钙离子对江浙蝮蛇毒酸性磷脂酶A2结构与功能的影响

钙离子在江浙蝮蛇毒酸性磷脂酶Ａ２中的作用是多方面的,它不仅能够引起酸性磷脂酶Ａ２在溶液中构象的变化,而且对该酶活性有较大的影响,Ｃａ＾２＋为酶活力所必需,当Ｃａ＾２＋浓度达到０．０６ｍｍｏｌ／Ｌ时,酶表现出很的活力;Ｃａ＾２＋浓度超过０．５ｍｍｏｌ／Ｌ时,催化反应出现一个明显的延滞期。化学修饰表明Ｈｉｓ４７在表现活力方面起重要作用,Ｃａ＾２＋的存在可降低其修饰反应的速度,提示这是由于Ｃａ＾２＋引起     

兔心肌细胞核钙转运

本研究在离体家兔心肌细胞核上,观察细胞核钙调节的特征。发现心肌细胞每毫克蛋白质的钱含量较细胞浆高２．６倍,而核总钙含量占细胞总钙含量的１／６。心肌细胞核上存在高新和力的Ｃａ－ＡＴＰａｓｅ,其活性具有Ｃａ＾２＋和ＡＴＰ依赖性,在２．０ｍｍｏｌ／ＬＡＴＰ时,其Ｃａ＾２＋依赖的Ｋａ＝２２６ｎｍｏｌ／Ｌ,Ｖｍａｘ＝３４６０ｎｍｌ／（ｈ．ｍｇ）;在４００ｎｍｏｌ／Ｌ的Ｃａ＾２＋时,其ＡＴＰ依赖的Ｋｍ＝３７６     

氨对脑细胞胞浆游离钙含量的影响

目的与方法：采用Ｆｕｒａ－２／ＡＭ探针技术观察ＮＨ４Ｃｌ对离体急性分离之Ｗｉｓｔａｒ乳鼠大脑细胞胞头游离钙「Ｃａ＾２＋」ｉ含量的影响。结果：ＮＨ４＾＋浓度为２．５ｍｍｏｌ／Ｌ时脑细胞内「Ｃａ＾２＋」ｉ含量升高。在一定范围内,随着ＮＨ４＾＋浓度的加大,细胞内Ｃａ＾２＋持续升高。ＮＨ４＾＋的升钙作用主要被Ｎｉｃａｒｄｉｐｉｎｅ所阴断,其变化特征类以ＫＣｌ。结论：ＮＨ４＾＋主要通过影响电压依赖性钙离子通     

LHRH—A2及无机离子促进草鱼脑垂体离体分泌GH的初步研究

用培养瓶（皿）培养法研究ＬＨＲＨ－Ａ２、Ｃａ＾２＋、Ｋ＾＋对草鱼脑垂体器官分泌ＧＨ的影响。结果表明，１ｎｍｏｌ／Ｌ、１０ｎｍｏｌ／Ｌ、１００ｎｍｏｌ／Ｌ和１０００ｎｍｏｌ／Ｌ的ＬＨＲＨ－Ａ２都能显著促进ＧＨ的分泌；随着Ｃａ＾２＋浓度（０．１、１、３ｍｍｏｌ／Ｌ）的增高ＧＨ的分泌增强，在１ｍｏｌ／Ｌ和３ｍｍｏｌ／Ｌ Ｃａ＾２＋条件下的ＧＨ分泌水平显著高于在０．１ｍｍｏｌ／Ｌ Ｃａ＾２＋条件下的ＧＨ分     

M型受体在大鼠肾上腺嗜铬细胞内钙库释放和激素分泌中的作用

在单个大鼠肾上腺嗜铬细胞上,用显微荧光测量和碳纤电极记录方法,测量可激活毒蕈碱（ｍｕｓｃａｒｉｎｅ,Ｍ）受体的激动剂乙酰甲胆碱（ｍｅｔｈａｃｈｏｌｉｎｅ,ＭＣｈ）对胞内游离钙浓度「Ｃａ＾２＋」ｉ和儿茶酚胺激素分泌的影响。在细胞外液含２ｍｍｏｌ／Ｌ Ｃａ＾２＋时,用含钙或不含钙的ＭＣｈ（１ｍｍｏｌ／Ｌ）刺激细胞,均引起「Ｃａ＾２＋」ｉ的升高或钙振荡,并诱发激素的分泌。     

钙离子对江浙蝮蛇毒酸性磷脂酶A_2结构与功能的影响

钙离子在江浙蝮蛇毒酸性磷脂酶Ａ_２中的作用是多方面的。它不仅能够引起酸性磷脂酶Ａ_２在溶液中构象的变化,而且对该酶活性有较大的影响。Ｃａ~（２＋）为酶活力所必需,当Ｃａ~（２＋）浓度达到０．０６ｍｍｏｌ／Ｌ时,酶表现出很高的活力;Ｃａ~（２＋）浓度超过０．５ｍｍｏｌ／Ｌ时,催化反应出现一个明显的延滞期。化学修饰表明Ｈｉｓ_（４７）在表现活力方面起重要作用,Ｃａ~（２＋）的存在可降低其修饰反应的速度,提示这是由于Ｃａ~（２＋）引起构象发生变化而造成的。     

糖皮质激素对肝细胞内游离钙的作用

应用钙离子荧光指示剂ｆｕｒａ－２,对糖皮质激素（Ｇｌｕｃｏｃｏｒｔｉｃｏｉｄ,ＧＣ）是否影响肝细胞内游离钙（Ｉｎｔｒａｃｅｌｌｕａｌａｒ ｆｒｅｅ ｃａｌｃｉｕｍ,［Ｃａ＾２＋］ｉ作了初步探讨,结果发现,ＧＣ在短期内能升高肝细胞［Ｃａ＾２＋］水入１．０μｍｏｌ／Ｌ的Ｃｏｒｔｉｓｏ０．２５ｍｉｎ即可引起肝细胞［Ｃａ＾２＋］的明显升高,到１０分钟时效应达高峰。此时与静息状态的肝细胞水平相比胞浆内游离钙     

体外培养新生大鼠大脑皮层星形神经元钾通道的特性及膜外钾离子浓度对其影响

用膜片钳技术中的细胞贴附方式和内面向外方式,首次在新生大鼠大脑皮层星形神经元胞体膜上记录到一类电压依赖性钾通道。此通道可被２０ｍｍｏｌ／Ｌ ＴＥＡ,５ｍｍｏｌ／Ｌ Ｂａ＾２＋,１４０ｍｍｏｌ／Ｌ Ｃｓ＾＋阻断,不受２０ｍｍｏｌ／Ｌ４－ＡＰ影响,其激活不依赖Ｃａ＾２＋。膜外钾离子浓度对通道的特性有显著的影响,逆转电位随Ｋ＾＋的增大而增大,并表现出一定的饱和现象,两者的对数呈线性关系;同一驱动电位下,     

NaCl（2 mol／L）处理PSⅡ颗粒的Ca^2＋重结合

回加Ｃａ＾２＋对ＮａＣｌ（２ ｍｏｌ／Ｌ）处理菠菜ＰＳⅡ颗粒放氧活恬的作用表现出有两种Ｋｍ值分别为０．０２１和０．５４５ｍｍｏｌ／Ｌ高亲和与低亲和的Ｃａ＾２＋重结合过程。高浓度ＮａＣｌ和低ｐＨ（３．０）处理支Ｃａ的ＰＳⅡ颗粒的光抑制放氧活性半衰期明显减小,重结合Ｃａ＾２＋后,虽然大部分丧失的放氧活性可恢复,但ＰＳⅡ颗粒放氧活性对光抑制的敏感性却并不随之恢复,ｔ１／２无明显改变。显然,重组Ｃａ＾２＋     

铝和铝＋钙对小麦幼苗根尖质膜,液泡膜微囊ATP酶和膜流动性？…

研究了铝和铝＋＿钙对小麦功苗根尖质膜、液泡膜微囊Ｈ＾＋－ＡＴＰ酶、Ｃａ＾２＋－ＡＴＰ酶、Ｍｇ＾２＋－ＡＴＰ酶活性及共动力学参数和膜流动性的影响。在质膜和液泡膜微囊制剂中加入１．０ｍｍｏｌ／Ｌ的ＡＩ＾３＋（ＡＩＣＩ３）时,Ｈ＾２＋－ＡＴＰ囊制剂中加入１．０ｍｍｏｌ／Ｌ的ＡＩ＾３＋（ＡＩＣＩ３）时,Ｈ＾＋－ＡＴＰ酶、Ｃａ＾２＋－ＡＴＰ酶、Ｍｇ＾２＋－ＡＴＰ酶活笥和酶促反应的Ｖｍａｘ及膜流动性下降,而酶     

钙离子对293细胞结团和生长的影响

分别在有血清和无血清条件下、方瓶和转瓶中考察了Ca²⁺ 对2 93细胞结团和生长的影响。通过实验发现,Ca²⁺ 浓度在0 1～1 0mmol L范围内对2 93细胞的贴壁和结团性质有显著影响,而对生长影响不大。结果表明:有血清贴壁培养时,较高的Ca²⁺ 浓度有利于细胞贴壁;无血清悬浮培养中,Ca²⁺ 浓度越高,细胞结团越严重,细胞结团达到平衡后的平均粒径(D ,μm)与Ca²⁺ 浓度(c,mmol L)在0.1～0.5mmol L范围内可用一次函数D =58.65c +16.96描述,细胞结团尺寸是可调控的;而细胞在不同的Ca²⁺ 浓度下有相似的生长规律。     

重组人BD3-BPI的内毒素中和活性及其在高盐环境中抗菌活性的稳定性

目的：验证重组人BD3-BPI（rhBD3-BPI）是否具有内毒素中和活性,研究其在高盐环境中是否能保持抗菌活性。方法：根据内毒素标准品绘制内毒素活性标准曲线,将100 μL梯度稀释的rhBD3-BPI-与100 μL 10EU／mL脂多糖（LPS）混匀,37℃水浴60min,同时设标准对照（只含10EU／mL LPS的标准品溶液）,并以无热源水作为空白对照,采用基质显色法进行鲎试验测定LPS的活性;将6×10^8 CFU／mL的革兰阳性和阴性标准菌株及临床分离的多药耐药菌株接种于含1mg／mL rhBD3-BPI和0～250mmol／L不同浓度NaCl的液体细菌培养基中,37℃培养3h后用10mmol／L磷酸钠按1：1～1：1000的比例稀释,铺LB培养基平板,37℃过夜培养,观察各平板菌落生长情况,计数并计算杀菌率。结果：在5EU／mL的标准内毒素体系中,当rhBD3-BPI的浓度高于4μg／mL时即开始表现出一定的内毒素中和活性,当rhBD3-BPI的浓度分别为16、32 μg／mL时,其内毒素中和率分别为23％和88％,随后rhBD3-BPI对内毒素的中和活性趋于平稳,50 μg／mL的rhBD3-BPI对所有受检菌均表现出100％的杀伤效应。当NaCl浓度低于150mmol／L时,rhBD3-BPI对各受检菌的杀菌活性均未受明显影响;NaCI浓度升高至150-200mmol/L,rhBD3-BPI对各受检菌的杀菌活性有所下降,但其杀伤率仍在90％以上;当NaCl浓度高于200mmol／L时,盐浓度对rhBD3-BPI杀菌活性的影响才较为明显,但即使NaCl浓度达到250mmol／L,rhBD3-BPI的杀菌活性仍保持在85％以上。结论：rh-BD3-BPI具有内毒素中和活性,在高盐环境中具有良好的抗菌活性稳定性。     

Dose-Response Relationship for Nicotine-Induced Up-Regulation of Rat Brain Nicotinic Receptors

Abstract: The chronic administration of nicotine to animals has been shown to result in an increase in brain nicotinic acetylcholine receptor (nAChR) density. It has been suggested that this agonist-induced receptor up-regulation is a consequence of long-term nAChR desensitization in vivo. In this study, the effects of different nicotine doses and administration schedules as well as the resulting blood and brain nicotine levels were determined to assess the effect of in vivo nicotine concentration on nAChR density in the brain. Rats with indwelling subcutaneous cannulas were infused for 10 days with 0.6–4.8 mg/kg/day nicotine either 2×, 4×, or 8×/day or by constant infusion. The nAChR density in cortical, striatal, and hippocampal tissue measured by [³H]cytisine binding as well as the corresponding plasma and brain nicotine levels measured by GC analysis were determined. The results showed a dose-dependent increase in nAChR density with significant increases achieved at 2.4 mg/kg/day in all three brain areas. It is surprising that at this dose there was little difference between the constant infusion of nicotine and twice-daily administration, whereas more frequent periodic injections were actually less effective at up-regulating nAChRs. An analysis of the blood and brain levels of nicotine compared with the concentrations that produce nAChR desensitization suggests that in vivo desensitization alone is not sufficient for nAChR up-regulation to occur.     

番茄碱对人红细胞膜Na+-K+-ATPase活性影响的研究

以低渗法从新鲜健康人红细胞中制得膜Na^ -K^ -ATPase,研究了番茄碱(tomatine)X~人红细胞膜Na^ -K^ -ATPase活性的影响。实验结果表明,反应体系中的tomatine浓度低于1mmol／L时,对不依赖钙调蛋白(CAM)激活的Na^ -K^ -ATPase(称之为基本酶活)影响不大,浓度达1mmol／L时,该酶活性仍保持在95％左右;而在此浓度范围内,tomatine对依赖CaM的Na^ -K^ -ATPase有明显的抑制作用,其IC50值为0．16mmol／L．说明tomatine对膜酶Na^ -K^ -ATPase活性的影响可能是通过阻断CaM激活的途径而起作用,从而为进一步研究番茄碱的作用机制奠定了基础。     

交联酶聚集体制备中钙离子的结构调控作用

以葡萄糖氧化酶(GOx)为研究对象,系统地研究了钙离子对交联酶聚集体(CLEA)粒子尺寸和微观结构的调控作用以及酶催化性能和实用性的影响。研究结果表明,GOx酶沉淀过程中引入钙离子可显著降低CLEA粒子尺寸并导致粒子内纳米孔道结构逐步消失。在0.1 mmol/L钙离子浓度下,GOx酶的CLEA仍保有清晰的纳米孔道结构。以葡萄糖为底物的GOx酶CLEA催化结果显示,该CLEA粒子的酶活性为对照CLEA粒子的2.69倍。即便1.0 mmol/L钙离子浓度下制备的CLEA粒子的GOx酶活性仍高出对照CLEA粒子约42%。此外,0.1 mmol/L钙离子浓度下制备的CLEA不仅具有更高的底物转化速率和很好的操作稳定性,而且CLEA中GOx酶的最大反应速度显著提高。这些实验结果明确了钙离子对CLEA粒子尺寸和微观结构的调控作用,为制备具有高效生物催化活性的CLEA粒子奠定了基础。     

体外构建组织工程化软骨的初步研究

观察用藻酸钠凝胶在体外长期培养软骨细胞的情况,为求进一步用藻酸钠凝胶在体外构建组织工程化软骨提供初步研究.将传代培养的、细胞终浓度为1×107/mL的软骨细胞复合藻酸钠凝胶,注入圆柱形模具中,将模具分别浸入100、200、300 mmol/L的固化剂氯化钙溶液中,固化15 min使其成形,于体外培养2、4、6周后,行HE染色、阿尔新蓝染色,了解软骨细胞的生长情况.2、4、6周时软骨细胞与藻酸钙凝胶复合良好并能在其中保持活性及分裂能力,且在6周时出现类软骨变化.因此藻酸钠凝胶是可用于体外构建组织工程化软骨的生物材料.     

钙提高玉米种子活力的作用研究

本文以玉米种子为材料,研究了钙对种子活力的影响．在０—４０ｍｍｏｌ／ＬＣａ（２＋）的浓度范围,低浓度的Ｃａ（２＋）提高种子的发芽率和活力,最适浓度为１０ｍｍｏｌ／Ｌ,超过此浓度Ｃａ（２＋）的促进作用减弱．Ｃａ（２＋）提高种子发芽率和活力的原因可能是Ｃａ（２＋）促进胚和胚乳中α－和β－淀粉酶的活性,加速胚乳中贮藏物质如淀粉和可溶性蛋白的动员．在种子萌发过程中,ＥＧＴＡ和ＥＤＴＡ能降低种子活力,２．５和１０ｍｍｏｌ／ＬＣａ（２＋）能部分解除ＥＧＴＡ和ＥＤＴＡ的抑制作用．     

Calcium transients accompany ooplasmic segregation in zebrafish embryos

Through the injection of f -aequorin (a calcium-specific luminescent reporter), and the use of an imaging photon detector, transient localized elevations of free cytosolic calcium in the forming blastodisc (BD) and animal hemisphere cortex were visualized that correlated with ooplasmic segregation. The introduction of an appropriate concentration of the weak (K_D= 1.5 μmol/L) calcium buffer 5,5'-dibromo-BAPTA results in the dissipation of these calcium domains, and inhibits cytoplasmic streaming and the subsequent formation of a BD at the animal pole. These inhibitory actions are dependent on the final cytosolic concentration of buffer within the egg: ≥ 1.3 mmol/L blocks ooplasmic streaming; < 1.3 mmol/L eggs segregate normally. Injection of 5,5'-dimethyl-BAPTA (K_D= 0.15 μmol/L) to a final concentration of 1.5 mmol/L as a control has no effect on ooplasmic streaming. These results suggest that localized domains of elevated free cytosolic calcium are essential for ooplasmic segregation in zebrafish. Furthermore, a hypothetical model is presented linking these calcium transients to the contraction of a cortically located actin microfilament network as a possible mechanism providing the driving force for segregation.     

Chlorophyll Biosynthetic Reactions during Senescence of Excised Barley (Hordeum vulgare L. cv IB 65) Leaves

The inhibitor sensitivity of the endoplasmic reticulum (ER) and plasma membrane (PM) calcium pumps of red beet (Beta vulgaris L.) were studied by measuring the ATP-driven accumulation of 45Ca2+ into isolated membrane vesicles. Both transporters were strongly inhibited by 50 [mu]mol m-3 erythrosin B, but only by 50% in the presence of 100 mmol m-3 vanadate. A number of inhibitors considered to be specific for the sarcoplasmic reticulum (SR)/ER-type calcium pump in animal cells were used to further characterize the PM and ER Ca2+-ATPases in red beet and were compared with their effect on the transport and hydrolytic activities of the PM and tonoplast H+-ATPases. The hydroquinones 2,5-di(tert-butyl)-1,4-benzohydroquinone and 2,5-di(tert-amyl)-1,4-benzohydroquinone produced around 20 and 40% inhibition of activity, respectively, of the PM and ER calcium pumps and the PM H+-ATPase when present at concentrations of 30 mmol m-3. In contrast, the vacuolar proton pump displayed a much higher sensitivity to these two compounds. Nonylphenol appeared to have a general inhibitory effect on all four membrane transport proteins and gave almost complete inhibition when present at a concentration of 100 mmol m-3. Thapsigargin and the structurally related compound trilobolide produced 50% inhibition of both the ER and PM calcium pumps at concentrations of 12.5 and 24 mmol m-3, respectively. The PM and tonoplast proton pumps were also sensitive to these compounds. The ER and PM calcium pumps were almost completely insensitive to cyclopiazonic acid (CPA) up to a concentration of 20 mmol m-3. When present at 100 mmol m-3 CPA caused 30% inhibition of the transport properties of all four ATPases. The high concentrations of all of the inhibitors of the SR/ER Ca-ATPase required to inhibit the red beet ER calcium pump, together with the similar effects on the PM calcium pump and the PM and tonoplast proton pumps, suggests that these hydrophobic compounds have a general nonselective action in red beet, possibly through disruption of membrane lipid-protein interactions.     

Muscarinic receptors couple to modulation of nicotinic ACh receptor desensitization in myenteric neurons

Signaling mechanisms coupled to activation of different neurotransmitter receptors interact in the enteric nervous system. ACh excites myenteric neurons by activating nicotinic ACh receptors (nAChRs) and muscarinic receptors expressed by the same neurons. These studies tested the hypothesis that muscarinic receptor activation alters the functional properties of nAChRs in guinea pig small intestinal myenteric neurons maintained in primary culture. Whole cell patch-clamp techniques were used to measure inward currents caused by ACh (1 mM) or nicotine (1 mM). Currents caused by ACh and nicotine were blocked by hexamethonium (100 microM) and showed complete cross desensitization. The rate and extent of nAChR desensitization was greater when recordings were obtained with ATP/GTP-containing compared with ATP/GTP-free pipette solutions. These data suggest that ATP/GTP-dependent mechanisms increase nAChR desensitization. The muscarinic receptor antagonist scopolamine (1 microM) decreased desensitization caused by ACh but not by nicotine, which does not activate muscarinic receptors. Phorbol 12,13-dibutyrate (10-100 nM), an activator of protein kinase C (PKC), but not 4-alpha-phorbol 12-myristate 13-acetate (a PKC inactive phorbol ester), increased nAChR desensitization caused by ACh and nicotine. Forskolin (1 microM), an activator of adenylate cyclase, increased nAChR desensitization, but this effect was mimicked by dideoxyforskolin, an adenylate cyclase inactive forskolin analog. These data indicate that simultaneous activation of nAChRs and muscarinic receptors increases nAChR desensitization. This effect may involve activation of a PKC-dependent pathway. These data also suggest that nAChRs and muscarinic receptors are coupled functionally through an intracellular signaling pathway in myenteric neurons.