白芷细胞外21kDCaMBP抗体制备

植物细胞外存在钙调素（CaM），并且胞外CaM具有促进白茫悬浮培养细胞增殖“’及原生质体第一次分裂、壁再生功能“‘．唐军”’首次在白花悬浮培养细胞外检测并纯化了一种与CaM结合依赖于Ca’“的分子量约为21kD的钙调素结合蛋白（CaMBP），那么，对此蛋白深入研究将有助于揭示胞外Ca’”·CaM信号分子的作用机制．由于ZIkDCaMBP主要存在于细胞壁区，含量较低，提纯困难，因此，对此蛋白的大量纯化及其抗体制备就成为对ZIkDCaMBP定量、定位及其生理功能深入研究的关键．本实验利用唐军”’建立的SephadexG—100凝胶过滤及CM…     

生长抑素对胰岛素分泌的调控机制研究

生长抑素(Somatostatins,SST)对胰腺β细胞胰岛素的分泌有重要的调节作用,这一调节作用与细胞内钙离子浓度变化相偶联.以大鼠胰腺β细胞为研究对象,采用显微荧光测钙技术和膜片钳技术,研究了胞外SST对胞内钙离子信号的影响,初步探讨了其作用机制.结果表明:在细胞外液有钙时,胞外SST可减少由去极化产生的胞外钙离子内流;而在细胞外液无钙时,胞外SST通过动员胞内钙库释放而引起胞浆内钙离子浓度显著增高,并触发胰岛素的分泌.     

悬浮培养红豆杉细胞团区域化与营养生理的关系

通过对悬浮培养的南方红豆杉（Ｔaxus chinensis var.Mairei)细胞团切片及胞内淀粉颗粒染色等形态学上的系统观察,发现细胞团从里向外呈现不同层次,淀粉颗粒主要分布在细胞外层。对胞内淀粉、可溶性蛋白、细胞生物量和紫杉醇产量进行动态测定,表明细胞干重与胞内淀 粉和蛋白量呈正相关,从对淀粉代谢情况看,可以初步认为细胞团不同层次细胞周围营养物质的浓度梯度是细胞形态与功能分化的重要原因。     

植物生物反应器细胞悬浮培养研究进展

利用生物反应器进行植物细胞悬浮培养可以缩短植物细胞的生长周期，但进行大规模植物细胞悬浮培养会受到植物自身特点和生物技术的限制。通过与微生物相比较，笔详细介绍了植物细胞悬浮培养的特点，分析了用于植物细胞悬浮培养的反应器类型及其特点和适用范围，并提出以植物快速繁育为主要目标的植物生物反应器的研究方向。     

溶氧浓度对东北红豆杉悬浮细胞的生理影响

研究了均匀溶氧体积浓度变化对东北红豆杉悬浮植物细胞的生理影响,结果表明纯氧（DO=350）和缺氧（DO=0和10）培养条件均抑制了细胞的呼吸,导致了细胞线粒体活性降低。缺氧培养条件下细胞内外可溶性蛋白含量变化不大,而在纯氧（DO=350）和DO=40的培养条件下,胞内可溶性蛋白呈现增高趋势。此外,纯氧培养和缺氧培养条件均导致了细胞外酚的积累。     

升高细胞外钙离子能抑制和促进细胞凋亡的发生

钙离子是细胞分裂,生长,死亡过程中重要的信号传导分子。为研究胞外钙离子与细胞凋亡的关系,将Ｃａ＾２＋（５￣７ｍｍｏｌ）加入无血清培养液观察钙离子对无血清诱导人胚肺二倍体细胞株ＫＭＢ１７凋亡的影响并将Ｃａ＾２＋（５￣７ｍｍｏｌ）加入含体积分数为２％血清的脊髓灰质炎病毒（ｐｏｌｉｏｖｉｒｉｕｓ）生长培养液中,研究胞外钙离子对ｐｏｌｉｏｖｉｒｕｓ诱导凋亡的影响,经光学显微镜,荧光显微镜,细胞流式仪,电子     

不同培养基对羌活植物细胞悬浮培养的影响

探究不同培养基基质对羌活植物细胞悬浮培养的影响,为建立羌活植物细胞悬浮培养体系摸索条件.通过比较不同基础培养基种类、蔗糖浓度、硝态氮与铵态氮的比值和总氮含量等因素对羌活植物悬浮细胞生长的影响,以确定羌活植物细胞悬浮培养的最佳培养基条件.实验结果显示,以MS为基本培养基,当蔗糖浓度为30g/L,NH4^+/NO3^-=1∶2,总氮量为1/2N时,最有利于羌活植物细胞的悬浮培养.实验结果表明,确定的羌活植物细胞悬浮培养的最佳培养基条件,可为后续的羌活植物细胞悬浮培养体系的建立提供依据.     

人工悬浮培养发菜胞外多糖对小鼠免疫功能的影响

研究人工悬浮培养的发菜胞外多糖对小鼠非特异性及特异性免疫能力的影响.体内实验表明:50~100,mg/(kg·d)的发菜胞外多糖能够显著提高小鼠单核巨噬细胞吞噬碳粒的能力和NK细胞的自然杀伤活性(P0.05),并呈现剂量依赖性,同时,发菜胞外多糖能在一定程度上提高腹腔巨噬细胞的吞噬能力,起到提高机体非特异性免疫水平的作用.与空白组相比,高、低剂量的发菜胞外多糖对小鼠脏器指数、二硝基氟苯诱导的迟发型超敏反应(DTH)、脾淋巴细胞的增殖均无显著性影响,表明人工培养发菜胞外多糖对小鼠特异性免疫系统无显著性效果.     

细胞外ATP调节NaCl胁迫诱导的烟草悬浮细胞死亡和呼吸抑制

本文以烟草悬浮细胞BY-2为材料,探讨了胞外ATP对NaCl诱导的细胞死亡和呼吸抑制的调节作用.实验表明,随着NaCl浓度的逐渐上升(50、100、200、400mmol/L),细胞的死亡水平逐渐上升,而胞外ATP含量和细胞呼吸速率则随NaCl浓度的上升而逐渐下降.本文在200mmol/L NaCl处理下的细胞中探索了胞外ATP对NaCl诱导的细胞死亡和呼吸抑制的调节作用.结果发现,较之200mmol/L NaCl胁迫下的细胞,对NaCl胁迫的细胞加入外源ATP(20μmol/L)其使得细胞死亡水平显著性降低,也使得胞外ATP含量和细胞呼吸速率均有所回升.上述实验观察表明,NaCl胁迫诱导的植物细胞的死亡和呼吸抑制可能和细胞外ATP水平的变化有关,并且胞外ATP对NaCl诱导的细胞死亡具有一定的调节作用.     

低温诱导及Ca^2＋信号对胡萝卜悬浮培养细胞抗冻蛋白的合成及细胞抗冻能力的影响

选择胡萝卜这种较抗冻的植物作为实验材料,在 MS 2.4D_1液体培养基中加入 Ca~(2 )阻断剂进行悬浮培养。对胞外提取物进行 SDS-PAGE 分析的结果表明,加 LaCl_3与加入 EGTA 处理的细胞样品共同存在一条低量表达的36 kD 多肽。且两者细胞的抗冻能力均有所下降。通过 Western-blotting 证实了这条36 kD 的多肽是一种抗冻蛋白,研究表明该抗冻多肽在低温条件下的合成、诱导与钙信号紧密相关。     

Activation of latent Ca2+ channels in renal epithelial cells by parathyroid hormone.

Calcium has an important role in regulating epithelial cell ion transport and is itself transported by tissues involved in the maintenance of extracellular Ca2+ homeostasis. Although the mechanism of Ca2+ entry in electrically excitable cells is well-documented little is known about it in epithelial cells. Calcium absorption in polarized epithelial cells is a two-step process in which Ca2+ enters cells across apical plasma membranes and is extruded across basolateral membranes. Efflux may be mediated by an energy-dependent Ca2(+)-ATPase or by Na+/Ca2+ exchange. We examined Ca2+ influx in single cultured cells from distal renal tubules sensitive to parathyroid hormone by measuring intracellular Ca2+. Our results demonstrate that parathyroid hormone activates dihydropyridine-sensitive channels responsible for Ca2+ entry. We also show that microtubule-dependent exocytosis stimulated by parathyroid hormone may be necessary for the insertion or activation of Ca2+ channels in these cells. Once inserted or activated, dihydropyridine-sensitive channels mediate Ca2+ entry into these Ca2(+)-transporting epithelial cells. Our results support the view that agonist-induced exocytosis may represent a general paradigm for modulation of transport in epithelial cells by delivery and incorporation of transport proteins to plasma membranes or by delivery to plasma membranes of factors regulating these proteins.     

动物体液中的胞外钙调素结合蛋白（快报）

利用以生物素标记钙调素为探针的凝胶覆盖技术检测动物体液中胞外钙调素结合蛋白（ＣａＭＢＰ）。结果，人的唾液中检测到至少３种分子量分别为１４ｋＤ，２４ｋＤ和５２ｋＤ的ＣａＭＢＰｓ。其中，５２ｋＤ蛋白与ＣａＭ的结合依赖于Ｃａ２＋的存在，而２４ｋＤ和１４ｋＤ蛋白则不依赖于Ｃａ２＋。在鸡血清里，检测到以９４ｋＤ，４４／４５ｋＤ蛋白为主的４～５种胞外ＣａＭＢＰｓ，所有的这些蛋白与ＣａＭ的结合都依赖于Ｃａ２－。此外，在牛奶中也检出胞外ＣａＭＢＰｓ。以上结果，证明了在动物中普遍存在胞外ＣａＭＢＰｓ，为胞外钙调素的作用机理提供了新线索。     

Lumenal gating mechanism revealed in calcium pump crystal structures with phosphate analogues

P-type ion transporting ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes. Transfer of bound cations to the lumenal or extracellular side occurs while the ATPase is phosphorylated. Here we report at 2.3 A resolution the structure of the calcium-ATPase of skeletal muscle sarcoplasmic reticulum, a representative P-type ATPase that is crystallized in the absence of Ca2+ but in the presence of magnesium fluoride, a stable phosphate analogue. This and other crystal structures determined previously provide atomic models for all four principal states in the reaction cycle. These structures show that the three cytoplasmic domains rearrange to move six out of ten transmembrane helices, thereby changing the affinity of the Ca2+-binding sites and the gating of the ion pathway. Release of ADP triggers the opening of the lumenal gate and release of phosphate its closure, effected mainly through movement of the A-domain, the actuator of transmembrane gates.     

A consensus amino-acid sequence repeat in Torpedo and mammalian Ca2+-dependent membrane-binding proteins

A group of calcium-binding proteins which bind to biomembranes has recently been identified in widely different cells and tissues (refs 1-7, reviewed in ref. 8). Three of these proteins (p70, p36 and p32.5) cross-react with antiserum to calelectrin, a Ca2+-binding protein (relative molecular mass 34,000 (34K] from the ray Torpedo marmorata, giving rise to their designation as calelectrin-related proteins. We now report that calelectrin, p36 and p32.5 contain a 17-amino-acid consensus sequence which is conserved and present in multiple copies. We suggest that this sequence may be common to other members of this new group of Ca2+-binding proteins and may underlie their unusual mode of combination with biomembranes.     

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).     

Naloxone-reversible effect of opioids on pinocytosis in Amoeba proteus

A characteristic feature of induced pinocytosis in Amoeba proteus is the formation of broad channels by invagination of the cell membrane. This process, which requires Ca2+, occurs in response to depolarising cations. High Ca2+ levels reduce pinocytosis induced by cations such as Na+ and Tris+, whereas pinocytosis induced by K+ is less affected by Ca2+ (ref. 4). Agents which interfere with the calcium metabolism of the amoeba will therefore either stimulate or inhibit pinocytosis induced by Na+ (ref. 5). Among the agents which are supposed to reduce Ca2+ influx across cell membranes or otherwise decrease cellular availability of Ca2+ are the opiates and opioid peptides, high doses of which have been reported to affect the amoeba. Accordingly, Met-enkephalin, morphine and codeine potentiate the inhibition of pinocytosis caused by Ca2+-binding agents and reverse the calcium blockade of pinocytosis mediated by caffeine. In this report we show that pinocytosis induced by Na+ or Tris+ is suppressed by beta-endorphin, Metenkephalin and morphine. These effects were abolished or diminished by an opiate receptor antagonist, (-)naloxone, by increasing the Na+ concentration, or by addition of Ca2+.     

Crystal structure of the calcium pump with a bound ATP analogue

P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across cell and organelle membranes. Here, we describe the crystal structure of the Ca2+ pump of skeletal muscle sarcoplasmic reticulum, a representative member of the P-type ATPase superfamily, with an ATP analogue, a Mg2+ and two Ca2+ ions in the respective binding sites. In this state, the ATP analogue reorganizes the three cytoplasmic domains (A, N and P), which are widely separated without nucleotide, by directly bridging the N and P domains. The structure of the P-domain itself is altered by the binding of the ATP analogue and Mg2+. As a result, the A-domain is tilted so that one of the transmembrane helices moves to lock the cytoplasmic gate of the transmembrane Ca2+-binding sites. This appears to be the mechanism for occluding the bound Ca2+ ions, before releasing them into the lumen of the sarcoplasmic reticulum.     

Synaptic function modulated by changes in the ratio of synaptotagmin I and IV.

Communication within the nervous system is mediated by Ca2+-triggered fusion of synaptic vesicles with the presynaptic plasma membrane. Genetic and biochemical evidence indicates that synaptotagmin I may function as a Ca2+ sensor in neuronal exocytosis because it can bind Ca2+ and penetrate into lipid bilayers. Chronic depolarization or seizure activity results in the upregulation of a distinct and unusual isoform of the synaptotagmin family, synaptotagmin IV. We have identified a Drosophila homologue of synaptotagmin IV that is enriched on synaptic vesicles and contains an evolutionarily conserved substitution of aspartate to serine that abolishes its ability to bind membranes in response to Ca2+ influx. Synaptotagmin IV forms hetero-oligomers with synaptotagmin I, resulting in synaptotagmin clusters that cannot effectively penetrate lipid bilayers and are less efficient at coupling Ca2+ to secretion in vivo: upregulation of synaptotagmin IV, but not synaptotagmin I, decreases evoked neurotransmission. These findings indicate that modulating the expression of synaptotagmins with different Ca2+-binding affinities can lead to heteromultimers that can regulate the efficiency of excitation-secretion coupling in vivo and represent a new molecular mechanism for synaptic plasticity.     

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.