Date.     

Non-histone chromosomal proteins easily extractible from chick erythrocytes.

Those non-histone chromosomal proteins which are easily extractible from chick erythrocytes differ substantially from proteins similarly extracted from other tissues of various species. Although a protein P1 was isolated along with histone H1 by extraction of calf thymus chromatin with HC1O4, the same procedure did not extract this protein from chick erythrocyte chromatin of either normal or regenerating blood. Likewise , non-histone proteins extracted with 0.35 M NaCl from calf thymus differed from those of normal chick erythrocytes, which were qualitatively identical but quantitatively inferior to those of regenerating blood. The major protein of about 25 000 molecular weight, totally extracted with 0.35 M NaCl from calf thymus, was not found in chick erythrocyte chromatin, but rather another major protein of about 35 000 molecular weight was partially extracted from erythrocytes.     

Thermal transition of core particle is not a two-state process

Thermal transition of core particle which occurs before melting of DNA and can be followed by circular dichroism is not a two-state process; it is the result of two processes which cannot be dissociated in static experiments: unfolding of core particles is immediately followed by their aggregation. It is thus impossible to get thermodynamic parameters of core particle unfolding from its thermal transition monitored by circular dichroism. Thermal denaturation kinetics of core particles gives some information about their stability. Finally core particle structure is more stable in chromatin than in its isolated state.