Incorporation of the transmembrane hydrophobic domain of glycophorin into small unilamellar phospholipid vesicles Ion Flux studies

Human erythrocyte glycophorin is one of the best characterized integral membrane proteins. Reconstitution of the membrane-spanning hydrophobic segment of glycophorin (the tryptic insoluble peptide released when glycophorin is treated with trypsin) with liposomes results in the production of freeze-fracture intrabilayer particles of 80 Å diameter (Segrest, J.P., Gulik-Krzywicki, T. and Sardet, C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3294–3298), with particles appearing at or above a tryptic insoluble peptide concentration of 4 mmol per mol phosphatidylcholine. In the present study, increasing concentrations of tryptic insoluble peptide were added to sonicated small unilamellar egg phosphatidylcholine vesicles and the rate of efflux of ²²Na⁺ was examined by rapid (30 s) gel filtration on Sephadex G-50. Below a concentation of 3–5 mmol tryptic insoluble peptide/mol phosphatidylcholine, ²²Na⁺ efflux occurs at a constant slow rate at given tryptic insoluble peptide concentrations. Above a concentration of 3–5 mM, the rate of efflux is biphasic at given tryptic insoluble peptide concentrations, exhibiting both an initial fast and a subsequent slow component. On the basis of graphic and computer curve-fitting analysis, with increasing tryptic insoluble peptide concentration, the rate of the slow component reaches a plateau at a tryptic insoluble peptide concentration of 3–5 mM and remains essentially constant until much higher concentrations are reached; the fast component increases linearly with increasing tryptic insoluble peptide concentration well beyond 5 mM. The most consistent interpretation of this data is as follows. The slow ²²Na⁺ efflux component is due to perturbations of small unilamellar vesicle integrity by tryptic insoluble peptide monomers. At a tryptic insoluble peptide concentration of 3–5 mmol/mol, a critical concentration is reached following which there is intrabilayer tryptic insoluble peptide self-association. The fast ²²Na⁺ efflux component is due to the increasing presence of tryptic insoluble peptide self-associated multimers the 80-Å particles seen by freeze-fracture electron microscopy) which results in a significantly larger bilayer defect than do tryptic insoluble peptide monomers. The failure of complete saturation of efflux by the fast component is ascribed to the presence of two populations of small unilamellar vesicles, some of which contain tryptic insoluble peptide multimers and some of which do not.Addition of cholesterol to the tryptic insoluble peptide/phosphatidylcholine vesicles decreases the rate of ²²Na⁺ efflux by inhibiting primarily the fast component. Freeze-fracture electron microscopy indicates that the presence of cholesterol has no effect on the size, number or distribution of 80-Å intra-bilayer particles in the tryptic insoluble peptide/phosphatidylcholine vesicles. These results are consistent with a mechanism to explain the fast Na⁺ efflux component involving protein-lipid boundary perturbations.Efflux of ⁴⁵Ca²⁺ from phosphatidylcholine vesicles is also enhanced by incorporation of tryptic insoluble peptide, but only if divalent cations (Ca²⁺ or Mg²⁺) are present in the external bathing media as well as inside the sonicated vesicles. If monovalent Na⁺ only is present in the bathing media no ⁴⁵Ca²⁺ efflux is seen. Under conditions where ⁴⁵Ca²⁺ efflux is seen, both a fast and a slow component are present, although both appear lower than corresponding rate constants for ²²Na⁺ efflux. These results suggest a coordinated mechanism for ion efflux induced by tryptic insoluble peptide and, together with the ²²Na⁺ efflux studies, may have mechanistic implications for the transbilayer phospholipid exchange (flip-flop) suggesed to be induced at glycophorin/phospholipid interfaces (de Kruiff, B., van Zoelen, E.J.J. and van Deenen, L.L.M. (1978) Biochim. Biophys. Acta 509, 537–542).     

格网尺度下典型旅游城市生态服务价值估算和时空分异特征——以三亚为例

以典型旅游城市三亚市为案例地,利用2006-2018年4期Landsat遥感影像数据,借助ENVI、ArcGIS平台定量识别土地利用演变特征,在1km×1km格网尺度下估算旅游地生态系统服务价值,并结合空间探索性数据分析揭示生态系统服务价值时空分异特征及其与旅游地发展的时空耦合关系。结果表明：（1）2006-2018年间,三亚市生态系统服务价值总量呈逐年下降趋势,由6.73×10⁹元降至5.76×10⁹元,累计减少9.78×10⁸元;（2）空间格局上,三亚市呈"南低北高"空间分异格局,2006-2018年增值区域连片分布于崖州区、天涯区、吉阳区南部区域,且呈逐年减少趋势,减值区域集聚于天涯区东北部、海棠区;（3）空间集聚上,生态系统服务价值截面各年份均呈显著空间正相关且相关性先降后增。高高集聚区位于天涯区北部区域,低低集聚区分布于沿海、海湾地区;（4）旅游发展与生态系统服务价值时空演化特征关联性较强。三亚市天涯区北部林地生态环境良好,生态系统服务价值略有下降但绝对数值稳定,是生态系统服务价值主要来源;旅游发展较为迅速的三亚湾、崖州湾以及海棠湾,相对增值区域较多,但绝对生态系统服务价值损失显著,严重滞后于其他区域。     

Enhancement of keratinocyte growth factor potential in inducing adipose‐derived stem cells differentiation into keratinocytes by collagen‐targeting

Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose‐derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen‐binding domain from Vibrio mimicus metalloprotease (vibrioCBD‐KGF). KGF and vibrioCBD‐KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK‐293 and MCF‐7 cells. vibrioCBD‐KGF demonstrated enhanced collagen‐binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD‐KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen‐coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin‐10 and Involucrin), and stem cell markers (Collagen‐I and Vimentin) by real‐time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD‐KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down‐regulation of Collagen‐I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD‐KGF. The present study showed that vibrioCBD‐KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen‐based biomaterials.     

全球气候治理新进展——区域碳排放权分配研究综述

碳排放权作为稀缺的公共资源,其实质是一种新型发展权。科学合理分配有限的碳排放权对实现《巴黎协定》温控目标至关重要。从原则、方法、尺度与方案等维度对碳排放权分配的文献成果进行了系统梳理与归纳。研究表明,国内外学者大多基于公平性和效率性原则探索碳排放权分配;分配方法分为指标法、博弈论法、数据包络分析法和综合法等,各有利弊和适用条件;分配尺度大多涉及国际和区际两个层面,前者由于各国不同的利益诉求较难形成共识性方案,后者主要关注省际分配,更小尺度的研究相对较少。未来碳排放权分配研究趋向于多原则兼顾、多方法联用,涵盖国际、省际、市际及行业、企业等不同尺度。本研究可为制定科学合理的碳排放权分配方案提供理论依据,为我国更为积极有效地参与全球气候治理提供决策参考。     

生态风险评价研究进展

20多年来,生态风险评价研究经历了从环境风险到生态风险到区域生态风险评价的发展历程,风险源由单一风险源扩展到多风险源,风险受体由单一受体发展到多受体,评价范围由局地扩展到区域景观水平.区域生态风险评价就是大尺度上研究复杂环境背景下包含多风险源、多风险受体的综合风险研究.目前,区域生态风险评价的理论框架已经搭建起来,统计方法多采用相对评价法.区域生态风险评价未来的发展方向为继续加强实验和野外调查,进一步减小不确定性,逐步解决尺度推移问题.区域生态风险评价必须与经济、社会、文化相结合,才能充分发挥它在管理决策中的作用.     

白冠长尾雉越冬期栖息地选择的多尺度分析

2000年至2002年冬季,在河南董寨国家级自然保护区对我国特有珍稀雉类白冠长尾雉（Syrmaticus reevesii）越冬期栖息地进行了调查,并结合RS和GIS在多个尺度上对其栖息地选择进行了分析.结果表明,不同尺度上影响白冠长尾雉越冬期栖息地选择的因素存在差异,影响因子之间还存在相互作用.在微生境上,影响因子主要是坡度、乔木盖度以及坡向余弦值与灌木高度的相互作用;在115 m尺度上,关键因子是灌木林、阔叶林和针叶林的面积;250m尺度上,主要因子是针叶林和阔叶林的面积及针叶林与阔叶林面积的相互作用;对于距离因素,到河漫滩和到农田的距离是影响白冠长尾雉越冬期栖息地选择的关键因子.根据回归分析和AIC及AICc值,115 m尺度上栖息地变量对白冠长尾雉越冬期的栖息地选择影响最大.综合分析发现,在较大的尺度上,影响白冠长尾雉越冬期栖息地选择的关键因子有针叶林面积、阔叶林面积、针叶林和灌丛面积的相互作用、到河漫滩的距离以及到农田的距离.     

鼎湖山植物群落α多样性与环境的关系

用样带取样法,来研究不同取样尺度和不同取样尺度条件下的多个环境因子与鼎湖山植物群落α多样性的关系.取样尺度分别为10、20、40、80m和160m.涵盖鼎湖山主要的植被类型：季风常绿阔叶林、针阔混交林、沟谷常绿阔叶林和针叶林.相关分析和主成分分析结果表明,环境因子对各层次的α多样性的影响程度各不相同,达到显著相关性的取样尺度也不一样,表现出较大的复杂性,同时也表明样带上的环境异质性较高.因此,用海拔梯度作为主要的环境梯度来研究鼎湖山植物群落多样性具有合理性.海拔高度与乔木层多样性的关系在所有取样尺度上都较密切,这说明海拔高度可能是影响乔木层α多样性的最重要环境因子.     

Simple But Efficacious Enrichment of Integral Membrane Proteins and Their Interactions for In-Depth Membrane Proteomics

Membrane proteins play essential roles in various cellular processes, such as nutrient transport, bioenergetic processes, cell adhesion, and signal transduction. Proteomics is one of the key approaches to exploring membrane proteins comprehensively. Bottom–up proteomics using LC–MS/MS has been widely used in membrane proteomics. However, the low abundance and hydrophobic features of membrane proteins, especially integral membrane proteins, make it difficult to handle the proteins and are the bottleneck for identification by LC–MS/MS. Herein, to improve the identification and quantification of membrane proteins, we have stepwisely evaluated methods of membrane enrichment for the sample preparation. The enrichment methods of membranes consisted of precipitation by ultracentrifugation and treatment by urea or alkaline solutions. The best enrichment method in the study, washing with urea after isolation of the membranes, resulted in the identification of almost twice as many membrane proteins compared with samples without the enrichment. Notably, the method significantly enhances the identified numbers of multispanning transmembrane proteins, such as solute carrier transporters, ABC transporters, and G-protein–coupled receptors, by almost sixfold. Using this method, we revealed the profiles of amino acid transport systems with the validation by functional assays and found more protein–protein interactions, including membrane protein complexes and clusters. Our protocol uses standard procedures in biochemistry, but the method was efficient for the in-depth analysis of membrane proteome in a wide range of samples.     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.     

C4-dicarboxylates sensing mechanism revealed by the crystal structures of DctB sensor domain

C₄-dicarboxylates are the major carbon and energy sources during the symbiotic growth of rhizobia. Responses to C₄-dicarboxylates depend on typical two-component systems (TCS) consisting of a transmembrane sensor histidine kinase and a cytoplasmic response regulator. The DctB-DctD system is the first identified TCS for C₄-dicarboxylates sensing. Direct ligand binding to the sensor domain of DctB is believed to be the first step of the sensing events. In this report, the water-soluble periplasmic sensor domain of Sinorhizobium meliloti DctB (DctBp) was studied, and three crystal structures were solved: the apo protein, a complex with C₄ succinate, and a complex with C₃ malonate. Different from the two structurally known CitA family of carboxylate sensor proteins CitA and DcuS, the structure of DctBp consists of two tandem Per-Arnt-Sim (PAS) domains and one N-terminal helical region. Only the membrane-distal PAS domain was found to bind the ligands, whereas the proximal PAS domain was empty. Comparison of DctB, CitA, and DcuS suggests a detailed stereochemistry of C₄-dicarboxylates ligand perception. The structures of the different ligand binding states of DctBp also revealed a series of conformational changes initiated upon ligand binding and propagated to the N-terminal domain responsible for dimerization, providing insights into understanding the detailed mechanism of the signal transduction of TCS histidine kinases.