人穿孔素羧基端肽段的表达纯化与活性鉴定

穿孔素,即成孔蛋白（pore forming protein,PFP),其溶细胞作用与免疫调节和自身免疫病以及其它多种疾病过程中的免疫性病理损伤相关。为得到足够量的PFP建立与之相关的免疫学研究手段用于基础和临床研究,在已克隆人PFP cDNA的基础上,用基因工程方法表达了人PFP C端124个氨基酸肽段（hPFP-C),并通过谷胱甘肽琼脂糖新和层析获得纯化的GST/hPFP-C融合蛋白,经凝血酶酶切和再次北极和层析去除GST部分,得到了纯化的hPFP-C蛋白。纯化的hPFP-C蛋白与兔红细胞共育,呈现钙依赖的溶血活性。     

Homology of perforin to the ninth component of complement (C9)

Perforin is one of the cytolytic factors present in the cytoplasmic granules of mouse cytotoxic T lymphocytes and natural killer cells. We have determined the sequence of the N-terminal amino acids of perforin purified from a mouse natural killer cell line, and, by using oligonucleotide probes corresponding to the amino acid residues, we have identified a complementary DNA encoding perforin from the cDNA library of a mouse cytotoxic T lymphocyte clone. As predicted from the functional similarities between perforin and the ninth component of the serum cytolytic system, complement (C9) (refs 4-8), the deduced primary structure of perforin has homology with C9 at their respective functionally conserved regions. We find that perforin is only expressed in killer cell lines, and not in helper T lymphocytes or other tumour cells tested. Thus we have provided direct molecular evidence that a killer-cell-specific protein evolutionally linked to C9 is involved in cell-mediated cytolysis.     

Structural/functional similarity between proteins involved in complement- and cytotoxic T-lymphocyte-mediated cytolysis

Cytolysis mediated by complement or cytolytic lymphocytes results in the formation of morphology similar lesions in the target membrane. These lesions, formed by the polymerization of C9 or perforin respectively, contribute the major killing action by causing osmotic lysis of the target cell. Following the suggestion of Mayer that the mechanisms of humoral and cell-mediated cytotoxicity might be related, studies into the morphology of the membrane lesions formed, and the proteins responsible for causing the lesions, have shown several similarities. While the lesion caused by natural and T-killer cells is a little larger than that caused by complement, its overall shape is similar and in both cases the cylindrical pore is formed by polymerization of a monomeric subunit, C9 (relative molecular mass, Mr = 71,000) for complement, and perforin (Mr = 66,000) for cell-mediated cytotoxicity. C9 has an absolute requirement for a receptor in the target membrane formed by the earlier membrane attack complex components, C5b, C6, C7 and C8 (ref. 8). For perforin, polymerization in a target membrane requires no receptor, specificity being derived from the specific recognition between killer and target cell. Both proteins can be made to polymerize in vitro by the addition of divalent cations (Zn2+ for C9 (ref. 16) and Ca2+ for perforin) and the resultant complexes closely resemble their physiological counterparts. Antibodies raised against lymphocyte-killed targets have also been shown to cross-react with complement proteins, but the antigenically related proteins were not determined in these studies. We show here using purified proteins that perforin, C9 and complexes involving C7 and C8 share a common antigenic determinant which is probably involved in polymerization.     

胜利油区碎屑岩纳米尺度孔隙结构特征

选取胜利油区的3个不同油田取样分析资料,在储层微观孔隙结构研究的基础上通过扫描电镜、透射电镜以及原子力显微镜对储层中纳米尺度的孔隙结构和孔喉配置关系进行研究。结果表明:碎屑岩储层中存在纳米级的孔隙和喉道,且孔隙类型多样,同样可分为原生孔隙和次生孔隙两大类;纳米级原生孔隙包括单晶片层间缝、矿物解理缝、晶内孔、粒间孔和晶间缝5种类型,纳米级次生孔隙包括粒内溶孔、溶蚀缝、构造缝;研究区纳米尺度下的孔喉配置关系归结为孔喉一致型、弯曲沙漏型、"X"形孔喉交叉型、"丰"字形孔隙交叉型和杯形交叉缝型5种类型。     

Conformational change and protein-protein interactions of the fusion protein of Semliki Forest virus

Fusion of biological membranes is mediated by specific lipid-interacting proteins that induce the formation and expansion of an initial fusion pore. Here we report the crystal structure of the ectodomain of the Semliki Forest virus fusion glycoprotein E1 in its low-pH-induced trimeric form. E1 adopts a folded-back conformation that, in the final post-fusion form of the full-length protein, would bring the fusion peptide loop and the transmembrane anchor to the same end of a stable protein rod. The observed conformation of the fusion peptide loop is compatible with interactions only with the outer leaflet of the lipid bilayer. Crystal contacts between fusion peptide loops of adjacent E1 trimers, together with electron microscopy observations, suggest that in an early step of membrane fusion, an intermediate assembly of five trimers creates two opposing nipple-like deformations in the viral and target membranes, leading to formation of the fusion pore.     

Serine esterase in cytolytic T lymphocytes

The mechanisms that enable cytotoxic T lymphocytes (Tc cells) to destroy target cells are only vaguely understood. However, recent studies have identified in Tc cells and natural killer cells cytoplasmic granules that contain perforin, a cytolytic protein that resembles the ninth component of complement (C9). Antigen-specific lysis of target cells, traditionally ascribed solely to Tc cells, has now also been demonstrated in some T-helper cell (Th cell) lines, referred to here as T helper-killer or Th/c cells. We recently found a novel serine esterase that is present at greatly elevated levels in cloned murine Tc cell lines and one Th/c cell line, but not in two non-cytolytic Th cell lines. These findings suggest that the serine esterase is involved in cytolytic activity and that a variety of effector cells share a common cytolytic mechanism. To explore the role of the serine esterase in this process, we have been studying additional properties of the enzyme in murine T cells. We show here that it is a membrane-associated, disulphide-linked dimer, it has trypsin-like properties but is not a general protease, in density gradient centrifugation it sediments with perforin, it is secreted by Tc cells during their cytolytic attack on target cells, and antiserum to Tc-cell serine esterase reacts with the enzyme in Th/c cells.     

Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Rab-GTPases, together with their cofactors, mediate the attachment step in the membrane fusion of vesicles. But how bilayer mixing--the subsequent core process of fusion--is catalysed remains unclear. Ca2+/calmodulin controls this terminal process in many intracellular fusion events. Here we identify V0, the membrane-integral sector of the vacuolar H+-ATPase, as a target of calmodulin on yeast vacuoles. Between docking and bilayer fusion, V0 sectors from opposing membranes form complexes. V0 trans-complex formation occurs downstream from trans-SNARE pairing, and depends on both the Rab-GTPase Ypt7 and calmodulin. The maintenance of existing complexes and completion of fusion are independent of trans-SNARE pairs. Reconstituted proteolipids form sealed channels, which can expand to form aqueous pores in a Ca2+/calmodulin-dependent fashion. V0 trans-complexes may therefore form a continuous, proteolipid-lined channel at the fusion site. We propose that radial expansion of such a protein pore may be a mechanism for intracellular membrane fusion.     

Structure and function of human perforin

Perforin (P1) is a cytolytic protein with similarity to complement component C9. P1 has been described as a unique component of murine cytolytic T-cell and rat natural killer cell granules Previous studies indicated that human granules and P1 differed from murine granules and P1 in that they appeared to be cytolytically less active and lacked the haemolytic activity characteristic of P1. It has been suggested that P1, like C9, is under the control of the homologous restriction factor. Here we determine the primary structure of human P1, re-examine its functional properties, and address the question of homologous restriction.     

毛竹与樟子松木材孔隙结构的比较

【目的】竹木材料孔隙结构特征是影响材料性能的重要因素。通过定量表征与直观观察相结合方式探索竹木材料内部孔隙结构特征。通过对比分析,建立竹木材料内部孔隙结构与组织构造的对应关系,分析竹木材料内部孔隙结构差异,研究孔隙结构对材料性能的影响。【方法】以毛竹和樟子松木材为试验材料,采用压汞法对材料的孔隙率、孔体积、孔径分布、比表面积等参数进行定量测试,分析材料的孔隙结构特征。采用扫描电镜对材料的组织结构(毛竹:导管、筛管、薄壁细胞和纹孔等部位。樟子松:管胞、射线薄壁细胞、纹孔等部位)进行观察,确定各组织结构所构成孔隙的孔径范围。【结果】孔隙率(毛竹47.58%、樟子松67.16%)及汞压入量(毛竹0.633 mL/g、樟子松1.596 mL/g)测试结果表明毛竹内部孔体积显著低于樟子松。总孔面积(毛竹82.04 m²/g、樟子松18.16 m²/g)及中孔孔径(毛竹33.8 nm、樟子松445.0 nm)对比结果表明毛竹中大部分孔隙集中在孔径较小区域(32.4 nm左右),而樟子松木材中孔隙孔径主要集中在226.7、7 082.3 nm左右,造成毛竹孔面积显著高于樟子松木材。结合扫描电镜观察结果可知,毛竹中孔径11.3～100 μm范围内孔隙主要对应导管、基本组织中的薄壁细胞及纤维细胞。而835.0 nm左右孔隙对应基本组织及纤维细胞上纹孔。樟子松木材中孔径20 μm左右孔隙对应樟子松木材管胞; 而7 082.3 nm左右孔隙则对应具缘纹孔的纹孔口和射线薄壁细胞。此外,毛竹和樟子松木材中孔径小于1 μm的孔隙结构(毛竹中32.4 nm左右,樟子松木材中226.7、749.9 nm左右)主要位于具缘纹孔塞缘及细胞壁上。【结论】采用压汞法和扫描电镜观察方法可以实现对毛竹及樟子松木材孔隙结构的表征分析,有助于分析竹木材料性能差异产生的原因。然而,在通过压汞测试材料孔隙结构参数时,受墨水瓶孔效应影响,部分孔径较大的孔隙被认为是小孔,影响测试结果的准确性。因此,后续研究应考虑竹木材料的孔隙形态,从而实现对竹木材料孔隙结构的全面准确表征。     

核磁共振研究致密砂岩孔隙结构的方法及应用

基于核磁共振的原理,推导T_2弛豫时间与孔隙半径的关系,由孔喉比将孔隙半径转换为喉道半径,结合压汞喉道半径分布,利用插值和最小二乘法,将岩心100%饱和水的核磁共振T_2谱转换为孔喉半径分布,并将核磁孔喉分布曲线应用到油田开发评价中。以鄂尔多斯盆地延长组致密储层为例,结合岩心驱替试验,利用转化的核磁孔喉分布对研究区块储层的孔隙结构、可动流体和可动油分布以及可动流体喉道半径下限进行研究。结果表明:研究区块孔隙结构复杂,发育微米级和纳米-亚微米级孔喉,孔喉半径均值在0.095~1.263μm,0.001~0.01μm的孔喉内束缚流体分布较多,可动流体主要分布在喉道半径大于0.01μm的孔隙内,水驱主要动用喉道半径大于0.1μm的孔隙内的油,研究区可动流体喉道半径截止值平均为0.013μm。     

纳米-滑溜水在致密砂岩中的滞留及其对微观孔隙结构的影响

纳米材料已被证明可以提高非常规油气采收率,但其在储层孔隙中的吸附与滞留机理尚未明确。本文以大庆油田上白垩统青山口组致密砂岩为研究对象,采用低温液氮吸附、润湿角测定、扫描电镜、核磁共振及离心实验方法,研究了纳米-滑溜水压裂液在孔隙中的吸附与滞留,以及其对微观孔隙结构参数的影响。结果表明,纳米滑溜水压裂液处理后,扫描电镜观察到纳米颗粒在孔隙中滞留,岩石润湿角降低30.28%~58.17%;孔隙结构由平板孔向墨水瓶孔过渡,比表面积及吸附量显著增加;微孔占比减小20%~25%,过渡孔占比增大21%~26%,总孔体积增大;分形维数变小更接近2,孔隙结构变简单。纳米颗粒在储层孔隙中的吸附与滞留,导致微观孔隙结构发生变化。实验结果与认识对纳米-滑溜水压裂液在致密砂岩储层中的应用具有重要意义。     

罗庞塬地区延长组孔隙结构特征研究

储层的孔隙结构研究是油藏精细描述、储层综合评价的重要内容,油气储集岩的微观孔隙结构对其物性具有重要的控制作用。采用铸体薄片、扫描电镜、高压压汞等多种技术手段,对罗庞塬地区延长组的孔隙结构进行了深入的分析研究。罗庞塬地区砂岩储层孔隙类型主要为粒间孔,其次为粒内孔、铸模孔、长石溶孔,另有少量裂隙孔,岩石类型为细粒一极细粒岩屑长石砂岩。铸体薄片、孔喉图像分析表明,研究区具有复杂的孔喉分布特征,不同微相孔喉分布及孔隙结构特征是造成油井产能差异的重要因素,所以研究孔隙结构是非常重要的。     

The twisted ion-permeation pathway of a resting voltage-sensing domain

Proteins containing voltage-sensing domains (VSDs) translate changes in membrane potential into changes in ion permeability or enzymatic activity. In channels, voltage change triggers a switch in conformation of the VSD, which drives gating in a separate pore domain, or, in channels lacking a pore domain, directly gates an ion pathway within the VSD. Neither mechanism is well understood. In the Shaker potassium channel, mutation of the first arginine residue of the S4 helix to a smaller uncharged residue makes the VSD permeable to ions ('omega current') in the resting conformation ('S4 down'). Here we perform a structure-guided perturbation analysis of the omega conductance to map its VSD permeation pathway. We find that there are four omega pores per channel, which is consistent with one conduction path per VSD. Permeating ions from the extracellular medium enter the VSD at its peripheral junction with the pore domain, and then plunge into the core of the VSD in a curved conduction pathway. Our results provide a model of the resting conformation of the VSD.     

Disulphide-isomerase-enabled shedding of tumour-associated NKG2D ligands

Tumour-associated ligands of the activating NKG2D (natural killer group 2, member D; also called KLRK1) receptor-which are induced by genotoxic or cellular stress-trigger activation of natural killer cells and co-stimulation of effector T cells, and may thus promote resistance to cancer. However, many progressing tumours in humans counter this anti-tumour activity by shedding the soluble major histocompatibility complex class-I-related ligand MICA, which induces internalization and degradation of NKG2D and stimulates population expansions of normally rare NKG2D+CD4+ T cells with negative regulatory functions. Here we show that on the surface of tumour cells, MICA associates with endoplasmic reticulum protein 5 (ERp5; also called PDIA6 or P5), which, similar to protein disulphide isomerase, usually assists in the folding of nascent proteins inside cells. Pharmacological inhibition of thioreductase activity and ERp5 gene silencing revealed that cell-surface ERp5 function is required for MICA shedding. ERp5 and membrane-anchored MICA form transitory mixed disulphide complexes from which soluble MICA is released after proteolytic cleavage near the cell membrane. Reduction of the seemingly inaccessible disulphide bond in the membrane-proximal alpha3 domain of MICA must involve a large conformational change that enables proteolytic cleavage. These results uncover a molecular mechanism whereby domain-specific deconstruction regulates MICA protein shedding, thereby promoting tumour immune evasion, and identify surface ERp5 as a strategic target for therapeutic intervention.     

Structure of the E. coli protein-conducting channel bound to a translating ribosome

Secreted and membrane proteins are translocated across or into cell membranes through a protein-conducting channel (PCC). Here we present a cryo-electron microscopy reconstruction of the Escherichia coli PCC, SecYEG, complexed with the ribosome and a nascent chain containing a signal anchor. This reconstruction shows a messenger RNA, three transfer RNAs, the nascent chain, and detailed features of both a translocating PCC and a second, non-translocating PCC bound to mRNA hairpins. The translocating PCC forms connections with ribosomal RNA hairpins on two sides and ribosomal proteins at the back, leaving a frontal opening. Normal mode-based flexible fitting of the archaeal SecYEbeta structure into the PCC electron microscopy densities favours a front-to-front arrangement of two SecYEG complexes in the PCC, and supports channel formation by the opening of two linked SecY halves during polypeptide translocation. On the basis of our observation in the translocating PCC of two segregated pores with different degrees of access to bulk lipid, we propose a model for co-translational protein translocation.     

川东南盆缘复杂构造区龙马溪组页岩孔隙结构及分形特征

川东南盆缘复杂构造区龙马溪组页岩总有机碳(total organic carbon, TOC)含量高、热演化成熟度高,但构造条件复杂,其微观孔隙结构特征及分形特征相关研究较少且与四川盆内页岩存在差异,亟需进一步深入研究。为更好地表征页岩孔隙结构非均质性及其对页岩气富集的影响,综合运用核磁共振、高压压汞及扫描电镜等技术,定量表征复杂构造区页岩微观孔隙结构特征。基于分形理论,利用高压压汞、核磁共振方法获得不同尺度孔隙的分形维数,并探讨分形维数与孔喉结构参数、TOC含量、矿物组分含量的关系及其地质意义。结果表明：川东南盆缘复杂构造区页岩主要发育有机孔、粒间孔和微裂缝。孔隙结构具有多重分形特征,不同尺度孔喉分形维数存在差异,大孔喉复杂程度高于小孔喉,孔隙总分形维数为2.470 2～2.819 1,均值为2.625 6,反映复杂构造区页岩发育更为复杂的孔隙结构,为页岩气提供大量吸附点位,对页岩气聚集具有积极作用。TOC含量和石英含量等因素的共同影响,造成研究区页岩的强非均质性和复杂的孔隙结构特征。与四川盆内页岩相比,研究区页岩孔径分布较广、分形维数偏低。综合分析页岩孔隙结构及分形特征可为昭通示范...     

蜀南地区五峰-龙马溪组页岩微观孔隙结构及分形特征

南方上奥陶统五峰组-下志留统龙马溪组海相页岩是中国页岩气主力开发层位,页岩微观孔隙结构特征的研究对于页岩含气性和开发储量的评价有重要意义。采用场发射扫描电镜和低温氮气吸附实验方法对蜀南地区长宁区块五峰-龙马溪组页岩微观孔隙结构进行了定性评价和定量表征。实验结果表明,蜀南地区五峰-龙马溪组页岩以有机质孔隙为主,局部可见粒间孔和粒内孔发育。氮气吸附回滞环属于H4型,对应纳米级孔隙类型为狭缝型;五峰-龙马溪组页岩平均比表面积17.35 m~2/g,平均孔体积16.70 mm~3/g,平均孔径9.82 nm;页岩纳米级孔隙表面具有分形特征,分形维数平均值为2.681;有机碳含量的增加使得纳米级孔隙数量增多,页岩分形维数增大,孔隙表面粗糙程度增大,页岩比表面积增大,页岩吸附能力增强。     

Crystal structure of a lectin-like natural killer cell receptor bound to its MHC class I ligand

Natural killer (NK) cell function is regulated by NK receptors that interact with MHC class I (MHC-I) molecules on target cells. The murine NK receptor Ly49A inhibits NK cell activity by interacting with H-2D(d) through its C-type-lectin-like NK receptor domain. Here we report the crystal structure of the complex between the Ly49A NK receptor domain and unglycosylated H-2D(d). The Ly49A dimer interacts extensively with two H-2D(d) molecules at distinct sites. At one interface, a single Ly49A subunit contacts one side of the MHC-I peptide-binding platform, presenting an open cavity towards the conserved glycosylation site on the H-2D(d) alpha2 domain. At a second, larger interface, the Ly49A dimer binds in a region overlapping the CD8-binding site. The smaller interface probably represents the interaction between Ly49A on the NK cell and MHC-I on the target cell, whereas the larger one suggests an interaction between Ly49A and MHC-I on the NK cell itself. Both Ly49A binding sites on MHC-I are spatially distinct from that of the T-cell receptor.     

Fractal classification and natural classification of coal pore structure based on migration of coal bed methane

According to the data of 146 coal samples measured by mercury penetration, coal pores are classified into two levels of <65 nm diffusion pore and >65 nm seeping pore by fractal method based on the characteristics of diffusion, seepage of coal bed methane(CBM) and on the research results of specific pore volume and pore structure. The diffusion pores are further divided into three categories: <8 nm micropore, 8-20 nm transitional pore, and 20-65 nm mini-pore based on the relationship between increment of specific surface area and diameter of pores, while seepage pores are further divided into three categories: 65-325 nm mesopore, 325-1000 nm transitional pore, and >1000 nm macropore based on the abrupt change in the increment of specific pore volume.