构建钙荧光探针细胞用于探究胞浆和线粒体钙对ATP刺激的响应

目的:构建表达基因编辑钙探针(GECIs)的细胞系HeLa-GECIs,探究细胞应答外界ATP刺激中钙离子在细胞内的响应和变化。方法:分别用能够直接通过荧光强度反映细胞胞浆内和线粒体内钙离子相对浓度的2种钙探针cyto-GCaMP6和4mt-GCaMP6感染HeLa细胞,获得2种表达钙离子探针的HeLa细胞系;在感染了2种腺病毒探针24 h后,用共聚焦荧光显微镜检测荧光探针在HeLa细胞内的表达情况;在表达2种钙探针的细胞的培养基中加入外源ATP,用Time-lapse成像动态观测技术观察HeLa细胞内钙离子对外环境中ATP的响应。结果:共聚焦荧光显微镜观察,确定95%以上的细胞表达了对应的钙离子指示荧光探针;Time-lapse成像动态观测技术观察发现,在细胞培养基中加入ATP后,细胞胞浆钙探针荧光强度瞬时(3～6 s)升至10倍,200 s后逐渐降低到基础水平;线粒体钙到达峰值(4倍)的时间稍滞后(5～8 s),并且回落更慢,300 s时至1.5倍。在ATP受体P2X7抑制剂A438079预处理的实验组,上述胞浆钙和线粒体钙浓度上升不明显。结论:构建了能在活体细胞内通过荧光探针实时监测钙离子响应胞外ATP刺激的细胞实验体系,为进一步深入探究ATP等危险信号导致细胞的炎性损伤机制奠定了基础。     

垂体腺苷环化酶激活多肽抑制β淀粉样蛋白对Neuro-2a细胞神经毒性作用的机制

通过MTT方法检测细胞活性 ,同时采用激光共聚焦显微镜技术检测细胞内游离钙离子的瞬时运动 ,研究了垂体腺苷环化酶激活多肽 (pituitaryadenylatecyclaseactivatingpolypeptide 2 7,PACAP2 7)通过调节细胞内钙对抗淀粉样蛋白Aβ2 5 35引起Neuro 2a细胞神经毒性作用的可能机制。结果表明 ,PACAP在一定浓度范围内 (<0 1μmol/L)可提高Neuro 2a细胞增殖能力并对抗Aβ引起的神经毒性 ,此作用可以被PACAP受体竞争性拮抗剂PACAP6 2 7所抑制。 2 5 μmol/LAβ使细胞内钙离子缓慢上升 ,并有一个较长时间的平台期。 0 1μmol/L的PACAP使细胞内钙离子迅速升高后下降 ,10min后回到接近基线水平 ,伴有较长时间的不应期。用PACAP预处理细胞 10min后Aβ引起细胞内钙的慢上升不再出现。推测 ,PACAP受体激活引起瞬时内向钙离子运动 ,而后伴随一个较长时间的不应期 ,可能是一个消除凋亡或阻止凋亡启动的保护机制。     

增强UV-B辐射对小麦根尖细胞游离钙离子分布的影响

以小麦根尖为材料,利用低温装载法在根尖细胞中成功地装载了酯化形式的钙离子荧光指示剂fluo-3/AM,利用激光共聚焦显微技术检测了增强UV-B辐射后小麦根尖细胞内游离钙离子荧光强度的分布,并对胞质内游离钙离子浓度进行了测定.结果表明:(1)对照组小麦根尖细胞内钙离子荧光主要分布于细胞胞质周缘;而经UV-B辐射处理后,细胞内钙离子荧光不仅分布在胞质周缘,且在细胞壁与胞间隙可观察到大量钙离子荧光.(2)对单个细胞内钙离子荧光强度进行测定,发现UV-B处理使细胞胞质内游离钙离子浓度明显升高.     

重症休克时平滑肌细胞内的钙动力学变化

使用荧光比率方法研究血管平滑肌细胞钙动力学变化在重症休克血管反应性降低中的作用．复制SD大鼠失血性休克模型,分离肠系膜细动脉血管平滑肌(ASMC)．使用荧光探针Fluo-3／AM、FuraRed双标记比率方法结合激光扫描共聚焦显微成像技术测定单个平滑肌细胞钙动力学变化,观察ATP敏感钾通道(KATP)特异性阻滞剂优降糖对血管反应性和钙动力学影响、实验结果发现休克后2h(失代偿期),血管反应性明显降低,norepinephrine作用带来的平滑肌细胞内钙离子浓度升高的程度明显减弱;加入优降糖可明显提高NE对平滑肌细胞内钙离子的升高作用,改善细动脉对NE的反应性．带来血管反应性部分恢复．     

凝血酶引致的血小板内钙,镁离子浓度及分布状态的变化

我们使用荧光探针ｆｕｒａ２、ｍａｇ－ｆｕｒａ２和ｆｌｕｏ３测定了凝血酶引致的血小板凝聚过程中细胞内钙、镁离子浓度的变化及分布状态。在０．５Ｕ／ｍｌ凝血酶作用下,血小板细胞内钙离子浓度呈双时相变化。血小板细胞群中细胞内钙离子浓度呈正态分布。伴随血小板凝聚时细胞内钙离子浓度增加,血小板细胞内游离镁离子浓度也明显增加,提示镁离子在血小板凝聚中有重要的作用。     

内质网钙池操控钙内流研究进展

细胞内的内质网钙库清空所引发的钙内流是细胞钙信号的重要组成,介导胞外钙离子进入细胞内,并参与细胞内一系列广泛的生理过程。该过程主要由内质网上的钙离子感受器STIM蛋白和细胞膜上的Orai钙离子通道的所介导的。对钙库操控性钙内流的研究进展进行了讨论,并展望了未来的研究方向,以期为相关研究提供参考。     

ATP 调控大鼠胰腺β细胞胰岛素分泌的双重作用

实验以大鼠胰腺β细胞为研究对象,采用荧光测钙和全细胞膜片钳膜电容测量技术,研究 ATP 对胞内钙离子信号和细胞分泌的影响,并初步探讨了其作用机制 . 实验表明：胞外 ATP 刺激通过动员细胞内 thapsigargin 敏感的钙库 Ca2+ 释放,使大鼠胰腺β细胞内的游离钙离子浓度显著升高,细胞外的 ATP 信号对β细胞胰岛素分泌有双向调节作用,其一,主要通过降低去极化引起的钙电流而对β细胞胰岛素分泌产生较弱的抑制作用,其二,细胞在静息状态下, ATP 通过动员胞内钙库的 Ca2+ 释放使胞浆中的钙离子浓度显著增加,触发β细胞强烈分泌胰岛素 . ATP 的这种双向调节可能对胰岛素分泌的精确调控具有重要的生理意义 .     

钙荧光探针Fura-2/AM对大肠杆菌细胞的负载行为

Fura-2/AM作为一类高效的钙离子荧光探针,在动物细胞中已得到较成功的应用,但在微生物细胞分析方面鲜见报道。该文系统研究了该探针的荧光光谱特征及其在大肠杆菌细胞内的负载行为,并考察了在大肠杆菌细胞中钙离子与Fura-2的结合情况。为利用其研究大肠杆菌等微生物细胞内钙离子浓度及钙离子跨膜行为奠定了基础。     

应用AR—CM—MIC阳离子测定系统检测单个神经元内游离钙

运用Ca~(2 )指示剂Fura-2作为细胞内钙离子的荧光探针,采用精密的AR-CM-MIC阳离子测定系统,检测了分离的单个神经细胞内游离钙离子浓度的动态变化,同时观察了钙离子载体、钙螯合剂等多种药物对细胞内钙浓度的影响,并追踪刺激前后的瞬间变化,探讨此项技术应用于检测细胞内游离钙的灵敏度及适用范围,取得了良好的效果。     

钙荧光探针Fura2/AM对大肠杆菌细胞的负载行为

Fura2/AM作为一类高效的钙离子荧光探针,在动物细胞中已得到较成功的应用,但在微生物细胞分析方面鲜见报道。该文系统研究了该探针的荧光光谱特征及其在大肠杆菌细胞内的负载行为,并考察了在大肠杆菌细胞中钙离子与Fura2的结合情况。为利用其研究大肠杆菌等微生物细胞内钙离子浓度及钙离子跨膜行为奠定了基础。     

A new approach of pellet formation of a filamentous fungus -Rhizopus oryzae

The effects of inoculum and medium composition (i.e. potato dextrose broth as carbon source, soybean peptone, calcium carbonate, and metal ions) on pellet formation of Rhizopus oryzae ATCC 20344 have been studied. Metal ions were found to have a significant negative effect on pellet formation while soybean peptone had a positive effect. In addition, potato dextrose broth and calcium carbonate were beneficial to R. oryzae for growing small, smooth pellets during the culture. The study also demonstrated that an inoculum size of less than 1.5x10(9)spores/L had no significant influence on pellet formation although it had large impacts on pellet growth. Thus, a new approach to form pellets has been developed using only potato dextrose broth, soybean peptone, and calcium carbonate allowing for pellet size to be controlled by adjusting inoculum size and the concentrations of potato dextrose broth, soybean peptone, and calcium carbonate in the medium.     

Calcium ion-dependent proliferation of L1210 cells in culture

Maximum growth of L1210 cells in culture required the presence of free extracellular calcium ions. Reducing the free extracellular calcium ion concentration with EGTA served to decrease the growth rate of the cells. The decrease in cell growth was not due to cell death but rather due to the "pile-up" of the L1210 cells in the GO/Gl phase of the cell cycle. With the readdition of excess calcium ions, there was a lag period of 3 to 6 hours before the L1210 cells initiated DNA synthesis or transited from the G0/G1 phase to S-phase. Cells enriched for S and G2/M phase by elutriation and which were incubated in EGTA-containing culture medium, continued through the cell cycle and were blocked in GO/Gl. These data indicate that the proliferation of L1210 cells in culture requires a calcium ion-dependent process to allow movement from the G0/G1 to S-phase of the cell cycle.     

Calcium ions are involved in the delay of plant cell cycle progression by abiotic stresses

Higher plants respond to environmental stresses by a sequence of reactions which include the reduction of growth by affecting cell division. It has been shown that calcium ions plays a role as a second messenger in mediating various defence responses under environmental stresses. In this study, the role of calcium ions on cell cycle progression under abiotic stresses has been examined in tobacco BY-2 suspension culture cells. Using synchronized BY-2 cells expressing the endogenous calcium sensor aequorin as experimental system, we could show that oxidative and hypoosmotic stress both induce an increase of intracellular calcium and cause a delay of the cell cycle. The inhibitory effect of these abiotic stress stimuli on cell cycle progression could be mimicked by increasing the intracellular calcium concentration via application of an external electrical field. Likewise, depletion of calcium ions in the culture medium suppressed the effect of the stimuli tested. These results demonstrate that calcium signalling is involved in the regulation of cell cycle progression in response to abiotic stress.     

A comparison of raw starch-digesting glucoamylase production in liquid and solid cultures of Rhizopus strains

Maximum growth for Rhizopus sp. A-11 was obtained at a zinc ion concentration of 0.7 ppm in a liquid medium. Glucoamylase (GA, EC 3.2.1.3) production in Rhizopus sp. A-11 was maximized at 710 U/ml, at the presence of 75 ppm for calcium and 0.7 ppm of zinc ions in liquid medium. Zinc ion is known as an essential biometal for Rhizopus growth; however, growth was inhibited by the zinc ion concentration, not maximized. Although calcium ion was not necessary to Rhizopus growth, GA production using Rhizopus sp. A-11 was markedly stimulated by calcium ion concentration over 75 ppm in the liquid medium. The GA productivity of the present liquid culture was about 4.4 times higher than that of the solid state culture, based on the unit starch amount in the liquid and solid media carbon source. The characteristics of the GA produced by the Rhizopus sp. A-11 liquid culture were interesting; that is, almost all the GA produced was classified as raw starch-digesting GA (GA-I). Secreted protein in the culture liquid after 30 h was nearly GA, and had a limited amount of impure protein. As a result, it was found that using a Rhizopus culture in a specified metal-ion regulated medium was an effective method for producing GA. Thus the present culture method was renamed the "metal-ion-regulated liquid culture method".     

Influence of potassium and calcium ions on the phosphatidylinositol and phosphatidylcholine metabolism in rat fibroblasts after growth stimulation by calf serum.

In secondary cultures of embryonic rat fibroblasts which were arrested in G1 (G0) by serum depletion and subsequently triggered into the cell cycle by readdition of growth factors isolated from fetal calf serum the influence of the potassium and calcium concentrations in the medium on phosphatidylinositol and phosphatidylcholine metabolism was investigated. The incorporation of inorganic [32P]phosphate into phosphatidylinositol is dependent on the potassium content of the culture medium. The specific activity of 32P in phosphatidylinositol is increased at K+ concentrations between 0.1 and 1 mM. Also calcium (between 0.01 and 2 mM) slightly stimulates phosphatidylinositol metabolism. Also the incorporation of myo-[3H]inositol is increased at potassium concentrations between 0.2 and 1 mM, whereas calcium is slightly inhibitory. The labelling of phosphatidylcholine with either [32P]phosphate or [3H]choline is not dependent on the potassium and calcium concentrations of the culture medium. Moreover, the phospholipid metabolism of permanently growing epithelioid and fibroblastoid cells lines, which were investigated, is considerably less dependent on the K+ and Ca2+ ions.     

Influence of the culture medium on the production of iturin A by Bacillus subtilis

The production of iturin A by Bacillus subtilis was studied with respect to the composition of the culture medium. Increasing phosphate concentrations did not modify the antibiotic yield. Fructose, sucrose and mannitol were better carbon sources than glucose for antibiotic production. The nature of the nitrogen source was an important factor in the production of antibiotic. Among the amino acids which are components of iturin A, L-asparagine was the best substrate for the biosynthesis of iturin A; L-glutamine and L-serine were rather poor substrates while L-proline and D-tyrosine gave no antibiotic. Ammonium salts permitted good synthesis of antibiotic but the addition of calcium ions to the culture medium inhibited the excretion of antibiotic from the cells.     

Biosynthesis of cephamycin by resting cells of Streptomyces lactamdurans L 2/6

In order to investigate the nutritional conditions of cephamycin biosynthesis independently of the biomass growth process, the nutrient limited-resting cell system was used. A replacement medium eliminating cell multiplication was developed. The presence of Mg2+, carbon source and nitrogen source was necessary for cephamycin production by resting cells of Streptomyces lactamdurans L 2/6. Maximum antibiotic production was obtained when maltose, saccharose, and fructose were used as carbon source, and L-asparagine as nitrogen source. An inhibitory effect on the process was exerted by the calcium ions. There was no visible inhibition of cephamycin biosynthesis by inorganic phosphate ions in concentration up to 100 mM.     

Effect of calcium ion uptake on Candida albicans morphology

In liquid culture using a synthetic medium, added magnesium but not calcium was required for exponential growth of Candida albicans yeast cells. However, medium without added divalent cations supported 2-3 generations of yeast growth or germ tube induction. The addition of calcium ions (1.0 mM) at any stage during the induction of germ tube formation caused reversion to a yeast mode of growth, in contrast to the effect of zinc and cobalt ions which were toxic to all growth. Inhibition of germ tube formation by calcium was not observed in the presence of either magnesium (10 microM) or manganese (100 microM). The presence of either of these ions caused inhibition of 45Ca uptake in yeast cultures. We conclude that unrestricted calcium uptake resulted in the specific inhibition of C. albicans mycelial growth, indicating a critical role for calcium in the regulation of C. albicans morphogenesis.     

Changes in cytosolic calcium, bleb formation, and cell death in neural crest cells treated with isotretinoin and 4-oxo-isotretinoin

Information regarding the cytopathologic mechanism of action of the retinoids [isotretinoin (IR) and 4-oxo-isotretinoin (4-OIR)] on neural crest cells (NCCs) in culture was sought. Those pathophysiologic alterations in cell metabolism studied were: cell blebbing (xieosis), free radical formation, cell viability, and cellular calcium homeostasis. Cells were treated with IR or 4-OIR in the presence of high (1.4 mM) and low (5.0 microM) levels of extracellular calcium ions. Recently developed techniques utilizing fluorescent molecular probes for calcium analyses, i.e., Fura 2AM, were used to study the effects of these drugs on the cytosolic calcium concentration of NCCs. The effects of IR and 4-OIR on NCC viability, [Ca++]int, were contrasted with the effects of certain sulfhydryl drugs (HgCl2, NEM, PCMBS) and calcium ionophores (ionomycin, A23187), agents known to perturb cell membranes, increase cytosolic calcium loads, and induce cell injury and subsequent cell death. Both retinoids were shown to induce an increase in the generation of superoxide radicals (SO) and increase the influx of calcium ions by the NCCs, thus increasing [Ca++]int by several hundred percent within 5 to 10 min. The liberation of SO was calcium dependent. These early effects were accompanied by an increase in cell blebbing activity. Also, a significant decrease in NCC viability was seen as early as 10 min after the addition of IR or 4-OIR to the incubation medium. 4-OIR proved to be the more potent of the two retinoids tested. The severity of these effects on NCC metabolism was dependent on medium calcium concentration with all changes being increased in the presence of the higher extracellular calcium levels. From the data presented it appears as though the retinoids cause a rapid elevation in cytosolic [Ca++]int possibly by purturbing the integrity of the cell membrane, denaturing membrane Ca-ATPase activity, or both. Retinoid-induced changes in membrane activity are evidenced by increased surface blebbing and superoxide formation. The prolonged elevation of intracellular [Ca++] may be directly related to depressed NCC viability and thus explain the known teratogenic effects of these drugs and their relationship to ectomesenchymal cell hypoplasia and craniofacial dysmorphogenesis.     

Deficiency of calcium and magnesium induces apoptosis via scavenger receptor BI

AimsCells undergo apoptosis in stressed status such as in intracellular calcium overload or extracellular calcium/magnesium deficiency. The mechanisms of how deficiency of the divalent metal ions induces apoptosis remain to be defined. Scavenger receptor BI (SR-BI) is a high density lipoprotein (HDL) receptor. Recent studies demonstrated that SR-BI is a stress response molecule which induces apoptosis upon serum deprivation. In this study, we assessed our hypothesis that the deficiency of calcium/magnesium induces apoptosis via SR-BI apoptotic pathway.Main methodsWe employed CHO cell lines expressing vector and SR-BI to test the effect of SR-BI on apoptosis induced by deficiency of calcium, magnesium and zinc in culture medium. The regain of different metal ions in deficient medium was also performed, respectively. Cell death was detected by morphological changes and quantified by LDH cytotoxicity assay. Apoptosis was also assessed by DNA ladder assay and DNA condensation assay. The SR-BIC323G mutant cells which lack the apoptotic activity of SR-BI were employed to verify the SR-BI-dependent effect on calcium/magnesium induced apoptosis.Key findingsThe deficiency of calcium/magnesium induced cell apoptosis in CHO-SR-BI cells, but not in CHO-vector cells. Moreover, no apoptotic cell death was observed in SR-BIC323G mutant cells, indicating that the deficiency of divalent metal ions induces apoptosis in a SR-BI-dependent manner. Furthermore, the restoration of calcium or magnesium, but not zinc, protected CHO-SR-BI cells from apoptotic cell death, in a dose-dependent fashion.SignificanceThese findings extend our understanding about how calcium and magnesium deficiency induces apoptosis.