Structural analysis of the GLUT1 facilitative glucose transporter

The structure of the human erythrocyte facilitative glucose transporter (GLUT1) has been intensively investigated using a wide array of chemical and biophysical approaches. Despite the lack of a crystal structure for any of the facilitative monosaccharide transport proteins, detailed information regarding primary and secondary structure, membrane topology, transport kinetics, and functionally important residues has allowed the construction of a sophisticated working model for GLUT1 tertiary structure. The existing data support the formation of a central aqueous channel formed by the juxtaposition of several amphipathic transmembrane-spanning α-helices. The results of extensive mutational analysis of GLUT1 have elucidated many of the structural determinants of the glucose permeation pathway. Continued application of currently available technologies will allow further refinement of this working model. In addition to providing insights into the molecular basis of both normal and disordered glucose homeostasis, this detailed understanding of structure/function relationships within GLUT1 can provide a basis for understanding transport carried out by othermembers of the major facilitator super family.     

The effect of Mg2+, nucleotides and ATPase inhibitors on the uptake of [3H]-cGMP to inside-out vesicles from human erythrocytes

An ATP-dependent transport system is responsible for the cellular extrusion of cGMP. The objective of the present study was to determine the effect of Mg2+, ATP and other nucleotides (2'-dATP, GTP and ADP), exogenous ATPase modulators (such as metavanadate, ouabain, EGTA, NEM, bafilomycin A1 and oligomycin A) on the cGMP transport. The uptake of [3H]-cGMP (1 mu M) at 37 C was studied in inside-out vesicles from human erythrocytes. Magnesium caused a maximal activation between 5 and 10 mM and the optimal ATP concentration was 1.25 mM with K50-values of 0.3-0.5 mM. Among other nucleotides tested, 2'-dATP (K50 of 0.7 mM) was nearly as effective as ATP, whereas cGMP accumulated slowly in the presence of GTP. ADP and metavanadate (P-type ATPase inhibitor) showed to be competitive inhibitors with Ki values of 0.15 mM and 10 mu m, respectively. NEM (a sulphydryl agent) reduced the ATP-dependent uptake in a concentration-dependent manner with a Ki value of 10 mu M. Ouabain (Na+/K+-ATPase inhibitor) had no effect. Bafilomycin A1 (V- type ATPase inhibitor) and oligomycin (F-type ATPase inhibitor) were the most potent inhibitors with Ki values of 0.7 and 1.8 mu M, respectively. The present study suggests that the cellular cGMP extrusion is energized by an ATPase with a unique inhibitor profile, which clearly differentiates it from the other major classes of membrane-bound ATPases.     

Cross-linking of dimeric CitS and GltS transport proteins

Abstract

CitS of Klebsiella pneumoniae and GltS of Escherichia coli are Na⁺-dependent secondary transporters from different families that are believed to share the same fold and quaternary structure. A 10 kDa protein tag (Biotin Acceptor Domain [BAD]) was fused to the N-terminus of both proteins (CitS-BAD1 and GltS-BAD1, respectively) and inserted in the central cytoplasmic loop that connects the two halves of the proteins (CitS-BAD260 and GltS-BAD206). Both CitS constructs and GltS-BAD206 were produced and shown to be active transporters, but GltS-BAD1 could not be detected in the membrane. Distance relationships in the complexes were studied by cross-linking studies. Both CitS constructs were shown to be in the dimeric state after purification in detergent by cross-linking with glutaraldehyde. The concentration of glutaraldehyde resulting in 50% cross-linking was significantly higher for CitS-BAD1 than for CitS and CitS-BAD260. Remarkably, GltS and GltS-BAD260 were not cross-linked by glutaraldehyde because of the lack of productive reactive sites. Cross-linking of GltS was observed when the N-terminal 46 residues of CitS with or without BAD at the N-terminus were added to the N-terminus of GltS. The stretch of 46 residues contains the first transmembrane segment of CitS that is missing in the GltS structure. The data support an orientation of the monomers in the dimer with the N-termini close to the dimer interface and the central cytoplasmic loops far away at the ends of the long axis of the dimer structure in a view perpendicular to the membrane.     

公立医院改革下护理管理的创新思路

公立医院改革是新医改的核心。加强护理工作是实现医改目标的重要措施之一,是构建和谐医患关系的客观要求。在推进公立医院改革的进程中,从医院层面开展“文化护理管理”、建立护理后勤、创新护理服务等方面的工作,探索公立医院护理管理创新思路。     

核小体及染色质修饰的基因组分布模式和染色质状态

染色质是真核DNA的存在方式,可以通过影响DNA的可及性调节基因转录,其基本单元为核小体,系由约147 bp的DNA缠绕在组蛋白八联体上形成的结构,核小体之间以连接DNA相连．核小体组蛋白上能发生甲基化和乙酰化等化学修饰．核小体位置、DNA的甲基化和组蛋白的修饰等对染色质状态(常染色质或异染色质)及基因组之间的长程相互作用有重要影响．近年,基于高通量测序技术,核小体位置和染色质修饰在多种细胞中的基因组分布已被测定．结果显示,这些标记的分布模式具有位点特异、动态变化、相互偶联和高度复杂的特征．本文详细回顾并评述了核小体位置和染色质修饰的分布模式、对应生物学功能、修饰之间的关联、实验测定技术、染色质状态的计算分析等内容．该工作对于深入认识和理解染色质的表观遗传调节机制有重要意义．     

Crystal structure of a Vip3B family insecticidal protein reveals a new fold and a unique tetrameric assembly

Vegetatively expressed insecticidal proteins (VIPs) produced by Bacillus thuringiensis fall into several classes of which the third, VIP3, is known for their activity against several key Lepidopteran pests of commercial broad acre crops and because their mode of action does not overlap with that of crystalline insecticidal proteins. The details of the VIP3 structure and mode of action have remained obscure for the quarter century that has passed since their discovery. In the present article, we report the first crystal structure of a full‐length VIP3 protein. Crystallization of this target required multiple rounds of construct optimization and screening—over 200 individual sequences were expressed and tested. This protein adopts a novel global fold that combines domains with hitherto unreported topology and containing elements seemingly borrowed from carbohydrate‐binding domains, lectins, or from other insecticidal proteins.     

微生物遗传学与育种课程教学改革与探索

微生物遗传学与育种是“生物工程专业卓越人才实验班”和“生物工程国际留学生班”的必修课程。然而,传统授课模式在内容选择、教学方法及手段和考核形式等方面均存在诸多不足。为了提高教学质量和效果,促进天津科技大学微生物学教学领域的进步和发展,更加高效地培养国家需要、满足国际需求的创新领军人才,文中对微生物遗传学与育种的教学内容、教学方法及手段、课程考核方式进行了改革与探索。借助最新科研进展、课前预习体系、视频展示、考核方式多样化等形式对授课模式进行创新性改革。不仅使学生掌握了微生物遗传学与育种的相关专业知识,更加锻炼了学生的主观能动性、团队合作意识和专业外语表达水平,培养了学生对微生物遗传学相关科学知识的兴趣。     

两种抗生素对携带Wolbachia的短管赤眼蜂生殖模式的影响

携带有Wolbachia的短管赤眼蜂为完全的产雌孤雌生殖。前期的实验表明,利用四环素对携带有Wolbachia的短管赤眼蜂进行除菌处理,但结果并不能获得100%恢复孤雌产雄。本实验拟用环丙沙星与磺胺嘧啶这两种抗生素对携带有Wolbachia的短管赤眼蜂进行除菌效果筛选。实验分3组药剂处理,分别为使用环丙沙星、磺胺嘧啶及环丙沙星与磺胺嘧啶1:1混配液进行除菌处理。各组实验分别进行浓度为0.1 mg/mL、1.0 mg/mL和10.0 mg/mL 3个浓度处理。以无抗生素蜂蜜水处理作为实验对照组。分别检测、记录各处理的F0 - F1代的寄生卵量,F1 - F2代羽化个体数、雌雄个体数,雌雄相嵌体的个体数。结果表明：磺胺嘧啶对短管赤眼蜂的F0代产卵量降低,但F1代恢复产卵量;仅有0.1 mg/mL环丙沙星与磺胺嘧啶1:1混配液对短管赤眼蜂F2代的卵羽化率具有抑制作用,其它的处理与对照组结果无差异。环丙沙星处理后的携带Wolbachia的短管赤眼蜂没出现雄性后代,浓度为0.1 mg/mL环丙沙星与磺胺嘧啶1:1混配液处理在F2代出现了雌雄相嵌体。浓度为1.0 mg/mL和10 mg/mL的环丙沙星与磺胺嘧啶1:1混配液处理和磺胺嘧啶处理在F2代出现了完全雄性后代。使用磺胺嘧啶处理携带Wolbachia的短管赤眼蜂,可更方便快捷获得100%雄性后代。     

Enhancing lipophilicity as a strategy to overcome resistance against platinum complexes?

Decreased influx represents one of the major resistance mechanisms of platinum complexes. In order to address the question if this mechanism of resistance can be overcome by enhancing the lipophilicity of platinum complexes, we investigated the influence of lipophilicity on cellular accumulation and cytotoxicity in a panel of oxaliplatin analogues with different carrier ligands. Cellular accumulation, DNA platination and cytotoxicity were measured in a cisplatin-sensitive and -resistant ovarian carcinoma (A2780/A2780cis) and in an oxaliplatin-sensitive and -resistant ileocecal colorectal adenocarcinoma (HCT-8/HCT-8ox) cell line pair. Platinum concentrations were determined by flameless atomic absorption spectrometry or adsorptive stripping voltammetry. Passive diffusion represented the main influx mechanism of oxaliplatin analogues during the first minutes of incubation as indicated by a correlation between lipophilicity and early influx rate. Afterwards, the predominant influx mechanism was lipophilicity-independent. More lipophilic complexes showed a reduced cytotoxic activity, although the early influx rate was increased. The resistance profiles of the two cell line pairs were found to be different: HCT-8ox cells were less resistant against more lipophilic complexes, whereas A2780cis cells exhibited a comparable degree of resistance against all investigated compounds. However, the reduction in resistance factor of HCT-8ox cells cannot be explained by increased influx suggesting that other resistance mechanisms are circumvented upon exposure to more lipophilic compounds. Though resistance against more lipophilic platinum complexes analogues is lower we conclude that enhancing lipophilicity is not a successful strategy to overcome platinum resistance as higher lipophilicity is also associated with lower cytotoxic activity.