The addition of calcium ion chelator,EGTA to thawing solution improves fertilizing ability in frozen–thawed boar sperm

In our previous study, seminal plasma effectively suppressed the induction of sperm to capacitation‐like status and acrosome loss during the thawing process. However, because boar seminal plasma is contaminated with various kinds of bacteria and/or viruses, it is necessary to develop a thawing solution without animal‐derived materials. In this study, we focused on the increase of intracellular Ca²⁺ ([Ca²⁺]_i) in sperm after thawing and the negative effects of sperm qualities. After thawing, the fluorescent intensity of [Ca²⁺]_i indicator, Fluo‐3/AM, and the level of phosphorylated tyrosine residue of protein were increased in the sperm. Next, we investigated whether the addition of Ca²⁺ chelators (ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA)) improved post‐thawed sperm motility. When the frozen–thawed sperm were treated with 6 mmol/L EDTA + 6 mmol/L EGTA, sperm motility was significantly increased as compared with control (6 mmol/L EDTA alone) at all incubation periods (P < 0.05). The combinational treatment significantly suppressed the elevation of [Ca²⁺]_i and the tyrosine phosphorylation, which improved the acrosomal status and fertilizing ability in vitro. Furthermore, when the thawing method was applied for artificial insemination, the fertilization rate was significantly higher than control (P < 0.05, 33% vs. 82%). Thus, we conclude that the addition of EDTA + EGTA to thawing solution is a beneficial tool for artificial insemination using frozen–thawed boar sperm.     

钙对赭曲霉毒素A(OTA)诱导拟南芥毒性的抑制作用

为丰富赭曲霉毒素A (ochratoxinA,OTA)的植物毒性及其作用机理,探究钙在OTA诱导的植物毒性调控作用机理,本研究采用添加外源Ca2+,钙离子螯合剂乙二醇双(2-氨基乙基醚)四乙酸(ethylenebis(oxyethylenenitrilo) tetraacetic acid,EGTA)及OTA等处理离体拟南芥(Arabidopsis thaliana)叶片,通过形态学观察,叶片相对电导率、活性氧含量(reactive oxygen species,ROS)和丙二醛(malondialdehyde,MDA)含量的测定等研究钙对OTA诱导的拟南芥毒性的影响,结果表明,在一定浓度范围内(0～50 mmol/L),外源Ca2+单独处理,拟南芥叶片形态学无明显变化,EGTA与OTA分别处理均会引起拟南芥叶片失绿、坏死病斑的形成,而Ca2+与OTA共同处理,能显著抑制OTA诱导的拟南芥叶片失绿、坏死病斑的形成,EGTA加剧OTA的毒性作用;OTA处理使拟南芥叶片相对电导率升高,细胞膜通透性增大,外源钙能显著抑制OTA引起的拟南芥叶片相对电导率升高(P＜0.05),20 mmol/L Ca2+的抑制作用最强,抑制率为61.32％.此外,OTA处理使拟南芥叶片细胞ROS的爆发及MDA的积累,对拟南芥造成氧化损伤,钙能显著抑制OTA诱导的拟南芥ROS爆发(P＜0.05),减少MDA的生成,抑制率分别为29.7％和71.4％.研究结果说明OTA能诱导拟南芥的植物毒性,钙能显著抑制OTA诱导的拟南芥植物毒性,钙在拟南芥受外界胁迫中起重要作用.     

硝酸钙对荔枝花粉萌发和花粉管生长的影响

采用不同浓度的硝酸钙及EGTA处理对‘妃子笑’荔枝的花粉进行离体培养,通过电镜观察并测定花粉的萌发率与花粉管的生长长度。结果表明,低浓度(0.5～2.0mmol/L)硝酸钙较对照(无钙处理)提高了花粉的萌发率,促进花粉管的生长,以2.0mmol/L硝酸钙处理的花粉萌发率最高(68.5%),与对照相比达到显著水平;而以1.5mmol/L硝酸钙处理的花粉管生长长度最长(255.4μm),比对照达到显著水平。但是加入1.0mmol/LCa2+鳌合剂EGTA时,花粉萌发率为0。以上结果表明钙信号可能调控荔枝的花粉萌发与花粉管生长。     

(Ca~(2 ),Mg~(2 ))-EGTA缓冲溶液中总钙浓度的计算

作者推导出一个计算(Ca~(2 ),Mg~(2 ))-EGTA缓冲溶液中某一指定低游离钙离子浓度([Ca~(2 )]≤1×10~(-6)mol/L)下所需加入总钙浓度[Ca~(2 )]_t的公式,可供实验工作者应用.     

外源Ca2+、W7和EGTA处理对低温胁迫下辣椒幼苗渗透物质的调节

研究了不同Ca2 浓度处理和Ca2 信号抑制剂对低温下辣椒幼苗叶片中3种主要渗透调节物质的影响。结果表明,外源10mmol/LCa2 能明显地增加辣椒幼苗叶片可溶性蛋白质、可溶性糖和脯氨酸含量。采用Ca2 螯合剂EGTA和钙调素拮抗剂W 7[N-(6-氨基己基)-5-氯-1-萘磺胺]对辣椒幼苗低温处理后,辣椒幼苗叶片中可溶性蛋白质、可溶性糖和脯氨酸含量降低。以上结果说明Ca2 信号系统参与了调节辣椒幼苗抗寒过程中渗透物质的合成。     

EGTA对‘秦冠’苹果花粉管Ca2+、微丝分布以及囊泡运输的影响

【目的】研究Ca~(2+)螯合剂EGTA对花粉萌发和生长过程中花粉管形态、Ca~(2+)分布、细胞骨架和胞内运输的影响。【方法】以Ca~(2+)螯合剂EGTA处理‘秦冠’苹果成熟花粉,显微镜观察其对花粉萌发、花粉管生长的影响。运用免疫荧光标记技术结合荧光显微镜及激光共聚焦扫描显微镜观察花粉管内Ca~(2+)分布、微丝分布和囊泡运输模式。【结果】发现低浓度的EGTA对花粉萌发和花粉管生长影响很小,甚至有促进作用。用较高浓度的EGTA处理花粉后,花粉萌发和花粉管生长均受到明显抑制,花粉粒甚至不萌发,花粉管有扭曲膨大短小等现象。用400μmol/L EGTA处理花粉后,花粉管顶端钙离子内流稍有增加,用2mmol/L EGTA处理后,花粉管顶端钙离子交换被抑制。用400μmol/L EGTA处理后,花粉管内游离Ca~(2+)在花粉管顶端聚集并呈现浓度梯度分布,与对照的分布模式一致,而用2mmol/L EGTA处理后顶端聚集模式消失。对照和400μmol/L EGTA处理后花粉管内微丝与花粉管生长方向平行排列,而用2 mmol/L EGTA处理后花粉管内微丝断裂、丝状结构不明显,无序分布在花粉管内。对照花粉管内囊泡运输在顶端以及近顶端区域聚集,用400μmol/L EGTA处理后分布模式变化不大,而用2mmol/L EGTA处理后,囊泡运输增加,布满整个花粉管内。【结论】钙可能作为一种信号因子,通过影响细胞骨架的构成和胞内囊泡的运输模式,从而调节苹果花粉的萌发及花粉管生长。     

The use of divalent cation chelating agents (EDTA/EGTA) to reduce non-specific serum protein interaction in enzyme immunoassay

An indirect enzyme immunoassay (ELISA) for detection of bovine antibody toBrucella abortus was modified by the addition of divalent chelating agents to the serum diluent. This addition resulted in an increase in specificity from 96.0% in the regular assay to 99.4% in the modified procedure. Of the 15 715 sera initially tested by the indirect ELISA, 691 that had given positive reactions were selected for retesting in the indirect ELISA with EDTA/EGTA added. The buffered plate antigen test (BPAT) correctly identified 98.6% of the samples as negative. The addition of chelating agents did not alter the sensitivity of the indirect ELISA, which correctly classified 609 sera from animals from whichB. abortus had been isolated as positive. The sensitivity of the BPAT was 97.8%.Abbreviations ABTS 2,2-azinobis(3-ethylbenzthiazoline sulphonic acid) - BPAT buffered plate antigen test - EDTA ethylenediaminetetraacetic acid disodium salt - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ELISA enzyme-linked immunosorbent assay - IgG₁ immuno- globulin G₁ - IgM immunoglobulin M - Tris tris(hydroxymethyl)aminomethane     

水分胁迫下钙对甘蔗幼苗抗氧化酶活性的影响

笔者选取甘蔗福农95-1702为材料,结合钙离子螯合剂EGTA预处理,对水分胁迫下Ca2+与甘蔗幼苗抗氧化酶类活性的关系进行研究,探讨逆境信号转导第二信使Ca2+在甘蔗抗旱性中的作用。结果表明,1~3mmol/L的EGTA预处理对正常生长条件下甘蔗幼苗的相对生长率、可溶性蛋白含量及抗氧化酶活性均无显著影响。但30%PEG处理下,1~3mmol/L的EGTA预处理降低了甘蔗幼苗的相对生长量,加剧了可溶性蛋白含量的下降：在处理的24小时,处理1E+D（1mmol/LEGTA+30%PEG）及3E+D(3mmol/LEGTA+30%PEG)中的可溶性蛋白含量分别比处理D（30%PEG）降低了40.2%和54.3%。同时,EGTA预处理抑制了PEG胁迫下超氧化物歧化酶（SOD）和过氧化物酶（POD）活性的升高,加剧了过氧化氢酶（CAT）活性的降低：在处理的24小时,处理1E+D及3E+D中的SOD活性分别比处理D降低了26.5%和29.9%;而POD活性则比处理D分别降低了31.3%和23.2%;处理3E+D的CAT酶活性比处理D降低了27.2%。这些结果暗示Ca2+可能参与了水分胁迫下甘蔗幼苗抗氧化酶系统的调节,Ca2+信号和活性氧（ROS）所介导的信号转导途径存在交叉互作     

水分胁迫下钙对甘蔗幼苗抗氧化酶活性的影响

选取甘蔗福农95-1702为材料,结合钙离子螯合剂EGTA预处理,对水分胁迫下Ca2 与甘蔗幼苗抗氧化酶类活性的关系进行研究,探讨逆境信号转导第二信使Ca2 在甘蔗抗旱性中的作用。结果表明,1￣3mmol/L的EGTA预处理对正常生长条件下甘蔗幼苗的相对生长率、可溶性蛋白含量及抗氧化酶活性均无显著影响。但30%PEG处理下,1￣3mmol/L的EGTA预处理降低了甘蔗幼苗的相对生长量,加剧了可溶性蛋白含量的下降:在处理的24小时,处理1E D(1mmol/LEGTA 30%PEG)及3E D(3mmol/LEGTA 30%PEG)中的可溶性蛋白含量分别比处理D(30%PEG)降低了40.2%和54.3%。同时,EGTA预处理抑制了PEG胁迫下超氧化物歧化酶(SOD)和过氧化物酶(POD)活性的升高,加剧了过氧化氢酶(CAT)活性的降低:在处理的24小时,处理1E D及3E D中的SOD活性分别比处理D降低了26.5%和29.9%;而POD活性则比处理D分别降低了31.3%和23.2%;处理3E D的CAT酶活性比处理D降低了27.2%。这些结果暗示Ca2 可能参与了水分胁迫下甘蔗幼苗抗氧化酶系统的调节,Ca2 信号和活性氧(ROS)所介导的信号转导途径存在交叉互作。     

A potential calcium antagonist and its antihypertensive effects

Imperatorin (Imp) as a hypotensive active ingredient, its hypotensive effect was evaluated in the SHRs, its calcium antagonism and affinity to L-type calcium channel was also confirmed. The results showed that the blood pressure was decreased in the SHRs treated with Imp, the aortic ring was relaxed with Imp, L-type calcium channel currents and intracellular calcium free ion rise was nearly disappeared when adding Imp. In addition, Imp displayed a chromatographic peak similar to nitrendipine and verapamil by the cell membrane chromatography, same results from protein–drug docking approaches. Hence, Imp target the L-type calcium channel, and may be used as a novel antihypertensive drug.