心肌细胞核钙调素Ⅰ介导的bcl-2转录调节在大鼠心肌肥厚中的作用

为探讨心肌细胞核钙调素Ⅰ（calmodulinⅠ,CaMⅠ）介导的bcl-2转录调节存人鼠心肌肥脬中的作用及其可能机制,实验随机分为对照组和心肌肥厚组,采用腹卡动脉缩窄法制备人鼠心肌肥厚模犁。模型复制成功后4周,以改良差速离心和密度梯度离心提取并纯化细胞核;蛋白印迹法测定心肌细胞核cAMP反应元件结合蛋白（cAMP response-element binding protein,CREB）及磷酸化CREB（phosphorylated cAMP response-element binding protein,pCREB）表达;免瘦组化法观察左审心肌组织CaMI蛋白表达及分布;延续转录分析法观察阻断CaMⅠ后心肌细胞核bcl-2 mRNA的变化。结果表明,心肌肥厚组pCREB蛋白表达较对照组明显增加（P〈0．05）,CREB蛋门表达无明显变化（P〉0．05）;CaMⅠ分布于细胞核及细胞浆,心肌肥厚组CaMⅠ蛋白表达较对照组明显增加（P〈0．05）;使用CaM抑制刺后心肌细胞核bcl-2 mRNA表达明显上调（P〈0．05）。结果提示,压力超负荷时心肌细胞核内CaMⅠ激活,抗凋亡基因bcl-2表达下调,核转录因子CREB磷酸化增加,但CREB在调节bcl-2基因转录过程中可能发挥次要作用。     

一种钙调素结合蛋白对小麦质膜H~ -ATP酶活性的调节

植物生长素参与植物生长和发育诸多方面的调节。研究表明,生长素的调节机制与Caz”的存在紧密相关。Caz”在植物激素的信号传导中起着信使作用（Hepler和Randy1985）,它与钙调素（Ca．－－－urr－ulin,CaM）结合参与了各种类型植物激素应答反应的调节。现已查明,CaM的诸多功能常受其内源性结合蛋白的调控。本研究组曾分离得到一种新的植物CaM结合蛋白——CaMBP－10o实验证明,CaMBP－10通过与CaM的特异性结合显著抑制了CaM对其靶酶的激活（尚克进等1991）。前期工作还发现CaMBP－10对生长素诱导的小麦芽鞘伸长和质子外排均有…     

外源钙调素对粟酒裂殖酵母细胞增殖的影响

外源钙调素（CaM）对粟酒裂殖酵母（Schizosaccharomycespombe）细胞增殖的影响。实验结果表明外源CaM能明显抑制粟酒裂殖酵母细胞的增殖,其作用方式是延长了粟酒裂殖酵母细胞生长的延滞期。抗粟酒裂殖酵母CaM抗体、TFP及Phenyl-SepharoseCL－4B能降低CaM对细胞生长的抑制作用,而Ca2+及Ca2+螫合剂EGTA对CaM的抑制作用均无影响。以上结果提示,外源CaM对粟酒裂殖酵母细胞增殖的抑制作用可能是由于胞外CaM激活了细胞膜上的Ca2+泵,使胞内Ca2+浓度降低所致。     

钙和钙调素对机械刺激诱导的烟草悬浮培养细胞耐热性的调控

机械刺激（mechanical stimulation,MS）可提高烟草悬浮培养细胞的钙调素（calmodulin,CaM）活性,诱导烟草悬浮培养细胞耐热性的形成,外源Ca2＋可加强,而Ca2＋螯合剂EGTA、质膜Ca2＋通道阻塞剂La3＋和胞内Ca2＋通道阻断剂钌红（RR）,以及CaM拮抗剂氯丙嗪（CPZ）和三氟拉嗪（TFP）则削弱这种耐热性的形成。这些暗示Ca2＋和CaM参与MS诱导的烟草悬浮培养细胞耐热性形成的调控。     

心肌细胞核钙调素I介导的bcl-2转录调节在大鼠心肌肥厚中的作用

为探讨心肌细胞核钙调素I(calmodulin I,CaM I)介导的bcl-2转录调节在大鼠心肌肥厚中的作用及其可能机制, 实验随机分为对照组和心肌肥厚组,采用腹主动脉缩窄法制备大鼠心肌肥厚模型。模型复制成功后4周,以改良差速离心和密度梯度离心提取并纯化细胞核;蛋白印迹法测定心肌细胞核cAMP反应元件结合蛋白(cAMP response-element binding protein,CREB)及磷酸化CREB(phosphorylated cAMP response-element binding protein,pCREB)表达;免疫组化法观察左室心肌组织CaM I蛋白表达及分布;延续转录分析法观察阻断CaM I后心肌细胞核bcl-2 mRNA的变化。结果表明,心肌肥厚组pCREB蛋白表达较对照组明显增加(P<0．05),CREB蛋白表达无明显变化(P>0．05);CaM I分布于细胞核及细胞浆,心肌肥厚组CaM I蛋白表达较对照组明显增加(P<0．05);使用CaM抑制剂后心肌细胞核bcl-2 mRNA表达明显上调(P<0．05)。结果提示,压力超负荷时心肌细胞核内CaM I激活,抗凋亡基因bcl-2表达下调,核转录因子CREB磷酸化增加,但CREB 在调节bcl-2基因转录过程中可能发挥次要作用。     

金标记植物钙调素的制备和应用

ThePreparationandAppicationofColloidalGold-LabeledPlantCalmodulinSONGChun－Fens，SUNDa－Ye（CellLaboratory,DepartmentofBiology,HebeiNormalUniversity,Shijiazhuang050016）LIShu-Rong（LaboratoryCenterofElectronMicroscopy,HebeiMedicalUniversity,Shijiazhuang050017）CaM是一种多功能的Ca2＋受体蛋白，广泛存在于各种真核、原核生物中，并参与众多生理功能的调控。但迄今尚未发现CaM具有任何酶活性，其调控作用是通过与之相结合的CaM结合蛋白（CaMBPs）实现的。因此，研究与CaM相结合的各种不同ChMBP…     

GM_1激活环核苷酸磷酸二酯酶的作用

神经节苷脂GM1能激活CaM依赖性环核苷酸磷酸二酯酶,其AC50为1．56μg／ml．这种作用并不一定依赖Ca2＋的存在．在有Ca2＋存在时,GM1对PDE的最大激活活性低于CaM,仅为CaM的80．4％;没有Ca2＋存在时,GM1与CaM对PDE有同样的最大激活活性（100％）．GM1能使CaM对PDE的激活曲线左移,AC50降低,但不改变CaM对PDE的最大激活活性．三氟啦嗪能使GM1激活的PDE的活性降低,其IC50为16．3μmol／L．     

植物钙调素研究进展

钙调素（Ｃａｌｍｏｄｕｌｉｎ,简称ＣａＭ）做为Ｃａ２＋的受体,在Ｃａ２＋信号系统传导中起着关键的作用,它通过和Ｃａ２＋的结合而激活一系列的靶酶和非酶蛋白质,从而调控生理代谢及基因表达。孙大业、郭艳林（１９９１）和龚明等人（１９９０）在各自的论著中对植物钙调素已作了介绍,本文除了涉及他们所没有提到的某些问题之外,重点介绍了植物ＣａＭ研究的新进展     

多功能的钙／钙调素依赖的蛋白激酶II介导的细胞信号传导

钙调素（Calmodulin,CaM）是一个特别的对钙敏感的蛋白,在钙信号传导通路中扮演重要角色钙／钙调素依赖性蛋白激酶（Calcium／calmodulin-dependent kinases（CaMKs））与荷尔蒙、神经迷质及其他信号引起的细胞反应相关、作为重要的第二信使,钙／钙调素依赖的蛋白激酶Ⅱ（CaM—KⅡ）是一类在细咆中无所不在的表达的蛋白激酶,能维持细胞内的钙浓度在很低的水平,再增加后续的特定的钙激动刺激。钙／钙调素依赖的簧白激酶Ⅱ独特的全酶结构和自我调节的性质使其对短暂的钙信号和胞内钙的变化能做出延长反应：本文从结构、合成、细胞分布、反应底物、生理功能等方面介绍了钙／钙调素依赖的蛋白激酶Ⅱ的激活对细胞信号传导的作用。     

钙调素对钙调素结合蛋白-10和玉米非特异性脂转移蛋白与脂质结合活性的影响

荧光标记的脂质结合实验表明,钙调素结合蛋白-10（CaMBP-10）具有典型的植物非特异性脂质转移蛋白与脂质结合的特性。进一步实验研究了钙调素（calmodulin,CaM）对CaMBP-10和玉米nsLTP与脂质结合的活性的影响,结果显示无论在有钙和无钙条件下,CaM对两者的影响均有不同之处,W-7和TFP能消除CaM的影响。提示CaM不仅与CaMBP-10和玉米nsLTP特异性相互作用,而且对2种脂转移蛋白可能具有不同的调节机制。     

Amino acid sequence around the active site cysteine residue of calcium-activated neutral protease (CANP)

We have examined hydrophobic properties of Tetrahymena CaM using the uncharged probe, n-phenyl-1-naphthylamine (NPN) fluorescence. The maximal fluorescence intensity of Tetrahymena calmodulin (CaM) is less than 1/12 of that of the bovine brain CaM. In the phosphodiesterase activation, the potency of Tetrahymena CaM, which was represented by reciprocals of the quantity of CaM required for half-maximal activation of enzyme was 22.7% respectively, of that of the bovine brain CaM. Here, Tetrahymena CaM had less hydrophobic groups exposed in the presence of Ca2+. Then Ca2+-CaM dependent enzymes require much amount of Tetrahymena CaM, comparing with the bovine brain CaM.     

Mode of activation of bovine brain inositol 1,4,5-trisphosphate 3-kinase by calmodulin and calcium.

The effect of Ca2+ and calmodulin (CaM) on the activation of purified bovine brain Ins(1,4,5)P3 kinase was quantified and interpreted according to the model of sequential equilibria generally used for other calmodulin-stimulated systems. Two main conclusions can be drawn. (i) CaM.Ca3 and CaM.Ca4 together are the biologically active species in vitro, as is the case for the great majority of other calmodulin targets. (ii) These species bind in a non-co-operative way to the enzyme with an affinity constant of 8.23 x 10(9) M-1, i.e. approx 10-fold higher than for most calmodulin-activated target enzymes. The dose-response curve of the activation of Ins(1,4,5)P3 kinase by calmodulin is not significantly impaired by melittin and trifluoperazine, whereas under very similar assay conditions the half-maximal activation of bovine brain cyclic AMP phosphodiesterase requires over 30-50-fold higher concentrations of CaM when 1 microM melittin or 20 microM-trifluoperazine is present in the assay medium. Similarly, 1 microM of the anti-calmodulin peptides seminalplasmin and gramicidin S, as well as 20 microM of N-(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide (W7), do not inhibit the activation process. These data suggest that binding and activation of Ins(1,4,5)P3 kinase require surface sites of calmodulin which are different from those involved in the binding of most other target enzymes or of model peptides.     

Cloning,Recombinant Expression and Characterization of Wild Type-105-Trp-Calmodulin of the Green Alga Mougeotia scalaris

The single calmodulin gene (CaM) of the green alga Mougeotia scalaris (Hassall) was cloned, sequenced and the CDNA inserted into the prokaryotic expression vector pGEX-2T. The recombinant calmodulin protein (CAM) was expressed as a fusion product together with glutathione S-transferase and isolated on glutathione sepharose. After cleavage and purification, the CaM was characterized by Ca²⁺-dependent shift in SDS-PAGE, by activation of cyclic 3′,5′nucleotide phosphodiesterase (PDE) and sensitivity to the inhibitors trifluoperazine and calmidazolium, with native Mougeotia CaM as control. Using Ca²⁺ buffers in the PDE test, affinity to Ca²⁺ of Mougeotia CaM was found to be diminished fivefold compared to maize or bovine brain CaMs. There was also a 20-fold increase of half maximal activation (K_act) in the PDE test for Mougeotia CaM relative to maize CaM, while the K_act of maize CaM to that of bovine brain CaM was almost the same. The derived amino acid sequences of CaM from Mougeotia and Zea mays revealed three major conservative amino acid exchanges, including unique 105-Trp (Mougeotia) → Leu (maize). In Mougeotia CaM the 105-Trp, including the neighbouring side chains of 92-Phe and 141-Phe, putatively form a hydrophobic ring interaction, as revealed by molecular modelling.     

Identification of a conserved calmodulin-binding motif in the sequence of F0F1 ATPsynthase inhibitor protein

The natural inhibitor proteins IF1 regulate mitochondrial F0F1 ATPsynthase in a wide range of species. We characterized the interaction of CaM with purified bovine IF1, two bovine IF1 synthetic peptides, as well as two homologous proteins from yeast, namely IF1 and STF1. Fluorometric analyses showed that bovine and yeast inhibitors bind CaM with a 1:1 stoichiometry in the pH range between 5 and 8 and that CaM-IF1 interaction is Ca2+-dependent. Bovine and yeast IF1 have intermediate binding affinity for CaM, while the Kd (dissociation constant) of the STF1-CaM interaction is slightly higher. Binding studies of CaM with bovine IF1 synthetic peptides allowed us to identify bovine IF1 sequence 33-42 as the putative CaM-binding region. Sequence alignment revealed that this region contains a hydrophobic motif for CaM binding, highly conserved in both yeast IF1 and STF1 sequences. In addition, the same region in bovine IF1 has an IQ motif for CaM binding, conserved as an IQ-like motif in yeast IF1 but not in STF1. Based on the pH and Ca2+ dependence of IF1 interaction with CaM, we suggest that the complex can be formed outside mitochondria, where CaM could regulate IF1 trafficking or additional IF1 roles not yet clarified.     

Calmodulin inhibits calcium influx current in vascular endothelium

Utilizing the whole-cell configuration of the patch-clamp technique the effect of calmodulin (CaM) on thapsigargin-induced Ca²⁺ current has been studied. Addition of several concentrations of CaM to the patch pipette induced concentration-dependent inhibition of thapsigargin-induced Ca²⁺ current in bovine aortic endothelial cells. The effect of CaM was Ca²⁺ dependent and was not observed when the intracellular Ca²⁺ was buffered to 1 nM with EGTA. CaM produced two major effects on the thapsigargin-induced Ca²⁺ current. First CaM slow down activation of the current by thapsigargin from a control value of 16 ± 5 to 31 ± 6 s with 1 μM CaM in the pipette solution. The second effect of CaM was to reduce the current amplitude in a concentration-dependent manner. The inhibition of Ca²⁺ current was observed at the peak of the current and at the sustained current level. The reduction of current at the sustained level was observed 15–20 s after onset of the thapsigargin response. The half inhibitory concentration determined from these experiments was 0.1 μM. These results indicate that CaM can modulate thapsigargin-induced Ca²⁺ current in this endothelium, suggesting a possible role for CaM in the regulation of store-operated Ca²⁺ influx.     

Development of a novel photoreactive calmodulin derivative: cross-linking of purified adenylate cyclase from bovine brain

A novel photoreactive calmodulin (CaM) derivative was developed and used to label the purified CaM-sensitive adenylate cyclase from bovine cortex. 125I-CaM was conjugated with the heterobifunctional cross-linking agent p-nitrophenyl 3-diazopyruvate (DAPpNP). Spectral data indicated that diazopyruvoyl (DAP) groups were incorporated into the CaM molecule. Iodo-CaM-DAPs behaved like native CaM with respect to (1) Ca2+-dependent enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and (2) Ca2+-dependent stimulation of adenylate cyclase activity. 125I-CaM-DAP photochemically cross-linked to CaM-binding proteins in a manner that was both Ca2+ dependent and CaM specific. Photolysis of forskolin-agarose-purified adenylate cyclase from bovine cortex with 125I-CaM-DAP produced a single cross-linked product which migrates on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of approximately 140,000.     

Role of CaM kinase II and ERK activation in thrombin-induced endothelial cell barrier dysfunction

We have previously shown that thrombin-induced endothelial cell barrier dysfunction involves cytoskeletal rearrangement and contraction, and we have elucidated the important role of endothelial cell myosin light chain kinase and the actin- and myosin-binding protein caldesmon. We evaluated the contribution of calmodulin (CaM) kinase II and extracellular signal-regulated kinase (ERK) activation in thrombin-mediated bovine pulmonary artery endothelial cell contraction and barrier dysfunction. Similar to thrombin, infection with a constitutively active adenoviral alpha-CaM kinase II construct induced significant ERK activation, indicating that CaM kinase II activation lies upstream of ERK. Thrombin-induced ERK-dependent caldesmon phosphorylation (Ser789) was inhibited by either KN-93, a specific CaM kinase II inhibitor, or U0126, an inhibitor of MEK activation. Immunofluorescence microscopy studies revealed phosphocaldesmon colocalization within thrombin-induced actin stress fibers. Pretreatment with either U0126 or KN-93 attenuated thrombin-mediated cytoskeletal rearrangement and evoked declines in transendothelial electrical resistance while reversing thrombin-induced dissociation of myosin from nondenaturing caldesmon immunoprecipitates. These results strongly suggest the involvement of CaM kinase II and ERK activities in thrombin-mediated caldesmon phosphorylation and both contractile and barrier regulation.     

The distribution of calmodulin and Ca^2＋—activated calmodulin in cell cycle of mouse erythroleukemia cells

Cell proliferation is accompanied with changing levels of intracellular calmodulin (CaM) and its activation.Prior data from synchronized cell population could not actually stand for various CaM levels in different phases of cell cycle.Here,based upon quantitative measurement of fluorescence in individual cells,a method was developed to investigate intracellular total CaM and Ca^2 -activated CaM contents. Intensity of CaM immunoflurescence gave total CaM level,and Ca^2 -activated CaM was measured by fluorescence intensity of CaM antagonist trifluoperazine (TFP).In mouse erythroleukemia (MEL) cells,total CaM level increased from G1 through S to G2M,reaching a maximum of 2-fold increase,then reduced to half amount after cell division.Meanwhile,Ca^2 -activated CaM also in creased through the cell cycle(G1,S,G2M).Increasing observed in G1 meant that the entry of cells from G1 into S phase may require CaM accumulation,and,equally or even more important,Ca^2 -dependent activation of CaM.Ca^2 -activated CaM decreased after cell division.The results suggested that CaM gene expression and C^2 -modulated CaM activation act synergistically to accomplish the cell cycle progression.     

Purification and properties of calmodulin from adrenal cortex

Calmodulin (CaM), a multifunctional calcium binding protein with no known enzymatic activity, has been purified to homogeneity from bovine adrenal cortex. The purification included anion exchange on DE-52 cellulose, ammonium sulfate precipitation, and separation by molecular sieving on Sephadex G-150. The yield of CaM from 900 g of whole adrenal was 150 mg. Adrenocortical CaM showed a molecular weight of 18,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, an isoelectric point of 4.1, and demonstrated a characteristic shift in mobility on polyacrylamide gels in the presence of calcium. The spectral properties of adrenocortical CaM differed slightly from those of CaM isolated from bovine brain. Minor differences were observed in peptide maps and amino acid composition between adrenocortical and brain CaM, but adrenocortical CaM contained a single trimethyl-lysine residue characteristic of all mammalian forms of CaM isolated to date. Adrenocortical CaM is biologically active in the stimulation of activator-deficient phosphodiesterase, and showed a half-maximal effective concentration (EC50) of 3 nM for stimulation of adenylate cyclase from Bordetella pertussis.