猪细小病毒VP2蛋白N端甘氨酸富集区的缺失对病毒样颗粒形成及免疫原性的影响

猪细小病毒(PPV)VP2蛋白N端连续9个甘氨酸富集的编码区是VP3蛋白的切割位点,常规PCR扩增容易导致该区段的缺失,为研究该缺失对PPV病毒样颗粒(VLPs)的影响,探索VP2病毒样颗粒上适合外源基因插入的位点,构建了该区段缺失的VP2的真核表达载体pCI-△VP2,并以完整VP2作为对照,采用脂质体介导法转染Vero细胞,通过生物信息学技术、SDS-PAGE、Western blotting、间接免疫荧光以及正染和免疫电镜对表达产物进行分析观察;进一步将重组质粒以核酸疫苗的方式直接肌注免疫小鼠,采用间接ELISA试验、淋巴细胞增殖试验和T细胞亚群流式细胞技术,分析免疫小鼠的体液和细胞免疫应答.结果显示,缺失△VP2和完整VP2在Vero细胞中均能自我装配成VLPs,并具有与完整病毒粒子类似血凝性,pCI-△VP2和pCI-VP2均可诱导小鼠产生较强的特异性体液免疫应答和良好的细胞免疫应答.结果表明,甘氨酸富集区的缺失不影响VP2病毒样颗粒的装配和免疫原性,△VP2同样可进行PPV VLPs疫苗和抗原转运载体的研制,为VLPs载体改造和修饰位点的探索提供了新方向,为VP2基因结构与蛋白质功能的关系提供了新的理论依据.     

Identification of curcumin derivatives as human glyoxalase I inhibitors: A combination of biological evaluation, molecular docking, 3D-QSAR and molecular dynamics simulation studies

Several recent developments suggest that the human glyoxalase I (GLO I) is a potential target for anti-tumor drug development. In present study, a series of curcumin derivatives with high inhibitory activity against human GLO I were discovered. Inhibition constant (K(i)) values of compounds 8, 9, 10, 11 and 13 to GLO I are 4.600μM, 2.600μM, 3.200μM, 3.600μM and 3.600μM, respectively. To elucidate the structural features of potent inhibitors, docking-based three-dimensional structure-activity relationship (3D-QSAR) analyses were performed. Satisfactory agreement between experiment and theory suggests that comparative molecular similarity index analysis (CoMSIA) modeling exhibit much better correlation and predictive power. The cross-validated q(2) value is 0.638 while no-validation r(2) value is 0.930. Integrated with docking-based 3D-QSAR CoMSIA modeling, molecular surface property (electrostatic and steric) mapping and molecular dynamics simulation, a set of receptor-ligand binding models and bio-affinity predictive models for rational design of more potent inhibitors of GLO I are established.     

Development of 3-substituted-androsterone derivatives as potent inhibitors of 17β-hydroxysteroid dehydrogenase type 3

17Beta-hydroxysteroid dehydrogenase type 3 (17β-HSD3) is a steroidogenic enzyme that catalyzes the transformation of 4-androstene-3,17-dione (Δ?-dione) into androgen testosterone (T). To provide effective inhibitors of androgen biosynthesis, we synthesized two different series (amines and carbamates) of 3β-substituted-androsterone derivatives and we tested their inhibitory activity on 17β-HSD3. From the results of our structure-activity relationship study, we identified a series of compounds producing a strong inhibition of 17β-HSD3 overexpressed in HEK-293 cells (homogenized cells). The most active compound when tested in intact HEK-293 transfected cells, namely (3α,5α)-3-{[trans-2,5-dimethyl-4-{[2-(trifluoromethyl)phenyl] sulfonyl}piperazin-1-yl]methyl}-3-hydroxyandrostan-17-one (15b), shows an IC?? value of 6 nM, this compound is thus eight times more active than our reference compound D-5-2 (IC??=51 nM). This new improved inhibitor did not stimulate the proliferation of androgen-sensitive Shionogi cells, suggesting a non-androgenic profile. Compound 15b is thus a good candidate for further in vivo studies on rodents.     

新型强脉冲光治疗雀斑临床疗效观察

目的:观察新型强脉冲光治疗雀斑的疗效。方法:采用飞顿辉煌激光360嫩肤系统(以色列飞顿公司),波长570～950nm,光斑面积10mm×30mm,脉宽10、12、15ms,能量14～19J/cm~2。治疗40例雀斑患者,每三周一次,共治疗5次,末次治疗后评价患者雀斑的疗效。结查:40例患者经过治疗后,18例(45%)基本完全消退,14例(35%)明显消退,8例(20%)好转,总有效率为100%。所有患者面部治疗区域皮肤质地较以前更光滑、细腻,无严重不良反应出现。结论:采用新型强脉冲光治疗雀斑疗效显著、安全,副作用少。     

Role of miR-1 and miR-133a in myocardial ischemic postconditioning

Background  

Ischemic postconditioning (IPost) has aroused much attention since 2003 when it was firstly reported. The role of microRNAs (miRNAs or miRs) in IPost has rarely been reported. The present study was undertaken to investigate whether miRNAs were involved in the protective effect of IPost against myocardial ischemia-reperfusion (IR) injury and the probable mechanisms involved.     

Serotonin transporter interacts with the PDGFβ receptor in PDGF-BB-induced signaling and mitogenesis in pulmonary artery smooth muscle cells

The serotonin transporter (SERT) and the platelet-derived growth factor receptor (PDGFR) have been implicated in both clinical and experimental pulmonary hypertension (PH) and the facilitation of pulmonary artery smooth muscle cell (PASMC) growth. To gain a better understanding of the possible relationship of these two cell surface molecules we have explored interactions between SERT and PDGFR. We have previously demonstrated that SERT transactivates PDGFRβ in serotonin-stimulated PASMC proliferation. We now provide evidence for a role for SERT in PDGF-BB signaling and PASMC proliferation by using pharmacological inhibitors, genetic ablation, and construct overexpression of SERT. The results show that four tested SERT blockers dose dependently inhibit PDGF-stimulated human and bovine PASMC proliferation with comparable efficacy to that of PDGFR inhibitors, whereas 5-HT1B or 5-HT2A receptor inhibitors had no effect. Combinations of the SERT and PDGFR inhibitors led to synergistic/additive inhibition. Similarly, PDGF-induced PASMC proliferation was attenuated by small interfering RNA downregulation of SERT. Inhibition of SERT in PASMCs attenuated PDGF-induced phosphorylation of PDGFRβ, Akt, and p38 but not Erk. Overexpression of SERT in HEK293 cells led to enhanced Akt phosphorylation by PDGF, which was blunted by a SERT PDZ motif mutant, indicating the mechanistic need for the PDZ motif of SERT in PDGF signaling. Furthermore, coimmunoprecipitation experiments showed that SERT and PDGFRβ become physically associated upon PDGF stimulation. In total, the data show for the first time an important interactive relationship between SERT and the PDGFRβ in the production of PASMC proliferation triggered by PDGF that may be important in PH.     

Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators

Modulator compounds intended to overcome disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) show significant promise in clinical testing for cystic fibrosis. However, the mechanism(s) of action underlying these compounds are not fully understood. Activation of CFTR ion transport requires PKA-regulated phosphorylation of the regulatory domain (R-D) and dimerization of the nucleotide binding domains. Using a newly developed assay, we evaluated nine compounds including both CFTR potentatiators and activators discovered via various high-throughput screening strategies to acutely augment CFTR activity. We found considerable differences in the effects on R-D phosphorylation. Some (including UC(CF)-152) stimulated robust phosphorylation, and others had little effect (e.g., VRT-532 and VX-770). We then compared CFTR activation by UC(CF)-152 and VRT-532 in Ussing chamber studies using two epithelial models, CFBE41o(-) and Fischer rat thyroid cells, expressing various CFTR forms. UC(CF)-152 activated wild-type-, G551D-, and rescued F508del-CFTR currents but did not potentiate cAMP-mediated CFTR activation. In contrast, VRT-532 moderately activated CFTR short-circuit current and strongly potentiated forskolin-mediated current. Combined with the result that UC(CF)-152, but not VRT-532 or VX-770, acts by increasing CFTR R-D phosphorylation, these findings indicate that potentiation of endogenous cAMP-mediated activation of mutant CFTR is not due to a pathway involving augmented R-D phosphorylation. This study presents an assay useful to distinguish preclinical compounds by a crucial mechanism underlying CFTR activation, delineates two types of compound able to acutely augment CFTR activity (e.g., activators and potentiators), and demonstrates that a number of different mechanisms can be successfully employed to activate mutant CFTR.     

RICE MORPHOLOGY DETERMINANT encodes the type II formin FH5 and regulates rice morphogenesis

Multicellular organisms contain a large number of formins; however, their physiological roles in plants remain poorly understood. Here, we reveal that formin homology 5 (FH5), a type II formin mutated in rice morphology determinant (rmd), plays a crucial role in determining rice (Oryza sativa) morphology. FH5/RMD encodes a formin-like protein consisting of an N-terminal phosphatase tensin (PTEN)-like domain, an FH1 domain, and an FH2 domain. The rmd mutants display a bending growth pattern in seedlings, are stunted as adult plants, and have aberrant inflorescence (panicle) and seed shape. Cytological analysis showed that rmd mutants have severe cell elongation defects and abnormal microtubule and microfilament arrays. FH5/RMD is ubiquitously expressed in rice tissues, and its protein localization to the chloroplast surface is mediated by the PTEN domain. Biochemical assays demonstrated that recombinant FH5 protein can nucleate actin polymerization from monomeric G-actin or actin/profilin complexes, cap the barbed end of actin filaments, and bundle actin filaments in vitro. Moreover, FH5 can directly bind to and bundle microtubules through its FH2 domain in vitro. Our findings suggest that the rice formin protein FH5 plays a critical role in determining plant morphology by regulating actin dynamics and proper spatial organization of microtubules and microfilaments.