Structural and functional insights into the heme-binding domain of the human soluble guanylate cyclase α2 subunit and heterodimeric α2β1

Soluble guanylate cyclase (sGC) mediates NO signaling for a wide range of physiological effects in the cardiovascular system and the central nervous system. The α1β1 isoform is ubiquitously distributed in cytosolic fractions of tissues, whereas α2β1 is mainly found in the brain. The major occurrence and the unique characteristic of human sGC α2β1 indicate a special role in the mediation of neuronal communication. We have efficiently purified and characterized the recombinant heme-binding domain of the human sGC α2 subunit (hsGC α2(H)) and heterodimeric α2β1 (hsGC β1(H)-α2(H)) by UV-vis spectroscopy, circular dichrosim spectroscopy, EPR spectroscopy, and homology modeling. The heme dissociation and related NO/CO binding/dissociation of both hsGC α2(H) and hsGC β1(H)-α2(H) were investigated. The two truncated proteins interact with heme noncovalently. The CO binding affinity of hsGC α2(H) is threefold greater than that of human sGC α1(H), whereas the dissociation constant k (1) for dissociation of NO from hsGC α2(H) is sevenfold larger than that for dissociation of NO from hsGC α1(H), although k (2) is almost identical. The results indicate that in comparison with the α1β1 isoform, the brain α2β1 isoform exhibits a distinctly different CO/NO affinity and binding rate in favor of NO signaling, and this is consistent with its physiological role in the activation and desensitization. Molecular modeling and sequence alignments are consistent with the hypothesis that His105 contributes to the different CO/NO binding properties of different isoforms. This valuable information is helpful to understand the molecular mechanism by which human sGC α2β1 mediates NO/CO signaling.     

Metal-Free DNA Linearized Nuclease Based on PASP-Polyamine Conjugates

Genome manipulation controlled by small metal complexes has attracted extensive interest because of their potential application in the fields of molecular biotechnology and drug development. However, their medicinal application is still limited due to the distinct toxicity of the free radicals generated by partial metal complexes based on oxidative cleaving processes. Thus, it is still a challenge for us to use metal free agent to cleave DNA. In this work, we showed that a family of polyamine-grafted PASP (poly(aspartic acid)) conjugates is able to rapidly induce DNA cleavage in the absence of metal ions, and obtain a high-yield linearization product via a hydrolytic path. From the results of detailed control experiments, it was revealed that the formation of polyamine cation/phosphate anion pair and free ungrafted nucleophilic groups would be the key factors to improve DNA linearization. Constructing polyamine conjugates based on short peptide such as polyamine-grafted PASP, as achieved here, could provide an attractive strategy for developing mild and efficient artificial nucleases as well as researching catalytic mechanisms on DNA chemistry.     

Thermophilic Thermotoga maritima ribose-5-phosphate isomerase RpiB: optimized heat treatment purification and basic characterization

The open reading frame TM1080 from Thermotoga maritima encoding ribose-5-phosphate isomerase type B (RpiB) was cloned and over-expressed in Escherichia coli BL21 (DE3). After optimization of cell culture conditions, more than 30% of intracellular proteins were soluble recombinant RpiB. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 70°C and pH 6.5-8.0. Under its suboptimal conditions (60°C and pH 7.0), k(cat) and K(m) values were 540s(-1) and 7.6mM, respectively; it had a half lifetime of 71h, resulting in its turn-over number of more than 2×10(8)mol of product per mol of enzyme. This study suggests that it is highly feasible to discover thermostable enzymes from exploding genomic DNA database of extremophiles with the desired stability suitable for in vitro synthetic biology projects and produce high-purity thermoenzymes at very low costs.     

Structural determinants of benzodiazepinedione/peptide-based p53-HDM2 inhibitors using 3D-QSAR, docking and molecular dynamics

As a tumor suppressor, p53 protein regulates the cell cycle and is involved in preventing tumorgenesis. The protein level of p53 is under the tight control of its negative regulator human double minute 2 (HDM₂) via ubiquitination. Therefore, the design of inhibitors of HDM₂ has attracted much interest of research on developing novel anticancer drugs. Presently, two classes of molecules, i.e., the 1,4-benzodiazepine-2,5-diones (BDPs) and N-Acylpolyamine (NAPA) derivatives were studied by three-dimensional quantitative structure–activity relationship (3D-QSAR) modeling approaches including the comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) as promising p53-HDM₂ inhibitors. Based on both the ligand-based and receptor-guided (docking) alignments, two optimal 3D-QSAR models were obtained with good predictive power of q ² = 0.41, r ² _pred = 0.60 for BDPs, and q ² = 0.414, r ² _pred = 0.69 for NAPA analogs, respectively. By analysis of the model and its related contour maps, it is revealed that the electrostatic interactions contributed much larger to the compound binding affinity than the steric effects. And the contour maps intuitively suggested where to modify the molecular structures in order to improve the binding affinity. In addition, molecular dynamics simulation (MD) study was also carried out on the dataset with purpose of exploring the detailed binding modes of ligand in the HDM₂ binding pocket. Based on the CoMFA contour maps and MD-based docking analyses, some key structural aspects responsible for inhibitory activity of these two classes of compounds were concluded as follows: For BDPs, the R₁ and R₃ regions should have small electronegativity groups; substituents R₂ and R₄ should be larger, and R₃ substituent mainly involves in H-bonds forming. For NAPA derivatives, bulky and electropositive groups in ring B and ring A, small substituent at region P is favorable for the inhibitory activity. The models and related information, we hope, may provide important insight into the inhibitor-p53-HDM₂ interactions and be helpful for facilitating the design of novel potent inhibitors.     

不同发育时期小麦粒重性状QTL的动态分析

利用6044×01-35构建的重组自交系(RIL)群体为试验材料,对小麦粒重性状进行发育动态QTL分析。结果表明,在小麦花后子粒灌浆的7个不同时期,两个试验点共检测到16个与粒重性状相关的QTL。其中开花后20d检测到的单穗粒重QTL位于2A染色体上,解释率达12%,遗传效应超过10;两环境下控制千粒重QTL在7个时期均被检测到。花后的各个时期均能在Xgwm448-Xgpw7399标记区间定位到千粒重QTL。其中花后10d检测到1个千粒重QTL,位于2A染色体的Xgwm448-Xgpw7399标记区间,解释较大的表型变异,达到18%。Qtl8、Qtl13和Qtl14均定位在Xgwm448-Xgpw7399标记区间的同一位置,共同解释11%的表型变异。花后20d和花后25d均检测到1个QTL,位于2A染色体的Xgwm372-Xgwm95标记区间的不同位点,均能解释4%的表型变异。花后40d检测到1个QTL,位于1D染色体的Xwmc93-Xgpw2224标记区间,解释1%的表型变异。从连锁群的位置上看,控制千粒重的QTL主要集中在2A染色体的Xgwm448-Xgpw7399标记区间,这是一个控制千粒重QTL的富集区域,以期进行精细定位和图位克隆。     

黏膜抗体在疫苗免疫效力评价中的前景

简单介绍目前疫苗效力检验的方法、黏膜抗体的功能及其在实验室疫苗效果效力评价中的应用,提出了黏膜抗体作为疫苗免疫效力试验的替代指标或免疫监测的主要抗体的建议。     

Clinic Observation of FGR Therapy by Using Fatty Emulsion and Amino Acid

FGR 临床病因可分为妊娠高血压疾病病因、胎盘因素、细菌病毒感染因素、孕妇营养因素、遗传因素等.笔者选取此类孕妇患者150例,按治疗意愿分为两组,治疗组予以脂肪乳联合氨基酸静脉滴注给药方式治疗,对照组为门诊膳食指导.结果发现治疗组相比对照组更能有效促进胎儿宫内生长,减少FGR并发症的发生(治疗组治疗有效率为91.76％,对照组治疗有效率为56.92％),从而提高新生儿出生质量,值得临床推广应用.     

一期前路病灶清除植骨融合内固定术治疗短节段腰椎结核

目的:对比观察一期前路病灶清除减压植骨融合内固定术与分期前后路联合手术针对短节段腰椎结核的疗效.方法:抽取兰州军区兰州总医院骨科中心脊柱外科30例住院治疗短节段腰椎结核的患者,随机化分为两组,每组15例,分别施行一期前路病灶清除减压植骨融合内固定术和分期前后联合手术,观察比较两组术前、术后6个月、12个月、18个月Frankle神经功能分级,术后12个月及18个月Birdwell植骨融合分级.结果:试验组及对照组术前、术后6个月Frankle分级差异有统计学意义(P＜0.05).两组间术后6个月,术后12个月Frankle分级差异无统计学意义(P＞0.05).两组间术后18个月Frankle分级差异有统计学意义(P＜0.05).两组间术后12个月Birdwell分级差异无统计学意义(P＞0.05),而两组间术后18个月Birdwell分级差异有统计学意义(P＜0.05).随访期间,对照组有两例发生断钉.结论:一期前路病灶清除、植骨融合内固定术治疗脊柱结核,病灶清除彻底、融合率高,远期效果良好,复发率低并能够最大限度恢复脊柱生物力学稳定性.具有重要的临床应用价值.     

几种食用菌菌丝体液体培养条件的研究

研究了鲍鱼菇、黑灵芝、猴头菇和羊肚菌等4种食用菌菌丝体的最佳液体培养条件。结果各食用菌适宜的培养条件为：鲍鱼菇：果糖20g／L,酵母浸膏2g／L,KHP040．5g／L,VC0．001g／L,适宜培养温度28℃,pH5;黑灵芝：葡萄糖20g／L,蛋白胨2g／L,K2HP040．5g／L,VB20．001g／L,适宜培养温度35℃,pH6;猴头菇：麦芽糖20g／L,酵母浸膏2g／L,K2HID040．5g／L,MgSO,0．5g／L,VB10．001g／L,适宜培养温度28℃,pH7;羊肚菌：蔗糖20g／L,蛋白胨2g／L,K2HP040．5g／L,VB10．001g／L,适宜培养温度28℃,pH7。