Ad-p27mt转染重组腺病毒治疗裸鼠内人胃癌的分子机制

目的:研究Ad-p27mt转染重组腺病毒对人胃癌细胞凋亡的作用及机制.方法:Ad-p27mt转染重组腺病毒导入胃癌细胞株SGC-7901内;流式细胞仪检测凋亡染色体亚二倍体峰值,了解Ad-p27mt对人胃癌组织凋亡的作用;TUNEL法检测DNA片断,在Ad-p27mt组和Ad-LacZ组中分析细胞的凋亡.结果:Ad-p27mt成功转入人胃癌细胞SGC-7901内,转化率达100%.流式细胞仪检测发现在感染后18h出现G1-S相前出现凋亡染色体亚二倍体峰值,并且DNA电泳出现凋亡特征性的条带;TUNEL法检测Ad-p27治疗组与对照组的凋亡率分别是92.3%±3.76%和2.01%±0.15%,两组的差异有显著性(P<0.01).结论:重组腺病毒转染的人p27突变基因能诱导裸鼠体内人胃癌细胞SGC-7901的凋亡.     

NERVOUS SYSTEM FUNCTION FOR THE TESTIS RNA-BINDING PROTEIN BOULE IN DROSOPHILA

The RNA-binding protein, Boule is conserved across species and is required for male fertility. Boule and the DAZ homologues in mice and humans appear specific to the testis. Boule functions in spermatogenesis by controlling the translation of the meiotic cell division cycle 25 (Cdc25) phosphatase, Twine. Here we show, for the first time, a function for the DAZ protein, Boule, outside of meiosis. We found that an isoform of Boule is expressed in the nervous system and when its expression is increased we observe mutant phenotypes in neural communication between the receptor and laminar cells of the fly eye, altered larval locomotion and when further overexpressed, viability. As in the germ line, genetic studies indicate that Boule functions in the Cdc25 phosphatase pathway in the nervous system. In a sensitized genetic background of Boule overexpression, we added a loss-of-function mutation of twine and demonstrated a role for Twine Cdc25, in the adult nervous system. Our results indicate that isoforms of boule are expressed outside of the male germ line and that these isoforms have a role in neural function, unlike the boule testis-specific isoform.     

ARE THE STRUCTURAL CHANGES IN ADULT DROSOPHILA MUSHROOM BODIES MEMORY TRACES? STUDIES ON BIOCHEMICAL LEARNING MUTANTS

The pre-imaginal development of Drosophila mushroom bodies is under the influence of an unknown variable which causes populations of wild-type flies at eclosion to differ in the average number of Kenyon cell fibers. During the first week of adult life the number adjusts to an intermediate level which depends upon the experience of the flies. Under olfactory deprivation or social isolation it reaches a lower level than under favorable rearing conditions (J. Neurogenet., 1 (1984) 113–126). The biochemical learning mutants dunce and rutabaga show no experince-dependent modulation of fiber number (Fig. 2). In both strains the mushroom bodies of young adults seem to develop abnormally; in dunce a loss of aboout 600 fibers is observed, in rutabaga fiber number is low at eclosion and does not increase (Fig. 1a). The following model for long-term memory is proposed: in mushroom bodies outgrowth and decay of Kenyon cell fibers occur simultaneously. The fibers randomly form transient synapses onto extrinsic output neurons of the mushroom bodies and receive synapses from modulating neurons. Experience consolidates certain synapses, thus prolonging survival of the respective Kenyon cell fibers and increasing the steady state level of fiber number (Fig. 3).     

Differentiation in Vitro of Neural Precursor Cells From Normal and Pecanex Mutant Drosophila Embryos

Early gastrula embryos, lacking both maternally and zygotically expressed activity of the neurogenic pecanex locus, are shown to contain a greater than wild-type number of stably determined neural precursor cells which can differentiate into neurons in culture.     

Three thioredoxin targets in the inner envelope membrane of chloroplasts function in protein import and chlorophyll metabolism

Thioredoxins (Trxs) are ubiquitous small proteins with a redox-active disulfide bridge. In their reduced form, they constitute very efficient protein disulfide oxidoreductases. In chloroplasts, two types of Trxs (f and m) coexist and play central roles in the regulation of the Calvin cycle and other processes. Here, we identified a class of Trx targets in the inner plastid envelope membrane of chloroplasts that share a CxxC motif approximately 73 aa from their carboxyl-terminal end. Members of this group belong to a superfamily of Rieske iron-sulfur proteins involved in protein translocation and chlorophyll metabolism. These proteins include the protein translocon protein TIC55, the precursor NADPH:protochlorophyllide oxidoreductase translocon protein PTC52, which operates as protochlorophyllide a-oxygenase, and the lethal leaf spot protein LLS1, which is identical with pheophorbide a oxygenase. The role of these proteins in dark/light regulation and oxidative control by the Trx system is discussed.     

QSAR of clinically important EGFR mutant L858R/T790M pyridinylimidazole inhibitors

EGFR is a well‐established therapeutic target of clinical relevance in cancer. However, acquisition of secondary mutation (T790M) makes first‐generation inhibitors ineffective. Therefore, to circumvent the problem of resistance, new T790M/L858R (TMLR) double mutant inhibitors are required. In this study, fragment‐based QSAR models (GQSAR) were generated for pyridinylimidazole derivatives having biological activity against TMLR mutants. The GQSAR model developed using partial least squares regression via stepwise forward–backward variable selection technique showed best results as judged using statistical parameters (r², q², and pred_r²). Additionally, applicability domain of the model was verified using Williams plot, which indicated that the predicted data are reliable. The GQSAR provided site‐specific clues wherein modifications related to decreasing lipophilic character and rotatable bonds and increasing SaaCHE‐index are required for improving inhibitory activity. Overall, the study indicated that the presence of acrylamide at R5 is essential for covalent bond formation with Cys797 and occurrence of aromatic residue at R2 is required for occupying hydrophobic region next to Met790 gatekeeper residue. Based on this information, new derivatives were designed that show better inhibitory activity than the experimentally reported most active molecules. Thus, the model developed can be used to design new pyridinylimidazole derivatives with improved TMLR bioactivity.     

联合IL-5可溶性受体与IL-4突变体对支气管哮喘小鼠气道高反应性及IgE、IFN-γ水平的影响

目的通过观察IL-5可溶性受体（sIL-5Ra）及IL-4突变体（IL-4mutant,IL-4MT）对哮喘小鼠气道高反应性（airwayhyperresponsiveness,AHR）及IgE、γ干扰素（IFN-γ）水平的影响,探讨其潜在的临床应用价值。方法随机将50只BALB／c雌性小鼠分成5组,分别为正常对照组、哮喘组、IL-4MT治疗组、sIL-5Ra治疗组和IL-4MT联合sIL-5Ra治疗组（简称联合治疗组）。采用腹腔注射卵蛋白（ovalbumin,OVA）与氢氧化铝混悬液对小鼠进行致敏,卵蛋白雾化,建立小鼠哮喘模型,其中正常对照组用生理盐水替代。雾化前30rain,IL-4MT治疗组、sIL-5Ra治疗组及联合治疗组分别腹腔注射IL-4MT100μg、sIL-5Ra100μg、IL-4MT和sIL～5Ra各100μg,正常对照组与哮喘组用生理盐水替代。末次激发24h后,用不同浓度的氯化乙酰胆碱激发各组小鼠,测定其肺阻力,ELISA法观察比较各组血清及BALFIgE、IFN-γ水平变化,肺组织行HE染色,观察病理变化。结果与正常对照组相比,哮喘组肺阻力显著增加（P〈0．01）;与哮喘组相比,IL-4MT组和联合治疗组肺阻力显著降低（P〈O．05）,联合治疗组下降更明显;与sIL-5Ra治疗组相比,联合治疗组肺阻力显著降低（P〈O．05）。与正常对照组相比,哮喘组血清及BALF中IgE含量显著升高,IFN-γ含量显著下降（P〈O．01）;与哮喘组相比,联合治疗组血清及BALF中IgE含量显著下降,IFN-γ含量显著升高（P〈O．01）;与单独治疗组相比,联合治疗组血清及BALF中IgE含量显著下降,IFN-γ含量显著升高（Pd0．05）。与哮喘组比,单独治疗组与联合治疗组肺组织炎症细胞渗出、浸润及对气道上皮的炎性损伤明显减轻,联合治疗组减轻更明显。结论联合应用sIL-5Ra与IL-4MT可以明显减轻哮喘小鼠AHR,降低血清及BALF中IgE含量,同时升高IFN-γ含量,减轻气道炎症。     

Local delivery of mutant CCL2 protein‐reduced orthopaedic implant wear particle‐induced osteolysis and inflammation in vivo

Total joint replacement (TJR) has been widely used as a standard treatment for late‐stage arthritis. One challenge for long‐term efficacy of TJR is the generation of ultra‐high molecular weight polyethylene wear particles from the implant surface that activates an inflammatory cascade which may lead to bone loss, prosthetic loosening and eventual failure of the procedure. Here, we investigate the efficacy of local administration of mutant CCL2 proteins, such as 7ND, on reducing wear particle‐induced inflammation and osteolysis in vivo using a mouse calvarial model. Mice were treated with local injection of 7ND or phosphate buffered saline (PBS) every other day for up to 14 days. Wear particle‐induced osteolysis and the effects of 7ND treatment were evaluated using micro‐CT, histology, and immunofluorescence staining. Compared with the PBS control, 7ND treatment significantly decreased wear particle‐induced osteolysis, which led to a higher bone volume fraction and bone mineral density. Furthermore, immunofluorescence staining showed 7ND treatment decreased the number of recruited inflammatory cells and osteoclasts. Together, our results support the feasibility of local delivery of 7ND for mitigating wear particle‐induced inflammation and osteolysis, which may offer a promising strategy for extending the life time of TJRs. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:58–64, 2016.     

Kv7.2-7.5 voltage-gated potassium channel (KCNQ2-5) opener, retigabine, reduces capsaicin-induced visceral pain in mice

The taiep rat is a myelin mutant in which immobility episodes (IEs) can be induced in adult males by gripping. EEG recordings during gripping-induced IEs show a rapid eye movement (REM) sleep-like pattern, similar to that reported for narcolepsy-cataplexy suggesting that IEs represent a disorder of REM-sleep. An alpha(2) adrenoceptor agonist increases gripping-induced IEs, whereas alpha(2) antagonists decrease these. We have studied the effect of prazosin on IEs and the levels of alpha(1) adrenoceptors were evaluated in cerebro-cortical homogenates of taiep and control rats. Systemic administration of prazosin results in a significant increase in both the frequency and duration of gripping-induced IEs. Our results show that cerebro-cortical tissue is not an adequate candidate for the expression of cataplexy-like symptoms, but prazosin, an alpha(1) antagonist, is a potent inducer of gripping-induced immobility episodes in taiep rats.     

Respiratory syncytial virus fusion nanoparticle vaccine immune responses target multiple neutralizing epitopes that contribute to protection against wild-type and palivizumab-resistant mutant virus challenge

Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in newborns, young children, elderly, and immune-compromised. The RSV fusion (F) glycoprotein is a major focus of vaccine development and the target of palivizumab (Synagis®) which is licensed as an immuno-prophylactic for use in newborn children at high risk of infection. However, clinical use of a narrowly targeted monoclonal antibodies leads to the generation of escape mutant strains that are fully resistant to neutralization by the antibody. Herein, we evaluated the RSV F nanoparticle vaccine (RSV F vaccine), produced as near-full-length, pre-fusogenic F trimers that form stable protein-detergent nanoparticles. The RSV F vaccine induces polyclonal antibodies that bind to antigenic site II as well as other epitopes known to be broadly neutralizing. Cotton rats immunized with the RSV F vaccine produced antibodies that were both neutralizing and protected against wild-type RSV infection, as well as against a palivizumab-resistant mutant virus. Use of aluminum phosphate adjuvant with the RSV F vaccine increased site II antibody avidity 100 to 1000-fold, which correlated with enhanced protection against challenge. The breadth of the vaccine-induced antibody response was demonstrated using competitive binding with monoclonal antibodies targeting antigenic sites Ø, II, IV, and VIII found on pre-fusion and post-fusion conformations of RSV F. In summary, we found the RSV F vaccine induced antibodies that bind to conserved epitopes including those defined as pre-fusion F specific; that use of adjuvant increased antibody avidity that correlated with enhanced protection in the cotton rat challenge model; and the polyclonal, high-avidity antibodies neutralized and protected against both wild-type and palivizumab-resistant mutant virus. These data support the ongoing clinical development of the aluminum phosphate adjuvanted RSV F nanoparticle vaccine.