Exposure of an antigen of chromaffin granules on cell surface during exocytosis

The synthesis rate of the membrane proteins of the catecholamine-storing vesicles (chromaffin granules) of the adrenal medulla is lower than that of the secretory proteins of the contents. Based on these results we proposed that after exocytosis the membranes of chromaffin granules are retrieved and are re-used for several secretion cycles (see also ref. 4). This concept of re-use of granule membranes has been further strengthened by the finding that exogenous markers which are taken up by secretory cells during stimulation can be traced to the Golgi region and to immature secretory organelles. However, one basic question remains: are the membranes of secretory organelles specifically and completely removed from the plasma membrane and if so, how fast is this process? By using an antiserum against a membrane glycoprotein of chromaffin granules we have now obtained quantitative data which demonstrate that during exocytosis this antigen becomes exposed on the cell surface and disappears again to a large degree within 30 min.     

Identification of a secretory granule-binding protein as caldesmon

Stimulation of adrenal chromaffin cells results in a rise in the concentration of intracellular free calcium which initiates catecholamine secretion by exocytosis. An understanding of the molecular basis of exocytosis will require knowledge of the sites of action of calcium. A role for calmodulin has been implicated in secretion from chromaffin cells, and isolated granule membranes bind both calmodulin and a series of cytosolic proteins in a calcium-dependent fashion. Here, we demonstrate that one of the cytosolic granule-binding proteins with a relative molecular mass (Mr) of 70,000 (70K) is a form of the calmodulin-regulated actin-binding protein caldesmon, first isolated from smooth muscle. Cytoplasmic gels assembled from an adrenal medullary extract in the absence of Ca2+ contained actin and the 70K protein. The association of both of these proteins with the cytoplasmic gel was inhibited by a micromolar concentration of Ca2+. In addition, we have demonstrated that the 70K protein is localized at the periphery of chromaffin cells. These results are consistent with the notion that 70K protein (caldesmon) has a role in regulating the organization of actin filaments of the cell periphery during the secretory process.     

Clathrin light chains and secretory vesicle binding proteins are distinct

Recently, several groups have initiated studies on cytosolic proteins that bind to isolated secretory vesicle membranes in the presence of Ca2+ in order to identify proteins that may regulate exocytosis. Two major chromaffin granule binding proteins, of molecular weights 32,000 (32K) and 34,000 (34K), were reported to have the same mobility on one-dimensional SDS gels as clathrin-associated light chains from the adrenal medulla, and the 34K granule binding protein the same one-dimensional peptide map as the 34K clathrin light chain. These observations support the hypothesis that Ca2+-dependent recruitment of soluble light chains to the vesicle membrane may nucleate the assembly of a clathrin coat and initiate endocytosis. Here we report that two-dimensional peptide maps of the clathrin light chains and of all chromaffin granule membrane binding proteins in the 30K range are distinct, and therefore fail to support this hypothesis. It has also been suggested that some or all of the vesicle binding proteins require calmodulin for their interaction with the membrane. However, we find that antagonism of calmodulin by trifluoperazine does not prevent the association of the other cytosolic proteins with the chromaffin granule membrane.     

Mechanism of membrane damage mediated by human eosinophil cationic protein

Recent evidence suggests a role for eosinophil granule proteins in contact-dependent antibody-mediated cytotoxicity. Cytolysis may involve a secretory phenomenon whereby granule proteins are released at the site of contact between eosinophil and target cells. Several basic proteins have been isolated from eosinophil granules, including the major basic protein, eosinophil cationic protein, eosinophil protein-X and eosinophil peroxidase. One of the major granule proteins of human eosinophils is the eosinophil cationic protein (ECP) which has been shown to damage schistosomula of Schistosoma mansoni at concentrations as low as 10(-7). Here, we describe the formation of functional channels by purified human ECP. The transmembrane pores formed by ECP are relatively voltage-insensitive and non-ion-selective, suggesting a role for channel formation by ECP in target cell damage mediated by eosinophils. Channel formation by granule proteins of immune effector cells may represent a general and effective mechanism of target cell killing.     

Recruitment of cytosolic proteins to a secretory granule membrane depends on Ca2+-calmodulin

An increase in free calcium triggers catecholamine secretion from chromaffin cells and calmodulin is strongly implicated as the intracellular Ca2+ receptor. In our recent studies of calmodulin action in the chromaffin cell, micromolar Ca2+ concentrations resulted in calmodulin and cytosolic proteins becoming bound to the chromaffin granule membranes. We now report that calmodulin is bound with high affinity to granule membrane proteins of molecular weights (Mrs) 25,000 and 22,000 (25K and 22K) at low Ca2+ (less than 10(-8) M) and to proteins with Mrs 69K and 50K at high Ca2+ (greater than 1 microM). Other cytosolic components (Mrs 70K, 36K, 34K and 32K) require calmodulin for their interfraction with membrane. These proteins separately bound to calmodulin-Sepharose at high Ca2+ concentrations. Although the functions of these adrenal proteins have not been established, the 34K and 32K Mr components co-migrate with clathrin light chains isolated from medullary coated vesicles and the Mr 34K components from both sources share the same one-dimensional peptide map. These interactions were observed at micromolar Ca2+ levels at 'intracellular' conditions of pH and ionic strength and would be expected to occur during secretion from the chromaffin cell.     

Phosphorylcholine acts as a Ca2+-dependent receptor molecule for lymphocyte perforin

Large granular lymphocytes and cytolytic T-lymphocytes (CTL) contain numerous cytoplasmic granules thought to be responsible, at least in part, for the cytolytic activity of these effector cells. Isolated granules are lytic for a variety of target cells and the granule proteins are specifically released upon target-cell interaction. Major proteins in mouse CTL granules are a family of seven serine proteases designated granzymes A to G, and a pore-forming protein called perforin (cytolysin). Purified perforin is cytolytic in the presence of Ca2+ and shows ultrastructural, immunological and amino-acid sequence similarities to complement component C9. Despite these similarities, perforin and C9 are clearly distinct in their mode of target-cell recognition. Whereas C9 insertion is absolutely dependent on a receptor moiety assembled from the complement proteins C5b, C6, C7, and C8 on the target-cell membrane, no requirement for a receptor molecule has been reported for perforin. Here, we demonstrate that phosphorylcholine acts as a specific, Ca2+-dependent receptor molecule for perforin.     

Aggregation of chromaffin granules by calpactin at micromolar levels of calcium

Several cytosolic proteins bind to secretory granule membranes in a Ca2+-dependent manner and thus may be involved in the mediation of membrane interactions during exocytosis. One of these proteins, calpactin, is a tetramer consisting of two heavy chains of relative molecular mass (Mr) 36K (p36) and two light chains of 10K (p10). We report here that calpactin promotes the Ca2+-dependent aggregation and fatty acid-dependent fusion of chromaffin granule membranes at a level of Ca2+ that is lower than that reported for other granule-aggregating proteins, and which parallels the Ca2+ requirement for secretion from permeabilized chromaffin cells. We found subunits of calpactin to be inactive in promoting granule aggregation. Two distinct 33K proteolytic fragments of p36, differing at their N termini, also promote granule aggregation but with different Ca2+ sensitivities from calpactin. These differences suggest that the N-terminal portion of p36 modulates the Ca2+/lipid binding sites in the core portion of p36 (ref.5).     

Anti-alpha-fodrin inhibits secretion from permeabilized chromaffin cells

Chromaffin cells release catecholamine- and peptide-containing granules by exocytosis, by a mechanism involving movement of secretory granules towards the cell membrane, their apposition to it and the fusion of the granule membrane with the plasma membrane. One of the two subunits of membrane-associated brain spectrin, alpha-fodrin is an actin-binding protein which is found at the periphery of chromaffin cells and may be involved in secretion. Because cultured chromaffin cells can be permeabilized with detergents, giving pores large enough to permit the entry of immunoglobulin molecules, we used permeabilized cells to test the effect of specific antibodies on secretory mechanisms. Incubation of permeabilized cells with polyclonal immunoaffinity-purified monospecific anti-alpha-fodrin antibody or its Fab fragments did not modify basal release but did specifically inhibit Ca2+-induced catecholamine release by exocytosis. Our observations indicate that fodrin and the cytoskeleton participate in the release mechanism.     

好氧颗粒污泥吸附孔雀绿研究

以蔗糖为碳源,在序批式间歇反应器中培养出沉降性能良好的黑色好氧颗粒污泥.以其作为吸附剂,研究颗粒污泥对染料孔雀绿的吸附作用.结果表明,Langmu ir吸附等温模型能较好地对吸附数据进行拟合,揭示好氧颗粒污泥对孔雀绿的吸附为颗粒污泥表面的单分子层吸附,最大吸附量为52.63 mg.gSS-1,且在pH为6时,吸附效果较好.准二级动力学模型能很好地拟合孔雀绿的吸附动力学过程.颗粒污泥对孔雀绿的平衡吸附量随污泥浓度的升高而减小.研究表明,好氧颗粒污泥可作为经济、有效的生物吸附剂用于染料废水的处理.     

短蛸血细胞的形态结构、类型、细胞化学特性及其吞噬活性研究

血细胞作为免疫防御的第一道防线，在软体动物的非特异性免疫系统中具有重要作用.为了揭示头足类动物血细胞的形态结构、生物学性质及其免疫学功能，实验利用活体染色、细胞化学和电镜技术等对短蛸血细胞的形态结构、类型、细胞化学性质及其弧菌吞噬活性进行了研究.研究结果显示：短蛸血细胞具有大透明细胞、小透明细胞、小颗粒细胞和大颗粒细胞4种类型.大透明细胞约占血细胞总数22.6%，平均直径为11.64±0.82?μm，胞质中没有或仅含有很少量的颗粒，细胞表面光滑无伪足伸出；小透明细胞约占血细胞总数的1.7%，平均直径为8.88±0.88?μm，胞质中仅含有少量颗粒，细胞核对台盼蓝呈阳性反应，核质比较大；小颗粒细胞约占血细胞总数的50.7%，平均直径为12.82±1.54?μm，胞质中具有许多大小较为均一的嗜碱性小颗粒，有些细胞内还含有小空泡，不为中性红所着色，疑为颗粒脱内容物所致，细胞表面有较短伪足伸出；大颗粒细胞约占血细胞总数的25.20%，平均直径为13.66±1.50?μm，胞质中具有大小不匀的许多嗜碱性颗粒，有些细胞内也含有不为中性红所着色的较大空泡，细胞表面有许多长伪足伸出.颗粒细胞中所含有的大量嗜碱性颗粒可能与蛋白质等分泌物的活跃合成有关.大颗粒细胞还具有全质分泌的特性，小透明细胞极可能是大颗粒细胞全质分泌后的一时性残余胞体.弧菌吞噬实验结果表明，两类透明细胞均没有弧菌吞噬活性，两类颗粒细胞均具有弧菌吞噬活性，暗示这两种颗粒细胞很可能是短蛸发挥细胞免疫功能的关键性细胞，不仅与某些物质的活跃合成与分泌有关，而且可能还直接参与了外来病原的吞噬及清除.研究结果为揭示短蛸血细胞在非特异性免疫防御中的作用奠定了基础，对于丰富软体动物免疫学也具有重要的科学价值.     

Chaperone release and unfolding of substrates in type III secretion

Type III protein secretion systems are essential virulence factors of many bacteria pathogenic to humans, animals and plants. These systems mediate the transfer of bacterial virulence proteins directly into the host cell cytoplasm. Proteins are thought to travel this pathway in a largely unfolded manner, and a family of customized cytoplasmic chaperones, which specifically bind cognate secreted proteins, are essential for secretion. Here we show that InvC, an ATPase associated with a Salmonella enterica type III secretion system, has a critical function in substrate recognition. Furthermore, InvC induces chaperone release from and unfolding of the cognate secreted protein in an ATP-dependent manner. Our results show a similarity between the mechanisms of substrate recognition by type III protein secretion systems and AAA + ATPase disassembly machines.     

Ankyrin binding to (Na+ + K+)ATPase and implications for the organization of membrane domains in polarized cells

The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.     

Inhibition of exocytosis by intracellularly applied antibodies against a chromaffin granule-binding protein

Exocytotic secretion requires the interaction and fusion of secretory vesicles with the plasma membrane. This process could be mediated by specific recognition molecules acting as intracellular, membrane-bound receptors and ligands. One possible component of such a recognition site on the plasma membrane is a protein of relative molecular mass (Mr) 51,000 (51K) that has been isolated from bovine adrenal chromaffin cells. This protein binds strongly to chromaffin granules, the secretory vesicles of these cells. To determine the function of this membrane-anchored chromaffin granule-binding protein in exocytosis, we tested the effect of intracellularly injected antibodies on secretion. Here we show, by two independent techniques in two different cell types, that antibodies against this protein inhibit exocytosis. In rat pheochromocytoma cell cultures, monospecific antibodies, applied by erythrocyte ghost fusion, impair the release of 3H-noradrenaline. The same antibodies, introduced into individual chromaffin cells through a patch pipette, block exocytosis, as revealed by the measurement of membrane capacitance. These results demonstrate the functional involvement in exocytosis of a plasma membrane protein with high affinity for secretory vesicles.     

柏薏颗粒对高尿酸血症大鼠的初步药效学分析

针对高尿酸血症大鼠模型开展柏薏颗粒的初步药效学研究,探究其作用机制.将72只雄性大鼠随机分为空白组、模型组、柏薏颗粒高、中、低剂量组及非布司他阳性对照组,采用氧嗪酸钾灌胃给药法建立高尿酸血症大鼠模型,连续给药治疗14 d,每天1次.采用酶联免疫吸附剂测定(ELISA)试剂盒测定血清尿酸(SUA)、肌酐(Cr)、尿素氮(BUN)水平及肝脏黄嘌呤氧化酶(XOD)活性,并用免疫印迹法测定肾脏中有机阴离子转运体1(OAT1)及葡萄糖转运体9(GLUT9)的表达.结果表明:与模型组相比,柏薏颗粒可剂量依赖性地降低SUA水平、肝脏XOD活性及GLUT9蛋白表达,并上调OAT1蛋白表达,且高剂量组与非布司他作用相当(P>0.05);柏薏颗粒组与空白组大鼠血清中Cr和BUN浓度无显著性差异,表明柏薏颗粒无肾损伤;柏薏颗粒具有明显的降血尿酸作用,其机制可能与抑制XOD活性和肾脏GLUT9蛋白表达,并上调OAT1蛋白表达有关.     

A role for calpactin in calcium-dependent exocytosis in adrenal chromaffin cells

Stimulation of bovine adrenal chromaffin cells results in a rise in the concentration of cytosolic calcium which triggers the release of catecholamines by exocytosis. Several cytosolic proteins that bind to secretory granule membranes in a calcium-dependent manner have been implicated in exocytosis and some belong to a family of calcium-binding proteins, the annexins. One of these, calpactin, is a tetramer consisting of two heavy and two light chains (relative molecular masses 36,000 and 10,000 respectively) and can aggregate and fuse membranes in vitro in the presence of arachidonic acid. Calpactin is found at the cell periphery and is phosphorylated when chromaffin cells are stimulated. We show here that both calpactin and calpactin heavy chain (p36) reconstitute secretion in permeabilized chromaffin cells in which secretion has been reduced as a result of leakage of cellular components. This effect is inhibited by an affinity-purified antibody against p36. Secretion from permeabilized cells is inhibited by a synthetic annexin-consensus peptide, but not by a nonspecific hydrophobic peptide; this inhibition is reversed by p36. Our results indicate that either calpactin or p36 is essential for exocytosis.     

贻贝棘尾虫休眠包囊胞质的电镜酶细胞化学

该文应用电镜酶细胞化学方法显示，贻贝棘尾虫休眠包囊中，在胞质嗜锇粒和拟糖原粒区，酸性磷酸酶反应颗粒聚集于具有消化泡状体上，葡萄糖－6－磷酸酶反应也定位在自噬泡膜位置。作者据所得结果推测，嗜锇粒可能是棘尾虫休眠包囊中用于自噬作用的主要结构成分，休眠细胞代谢过程中质膜结构也具有补充形成消化泡膜的作用。     

Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase

In physiological settings, nucleic-acid translocases must act on substrates occupied by other proteins, and an increasingly appreciated role of translocases is to catalyse protein displacement from RNA and DNA. However, little is known regarding the inevitable collisions that must occur, and the fate of protein obstacles and the mechanisms by which they are evicted from DNA remain unexplored. Here we sought to establish the mechanistic basis for protein displacement from DNA using RecBCD as a model system. Using nanofabricated curtains of DNA and multicolour single-molecule microscopy, we visualized collisions between a model translocase and different DNA-bound proteins in real time. We show that the DNA translocase RecBCD can disrupt core RNA polymerase, holoenzymes, stalled elongation complexes and transcribing RNA polymerases in either head-to-head or head-to-tail orientations, as well as EcoRI(E111Q), lac repressor and even nucleosomes. RecBCD did not pause during collisions and often pushed proteins thousands of base pairs before evicting them from DNA. We conclude that RecBCD overwhelms obstacles through direct transduction of chemomechanical force with no need for specific protein-protein interactions, and that proteins can be removed from DNA through active disruption mechanisms that act on a transition state intermediate as they are pushed from one nonspecific site to the next.     

Microtubule-associated protein 1C from brain is a two-headed cytosolic dynein

Dynein, an ATPase, is the force-generating protein in cilia and flagella. It has long been speculated that cytoplasmic microtubules contain a related enzyme involved in cell division or in intracellular organelle transport. A 'cytoplasmic dynein' has been described in sea urchin eggs, but because the egg stockpiles precursors for both cytoplasmic and ciliary microtubules, the role of this enzyme in the cell has remained unresolved. We recently found that the microtubule-associated protein (MAP) 1C (ref. 6) from brain is a microtubule-activated ATPase that produces force in the direction corresponding to retrograde organelle transport in the cell. MAP 1C has several similar properties to ciliary and flagellar dynein. Here we show directly, using scanning transmission electron microscopy, that MAP 1C is structurally equivalent to the ciliary and flagellar enzyme and is the long-sought cytoplasmic analogue of this enzyme.     

Exo1 and Exo2 proteins stimulate calcium-dependent exocytosis in permeabilized adrenal chromaffin cells.

In many cell types an increase in cytosolic calcium is the main signal for the exocytotic release of stored secretory components such as hormones and neurotransmitters. The site of action of calcium in exocytosis is not known, neither are the participating molecules. In the case of the intracellular membrane fusions that occur during transport through early stages of the secretory pathway, several cytosolic and peripheral membrane proteins are necessary. Permeabilized cells have been useful in understanding the requirements for calcium and nucleotides in regulated exocytosis and under certain conditions there is leakage of soluble protein components and run-down of the exocytotic response. This system can be used to identify the soluble proteins involved in exocytosis, one candidate in chromaffin cells being annexin II (calpactin). Here we use this assay to identify two other cytosolic protein factors that regulate exocytosis in permeabilized adrenal chromaffin cells, which we term Exo1 and Exo2. Exo1 from brain cytosol resolves on electrophoresis in SDS-polyacrylamide gels as a group of polypeptides of relative molecular mass approximately 30,000 and shares sequence homology with the 14-3-3 family of proteins. The ability of Exo1 to reactivate exocytosis is potentiated by protein kinase C activation and therefore Exo1 may influence the protein kinase C-mediated control of Ca(2+)-dependent exocytosis.     

A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans

Messenger RNA regulation is a critical mode of controlling gene expression. Regulation of mRNA stability and translation is linked to controls of poly(A) tail length. Poly(A) lengthening can stabilize and translationally activate mRNAs, whereas poly(A) removal can trigger degradation and translational repression. Germline granules (for example, polar granules in flies, P granules in worms) are ribonucleoprotein particles implicated in translational control. Here we report that the Caenorhabditis elegans gene gld-2, a regulator of mitosis/meiosis decision and other germline events, encodes the catalytic moiety of a cytoplasmic poly(A) polymerase (PAP) that is associated with P granules in early embryos. Importantly, the GLD-2 protein sequence has diverged substantially from that of conventional eukaryotic PAPs, and lacks a recognizable RRM (RNA recognition motif)-like domain. GLD-2 has little PAP activity on its own, but is stimulated in vitro by GLD-3. GLD-3 is also a developmental regulator, and belongs to the Bicaudal-C family of RNA binding proteins. We suggest that GLD-2 is the prototype for a class of regulatory cytoplasmic PAPs that are recruited to specific mRNAs by a binding partner, thereby targeting those mRNAs for polyadenylation and increased expression.