EGTA和La3+对几种逆境诱导辣椒样半萜 环化酶基因表达的影响

用钙离子螯合剂EGTA及细胞膜钙离子通道拮抗剂La     

EGTA和La~(3 )对几种逆境诱导辣椒样半萜环化酶基因表达的影响

用钙离子螯合剂EGTA及细胞膜钙离子通道拮抗剂La3 预处理辣椒叶片 ,以破坏辣椒叶片中的钙信使系统 ,再用紫外线、CuCl2 、HgCl2 处理辣椒叶片 ,研究表明EGTA和La3 预处理未能降低CuCl2 、HgCl2 、UV诱导辣椒倍半萜环化酶活化的作用 ,EGTA预处理反而对CuCl2 、HgCl2 、UV的诱导辣椒倍半萜环化酶活性作用有一定的促进效应 .单独用EGTA处理也能诱导离体辣椒叶片表现出倍半萜环化酶活性 .NorthenBlot分析结果表明 ,EGTA能诱导辣椒倍半萜环化酶基因转录 .研究表明 ,在辣椒倍半萜环化酶基因表达过程中 ,还存在钙信使系统以外的信号传递途径 ;非生物诱发因子对倍半萜环化酶基因表达诱导作用与生物Elicitor的诱导作用在信号传递上有差异 .     

杭州沼泽性水域原生动物群落变化规律

2005年4月-2006年3月调查了杭州市郊下沙区一个沼泽性水域原生动物群落的变化规律。共观察到各类原生动物265种,其中鞭毛虫114种,占原生动物总数的43%,其中植物性鞭毛虫占91%,动物性鞭毛虫仅占9%;肉足虫34种,占原生动物总数的13%,纤毛虫117种,占原生动物总数的44%。原生动物在此水域1个周年内的分布特征表现为植物性鞭毛虫种类多于动物性鞭毛虫,纤毛虫种类多于肉足虫种类。原生动物群落的结构参数(种类组成、个体丰度和多样性指数)因水质的变化而变化。此水域原生动物的优势种为梨形扁裸藻(Phacus pyrum)、旋转囊裸藻(Trachelomonas volvocina)、绿色裸藻(Euglenaviridis)和刺鱼状裸藻(Euglena gasterosteus),常年居留种为裸藻(Euglenasp.)、奇形扁裸藻(Phacus anomalus)、钟形虫(Vorticellasp.)、尾草履虫(Paramecium caudatum)和多态喇叭虫(Stentor polymorphrus)。全年3个采样站的Margalef生物多样性指数为1.056～6.054。     

云南不同海拔花椒园昆虫群落结构及动态

调查了4种不同海拔花椒园昆虫群落组成及结构的周年变化,应用群落特征参数及主分量分析方法分析了昆虫群落特征及其在时间过程中的主导因素和时间格局.结果表明,随着海拔的逐渐上升,昆虫群落中种类数量逐渐减少,种群数量相对增加,群落多样性、丰富度及均匀度等指数逐渐下降,随时间的变化而波动的趋势逐渐明显,主导昆虫群落变化的最主要因子由天敌亚群落的物种数和个体数量综合因子逐渐转向害虫亚群落的物种数和个体数量综合因子.     

外源水杨酸对辣椒倍半萜环化酶基因表达及抗氧化酶系的作用

分析了外源水杨酸对辣椒叶片倍半萜环化酶基因表达及抗氧化酶系的作用 .结果表明 ,在 0 .5～4mmol·L-1的浓度范围内 ,SA处理均能不同程度地诱导辣椒叶片中倍半萜环化酶基因转录并表达酶活性 ,但是酶活性较低且在SA处理 36h后才出现 ;SA处理后 ,辣椒叶片SOD和POD酶活性较对照增高 ,CAT酶活性较对照降低 ,相应地 ,H2 O2 浓度升高 .H2 O2 含量的升高与SA对辣椒叶片抗氧化酶活性的综合影响有关     

Cu2+作用下辣椒膜脂过氧化及倍半萜环化酶基因转录

分析了在CuCl2作用下辣椒叶片倍半萜环化酶活性,倍半萜环化酶mRNA表达,细胞GSH和GSSG代谢及膜脂过氧化,结果表明,Cu^2 能诱导辣椒叶片表达倍半萜环化酶活性,酶活性的表达与相应的基因转录有关,辣椒叶片在CuCl2作用下合成了含－SH的螯合肽,相应地GSH含量下降,GSSG的含量有所上升,细胞膜过氧化作用加剧,推测细胞膜脂过氧化产物或GSH氧化产物可能参与了CuCl2诱导辣椒倍半萜环化酶基因表达的信号传递作用。     

介质Ca2+和La3+对酿酒酵母生长的影响

采用正交实验研究了外加Ｃａ^２＋和Ｌａ^３＋对酿酒酵母生长的影响。结果表明：外加Ｃａ^２＋和Ｌａ^３＋对酿酒酵母的生长均有显著的影响,都呈现出低浓度时正效应和高浓度时负效应,当Ｃａ^２＋浓度为１ｍｍｏｌ／Ｌ及Ｌａ^３＋浓度为１５μｍｏｌ／Ｌ时酿酒酵母生长最好。     

不同盐度和Fe3+浓度对小球藻生长、硝酸还原酶活性及基因表达的影响

论文以一株紫外诱变获得的蛋白核小球藻F-9-3为材料,研究了4个盐度和5个Fe3+浓度对其生长和硝酸还原酶(NR)活性及其基因表达的影响。盐度实验结果表明在0～0.30 mol L-1NaCl浓度范围内,小球藻生长较快;NR酶活性及其基因表达量都是在0.15和0.30 mol L-1NaCl较高。铁浓度实验结果表明在0.03和0.06 mmol L-1Fe3+浓度培养小球藻生长较快,NR酶活性和基因表达量较高;而高铁浓度组(0.12 mmol L-1)其生长受抑制,缺铁情况下nr基因表达量最高。因此适合该小球藻生长的最适盐度范围是0～0.30 mol L-1NaCl和0.03～0.06 mmol L-1Fe3+浓度     

La3+对墨兰根状茎某些细胞器发育的影响

以１／２ＭＳ＋ＢＡ（１ｍｇＬ－１）＋ＮＡＡ（１ｍｇＬ－１）＋Ｌａ３＋（１０ｍｇＬ－１）培养墨兰根状茎３０ｄ后,取样观察根状茎细胞叶绿体、线粒体和细胞核的发育特点并与对照进行比较,发现：①处理组叶绿体发育较快,有较丰富的片层结构和嗜锇颗粒,而对照组的发育慢,含大型的淀粉粒,无明显的片层结构．②处理组线粒体体积较大,内含物较丰富,而对照组的较小,内含物较少。③处理组细胞核内的染色质较对照组丰富．上述结果表明,ｔａ３＋对墨兰根状茎细胞器的发育有一定的调节作用。     

热胁迫对辣椒花药发育过程中Ca2+分布的影响

运用焦锑酸钾沉淀法,研究了不同热胁迫时间对辣椒（Capsicum annuum L.)小孢子发生和花粉发育过程中Ca^2 分布的影响。在小孢子母细胞中,细胞表面有少量Ca^2 分布,细胞核中基本上观察不到Ca^2 ,热胁迫12h后,细胞质和细胞核中Ca^2 明显增多,液泡膜内侧也有许多Ca^2 分布,热胁迫24h后,大量的Ca^2 分布在细胞质中,液泡膜上和液泡内;在四分体时期,与小孢子母细胞相比,四分小孢子表面和细胞质中Ca^2 数量明显增加,热胁迫24h后,细胞质和细胞核中Ca^2 更多;在小孢子中,大量Ca^2 分布在壁上,质膜内侧,液泡膜上,少量分布在细胞质和细胞核中,热胁迫12h后,质膜上Ca^2 增多,24h后,细胞质,细胞核中,质膜内侧的Ca^2 继续增多,热胁迫对成熟花粉中Ca^2 的分布无明显影响。     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Role of Na+-Ca2+ Exchanger in Agonist-Induced Ca2+ Signaling in Cultured Rat Astrocytes

Abstract: We have previously demonstrated that activation of the Na⁺-Ca²⁺ exchanger in the reverse mode causes Ca²⁺ influx in astrocytes. In addition, we showed that the exchange activity was stimulated by nitric oxide (NO)/cyclic GMP and inhibited by ascorbic acid. The present study demonstrates that the Na⁺-Ca²⁺ exchanger is involved in agonist-induced Ca²⁺ signaling in cultured rat astrocytes. The astrocytic intracellular Ca²⁺ concentration ([Ca²⁺]_i) was increased by l -glutamate, noradrenaline (NA), and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced the ionomycin-induced increase in [Ca²⁺]_i. The Na-induced Ca²⁺ signal was also attenuated by S-nitroso-l -cysteine and 8-bromo cyclic GMP, whereas it was enhanced by 3,4-dichlorobenzamil, an inhibitor of the Na⁺-Ca²⁺ exchanger. Treatment of astrocytes with antisense, but not sense, deoxynucleotides to the sequence encoding the Na⁺-Ca²⁺ exchanger enhanced the ionomycin-induced increase in [Ca²⁺]_i and blocked the effects of SNP and 8-bromo cyclic GMP in reducing the NA-induced Ca²⁺ signal. Furthermore, the ionomycin-induced Ca²⁺ signal was enhanced by removal of extracellular Na⁺ and pretreatment with ascorbic acid. These findings indicate that the Na⁺-Ca²⁺ exchanger is a target for NO modulation of elevated [Ca²⁺]_i and that the exchanger plays a role in Ca²⁺ efflux when [Ca²⁺]_i is raised above basal levels in astrocytes.     

Alteration of Ca2+ Fluxes in Brain Microsomes by K+ and Na+: Modulation by Sulfated Polysaccharides and Trifluoperazine

Abstract: Rat brain microsomes accumulate Ca²⁺ at the expense of ATP hydrolysis. The rate of transport is not modulated by the monovalent cations K⁺, Na⁺, or Li⁺. Both the Ca²⁺ uptake and the Ca²⁺-dependent ATPase activity of microsomes are inhibited by the sulfated polysaccharides heparin, fucosylated chondroitin sulfate, and dextran sulfate. Half-maximal inhibition is observed with sulfated polysaccharide concentrations ranging from 0.5 to 8.0 µg/ml. The inhibition is antagonized by KCl and NaCl but not by LiCl. As a result, Ca²⁺ transport by the native vesicles, which in the absence of polysaccharides is not modulated by monovalent cations, becomes highly sensitive to these ions. Trifluoperazine has a dual effect on the Ca²⁺ pump of brain microsomes. At low concentrations (20–80 µM) it stimulates the rate of Ca²⁺ influx, and at concentrations >100 µM it inhibits both the Ca²⁺ uptake and the ATPase activity. The activation observed at low trifluoperazine concentrations is specific for the brain Ca²⁺-ATPase; for the Ca²⁺-ATPases found in blood platelets and in the sarcoplasmic reticulum of skeletal muscle, trifluoperazine causes only a concentration-dependent inhibition of Ca²⁺ uptake. Passive Ca²⁺ efflux from brain microsomes preloaded with Ca²⁺ is increased by trifluoperazine (50–150 µM), and this effect is potentiated by heparin (10 µg/ml), even in the presence of KCl. It is proposed that the Ca²⁺-ATPase isoform from brain microsomes is modulated differently by polysaccharides and trifluoperazine when compared with skeletal muscle and platelet isoforms.     

Effect of Ca2+ on In Vitro Astrocyte Injury

Abstract: Current literature suggests that a massive influx of Ca²⁺ into the cells of the CNS induces cell damage associated with traumatic brain injury (TBI). Using an in vitro model for stretch-induced cell injury developed by our laboratory, we have investigated the role of extracellular Ca²⁺ in astrocyte injury. The degree of injury was assessed by measurement of propidium iodide uptake and release of lactate dehydrogenase. Based on results of in vivo models of TBI developed by others, our initial hypothesis was that decreasing extracellular Ca²⁺ would result in a reduction in astrocyte injury. Quite unexpectedly, our results indicate that decreasing extracellular Ca²⁺ to levels observed after in vivo TBI increased astrocyte injury. Elevating the extracellular Ca²⁺ content to twofold above physiological levels (2 m M ) produced a reduction in cell injury. The reduction in injury afforded by Ca²⁺ could not be mimicked with Ba²⁺, Mn²⁺, Zn²⁺, or Mg²⁺, suggesting that a Ca²⁺-specific mechanism is involved. Using ⁴⁵Ca²⁺, we demonstrate that injury induces a rapid influx of extracellular Ca²⁺ into the astrocyte, achieving an elevation in total cell-associated Ca²⁺ content two- to threefold above basal levels. Pharmacological elevation of intracellular Ca²⁺ levels with the Ca²⁺ ionophore A23187 or thapsigargin before injury dramatically reduced astrocyte injury. Our data suggest that, contrary to popular assumptions, an elevation of total cell-associated Ca²⁺ reduces astrocyte injury produced by a traumatic insult.     

Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes

The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.     

Possible Regulation of Caffeine-Induced Intracellular Ca2+ Mobilization by Intracellular Free Na+

To gain some understanding of the regulatory mechanism involved in caffeine-induced Ca2+ release in adrenal chromaffin cells, we took advantage of the paradoxical observation that removal of divalent cations potentiated the secretory response to caffeine. We measured the concentration of cytosolic free Ca2+ ([Ca]in) in isolated cat chromaffin cells, by fura-2 microfluorometry, to see whether there was any correlation between the secretory response and the rise in [Ca]in. The caffeine-induced [Ca]in rise and catecholamine secretion were increased by treatment of cells with a divalent cation-deficient solution. These potentiated responses were strongly inhibited either by pretreatment with ryanodine, by the reduction of the external Na+ concentration, or by the addition of Ca2+ channel blockers. Removal of divalent cations caused a large rise in the cytosolic free Na+ concentration ([Na]in), which was measured using SBFI microfluorometry. This rise in [Na]in was reduced either by adding Ca2+ channel blockers or by reducing the external Na+ concentration. These results show a good correlation between caffeine-induced Ca2+ release and [Na]in at the time of stimulation, suggesting that caffeine-induced Ca2+ release is regulated by [Na]in.     

Kinetic Characterization of Ca2+ Transport in Synaptic Membranes

Lysed synaptosomal membranes were prepared from brain cortices of HA/ICR Swiss mice, and the ATP-stimulated Ca2+ uptake, Ca2+-stimulated Mg2+-dependent ATPase activity, and the Ca2+-stimulated acyl phosphorylation of these membranes were studied. The Km values for free calcium concentrations ([Ca2+]f) for these processes were 0.50 microM, 0.40 microM, and 0.31 microM, respectively. Two kinetically distinct binding sites for ATP were observed for the ATP-stimulated Ca2+ uptake and the Ca2+-stimulated Mg2+-ATPase activity. The high-affinity Km values for ATP for these two processes were 16.3 microM and 28 microM, respectively. These results indicate that the processes studied operate in similar physiological concentration ranges for the substrates [Ca2+]f and ATP under identical assay conditions and, further, that these processes may be functionally coupled in the membrane.