Adenosine A2A Receptor Signaling and Golf Assembly Show a Specific Requirement for the γ7 Subtype in the Striatum

The adenosine A_2A receptor (A_2AR) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein α_olf subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ₇ subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ₇ protein controls the hierarchial assembly of a specific G-protein α_olfβ₂γ₇ heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A_2AR activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ₇ subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A_2AR G-protein α_olfβ₂γ₇ interface as a possible therapeutic target for Parkinson disease.     

表皮生长因子受体基因突变检测方法改进及初步临床验证

目的:改进现有的检测表皮生长因子受体(EGFR)基因突变的荧光PCR法并开发出新的试剂盒,将其与直接测序法和ARMS法进行对比,验证该试剂盒用于临床诊断的敏感性、特异性和准确性。方法:收集2013年6月至2015年8月手术确诊的141例非小细胞肺癌(NSCLC)的石蜡包埋组织标本。采用盲法分别使用直接测序法、ARMS法和新试剂盒检测EGFR突变,比较新试剂盒与其他两种检测方法的差异,结果不一致时采用三种方法分别重复检验一次。结果:三种方法检测成功率均为100%,新试剂盒与直接测序法测得结果完全一致的比率达75.9%(107/141),在直接测序法测得的96例突变阳性中,92例在新试剂盒检测中得到验证(95.8%)。而直接测序法显示突变阴性的45例中,新试剂盒检测发现了23例突变阳性,两种检测方法的结果存在统计学差异(x2=40.745,P0.05)。与直接测序法进行比较,新试剂盒检测EGFR突变的敏感性、特异性分别为95.8%、48.9%,阳性预测值、阴性预测值分别为80.0%、84.6%,检测准确度为80.9%。以ARMS检测法为金标准,新试剂盒测得结果完全一致的比率达84.4%(119/141),两者的一致性比较好(K=0.749,P0.05),敏感性、特异性分别为94.1%、86.4%。结论:改进后EGFR基因突变检测的试剂盒在技术上较好地控制了检测结果的假阳性和假阴性,该检测方法较直接测序法具有更好的敏感性和准确性,与现有的ARMS法一致性较高。     

Rabbit small intestine does not contain an annexin II/caveolin 1 complex as a target for 2-azetidinone cholesterol absorption inhibitors

Intestinal cholesterol absorption is specifically inhibited by the 2-azetidinone cholesterol absorption inhibitor ezetimibe. Photoreactive ezetimibe analogues specifically label a 145-kDa protein in the brush border membrane of enterocytes from rabbit small intestine identified as aminopeptidase N (CD13). In zebrafish and mouse small intestinal cytosol, a heterocomplex of M_r 52 kDa between annexin II and caveolin 1 was suggested as a target of ezetimibe. In contrast, in the cytosol and brush border membrane vesicles (BBMV) from rabbit small intestine of control animals or rabbits treated with the nonabsorbable cholesterol absorption inhibitor AVE 5530, both annexin II and caveolin 1 were exclusively present as monomers without any heterocomplex formation. Upon immunoprecipitation with annexin II a 52-kDa band was observed after immunostaining with annexin II antibodies, whereas no staining of a 52-kDa band occurred with anti-caveolin 1 antibodies. Vice versa, a 52-kDa band obtained by immunoprecipitation with caveolin 1 antibodies did not stain with annexin II-antibodies. The intensity of the 52-kDa band was dependent on the amount of antibody and was also observed with anti-actin or anti-APN antibodies suggesting that the 52-kDa band is a biochemical artefact. After incubation of cytosol or BBMV with radioactively labelled ezetimibe analogues, no significant amounts of the ezetimibe analogues could be detected in the immunoprecipitate with caveolin-1 or annexin II antibodies. Photoaffinity labelling of rabbit small intestinal BBMV with ezetimibe analogues did not result in labelling of proteins being immunoreactive with annexin II, caveolin 1 or a 52-kDa heterocomplex. These findings indicate that the rabbit small intestine does not contain an annexin II/caveolin 1 heterocomplex as a target for ezetimibe.     

On measurements of the higher harmonics of transmembrane current

The higher harmonics of the current caused by an alternating voltage applied to bilayer lipid membranes made of diphytanoyl phosphatidylcholine (DPhPC) in decane and tetradecane were measured. A universal relation between the amplitudes of harmonics was proposed and experimentally verified. This allowed the coefficients of expansion of the capacitance in even powers of voltage to be calculated for the DPhPC membrane in tetradecane; it also permitted comparison of the inhomogeneity in the thickness of the DPhPC membranes in decane and tetradecane.     

Comparison of basic peptides- and lipid-based strategies for the delivery of splice correcting oligonucleotides

Expression of alternatively spliced mRNA variants at specific stages of development or in specific cells and tissues contributes to the functional diversity of the human genome. Aberrations in alternative splicing were found as a cause or a contributing factor to the development, progression, or maintenance of numerous diseases. The use of antisense oligonucleotides (ON) to modify aberrant expression patterns of alternatively spliced mRNAs is a novel means of potentially controlling such diseases. Oligonucleotides can be designed to repair genetic mutations, to modify genomic sequences in order to compensate for gene deletions, or to modify RNA processing in order to improve the effects of the underlying gene mutation. Steric block ON approach have proven to be effective in experimental model for various diseases. Here, we describe our experience in investigating two strategies for ON delivery: ON conjugation with basic peptides and lipid-based particulate system (lipoplex). Basic peptides or Cell Penetrating Peptides (CPP) such as the TAT-derived peptide appear to circumvent many problems associated with ON and drug delivery. This strategy may represent the next paradigm in our ability to modulate cell function and offers a unique avenue for the treatment of disease. Lipoplexes result from the intimate interaction of ON with cationic lipids leading to ON carrying particles able to be taken up by cells and to release ON in the cytoplasm. We have used as an experimental model the correction of a splicing alteration of the mutated β-globin intron causing thalassemia. Data on cell penetration and efficacy of correction of specific steric block ON delivered either by basic peptides or lipoplex are described. A comparison of the properties of both delivery systems is made respective to the use of this new class of therapeutic molecules.     

Transbilayer Movement of Sphingomyelin Precedes Catastrophic Breakage of Enterobacteria-Containing Vacuoles

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β-Carboline copper complex as a potential mitochondrial-targeted anticancer chemotherapeutic agent: Favorable attenuation of human breast cancer MCF7 cells via apoptosis

The development of preferentially selective cancer chemotherapeutics is a new trend in drug research. Thus, we designed and synthesized novel ternary complexes, [Cu(tryp)(Hnor)₂(DMSO)]NO₃ (1) and [Zn(tryp)(Hnor)₂(DMSO)]NO₃ (2) (tryp = DL-Tryptophane; Hnor = Norharmane, β-carboline; DMSO = Dimethyl sulfoxide), characterized with elemental analysis, FTIR, UV–vis, FL, NMR, ESI-MS, and molar conductivity. Furthermore, the TD-DFT studies with UV–vis and FTIR validated the proposed structures of 1 and 2. Moreover, we evaluated the HOMO-LUMO energy gap and found that 1 has a smaller energy gap than 2. Then, 1 and 2 were assessed for anticancer chemotherapeutic potential against cancer cell lines MCF7 (human breast cancer) and HepG2 (human liver hepatocellular carcinoma) as well as the non-tumorigenic HEK293 (human embryonic kidney) cells. The MTT assay illustrated the preferentially cytotoxic behavior of 1 when compared with that of 2 and cisplatin (standard drug) against MCF7 cells. Moreover, 1 was exposed to MCF7 cells, and the results indicated the arrest of the G2/M phases, which followed the apoptotic pathway predominantly. Generation of ROS, GSH depletion, and elevation in LPO validated the redox changes prompted by 1. These studies establish the great potential of 1 as a candidate for anticancer therapeutics.     

Cell surface energy,contact angles and phase partition III. Adhesion of bacterial cells to hydrophobic surfaces

The densities of adhesion of Staphylococcus epidermis, Staphylococcus aureus and Serratia marcescens to five types of plastics were studied in relation to interfacial free energies at the aqueous interfaces of both the bacteria and the plastics. The free energy of adhesion of bacteria to plastic in an aqueous medium is a linear function of partition of the bacteria between the solid surface and the liquid phase. These results show that the thermodynamics of the partitioning of a suspended particle between two immiscible liquid phases also apply to partitioning between a liquid and a solid phase.