一氧化氮供体对弓形虫速殖子DNA含量的影响

目的　探讨亚硝基铁氰化钠 (SNP)对弓形虫速殖子DNA含量的影响。　方法　采用SNP与影响胞内 /胞外钙离子浓度的试剂作用于RH株刚地弓形虫 (Toxoplasmagondii)速殖子 ,流式细胞仪检测其DNA含量的变化。　结果　①SNP导致弓形虫速殖子DNA的损伤 ,并呈时间和剂量依赖性 ;② 2mmol/L胞外钙离子螯合剂EGTA ,2 5 μmol/L胞内钙离子螯合剂BAPTA/AM及 5 0 μmol/L钙离子拮抗剂verapamil单独处理弓形虫速殖子 ,其DNA含量与对照组相比无显著变化 ;③ 2mmol/LEGTA ,2 5 μmol/LBAPTA/AM及 5 0 μmol/Lverapamil明显减少 2mmol/LSNP所致的速殖子亚二倍体峰的比例。　结论　SNP通过改变弓形虫速殖子胞浆游离钙离子浓度 ,诱导其亚二倍体峰的出现 ,造成虫体的损伤     

外源性一氧化氮对弓形虫速殖子凋亡的诱导作用

目的　探讨一氧化氮 (NO)是否能诱导弓形虫速殖子凋亡。方法　采用末端脱氧核苷酸转移酶(TdT)介导的原位末端标记法 (TUNEL)、透射电镜和琼脂糖凝胶电泳检测弓形虫速殖子凋亡的特征。结果　TUNEL标记法检测表明 ,NO供体亚硝基铁氰化钠 (SNP)可诱导弓形虫速殖子凋亡 ,并呈剂量和时间依赖性 ;NO清除剂 ,N 乙酰半胱氨酸能明显抑制SNP诱导的弓形虫速殖子凋亡 ;不含NO的SNP类似物 ,铁氰化钾不能诱导弓形虫速殖子凋亡。透射电镜下见SNP处理 15～ 2 0h的速殖子具有凋亡的典型形态学特征 :核染色质凝集、核固缩、核碎裂和凋亡小体形成。琼脂糖凝胶电泳显示SNP处理的弓形虫速殖子DNA片段呈现凋亡特征性的梯形条带。结论　从形态学和生化特征证明由SNP释出的NO可诱导弓形虫速殖子凋亡     

亚硝基铁氰化钠处理的弓形虫速殖子的超微结构特征

目的 观察一氧化氮（NO）供体亚硝基铁氰化钠（SNP）处理的弓形虫速殖子的超微结构特征。方法 应用透射电镜观察SNP处理的速殖子的超微结构。结果 发现SNP处理的速殖子大部分具有凋亡的典型形态学特征：核染色质凝集，核固缩，核碎裂，凋亡小体形成（个别速殖子形态表现为胀亡细胞、溶解性坏死细胞和坏死型凋亡细胞特征）。结论 从形态学上证明NO供体SNP可诱导弓形虫速殖子凋亡及坏死。     

L-精氨酸和L-瓜氨酸在体外活化巨噬细胞抗弓形虫感染中的作用

目的：探讨增加细胞外Ｌ－精氨酸（一氧化氮生物合成的底物）或Ｌ－瓜氨酸（通过再循环生成Ｌ－精氨酸）浓度是否可提高活化巨噬细胞（ｍａｃｒｏｐｈａｇｅ，Ｍ）诱生一氧化氮（ＮＯ）的产量及其对细胞内抑制或杀伤弓形虫速殖子的能力。方法：用ＲＨ株刚地弓形虫（Ｔｏｘｏｐｌａｓｍａｇｏｎｄｉ）速殖子感染活化Ｍ后，观察活化Ｍ在含不同浓度Ｌ－精氨酸和Ｌ－瓜氨酸培养基中培养１８ｈ后的ＮＯ产量、感染率以及纳虫泡内弓形虫速殖子数。结果：（１）活化Ｍ在生理水平的Ｌ－精氨酸浓度（８０μｍｏｌ／Ｌ）下可明显抑制细胞内弓形虫速殖子的增殖，并依赖于ＮＯ的产量；（２）增加细胞外源的Ｌ－精氨酸或Ｌ－瓜氨酸可明显提高Ｍ诱生的ＮＯ产量，进而降低Ｍ的感染率，并抑制细胞内弓形虫速殖子的增殖；（３）Ｌ－瓜氨酸可完全替代Ｌ－精氨酸提高Ｍ诱生的ＮＯ产量及其抑制或杀伤弓形虫速殖子的能力。结论：活化Ｍ胞浆内生理水平的Ｌ－精氨酸诱生的内源性ＮＯ产量足以抑制细胞内弓形虫速殖子的繁殖，但不能防止细胞感染，增加细胞外生物合成ＮＯ的底物浓度可提高Ｍ抑制或杀伤细胞内弓形虫速殖子的能力。     

急性脑血管病患者血小板胞浆游离钙浓度测定的意义

采用钙荧光指示剂Fura-2AM，对32名健康对照组和53例急性脑血管病患血小板静息状态胞浆游离钙浓度([Ca^2 ]i)进行测定。结果显示：脑出血和脑梗塞急性期患血小板[Ca^2 ]i明显高于恢复期和健康对照组(P<0．01)；脑出血和脑梗塞急性期患血小板[Ca^2 ]i分别与平均动脉压和梗塞灶体积呈高度正相关(r=0．926和0．906，P<0．01)。提示脑出血患的血压升高和维持可能与血小板[Ca^2 ]i升高有关；脑梗塞患存在血小板的活化过程，降低血小板[Ca^2 ]i可能有利于控制梗塞灶的发生和发展。     

激光照射对弓形虫速殖子作用的流式细胞分析

用流式细胞术（ＦＣＭ）检测分析了激光照射对弓形虫ＲＨ株速殖子的作用。结果显示，激光照射后弓形虫速殖子群体ＤＮＡ含量分布发生改变，ＤＮＡ含量的第１道数峰值（平均值）左移，第１峰值分布范围内速殖子数占总速殖子数百分率啬，３．５Ｗ和４．５Ｗ剂量组和对照组相比差异存在显著性（Ｐ〈０．０５）。提示激光照射能抑制弓形虫ＤＮＡ的合成，减少弓形虫群体ＤＮＡ的含量。     

锁阳对致衰小鼠神经元内NO、NOS及bcl-2、bax基因表达的实验研究

目的探讨老年小鼠神经元内自由基的变化及与细胞凋亡相关基因bcl-2、bax的表达。方法选用D-半乳糖致衰小鼠，以锁阳水煎剂、锁阳纳米微粒及其水煎剂灌胃，30d后分别检测不同月龄小鼠神经元内一氧化氮(NO)含量、一氧化氮合酶(nNOS)和超氧化物歧化酶(SOD)活性，游离钙离子浓度([Ca^2 ]i)及bcl-2、bax基因表达情况。结果小鼠神经元内NO、nNOS及[Ca^2 ]i、bax基因表达随增龄明显增加；而SOD及bcl-2基因表达随增龄明显降低，锁阳三种剂型均可不同程度抑制各衰老变化，且以锁阳纳米水提液的作用效果最为明显。结论自由基可诱导神经元细胞凋亡的发生，锁阳有显著抑制神经元细胞凋亡及延缓衰老的作用，且以锁阳纳米水提液作用最为明显。     

刚地弓形虫RH株速殖子协同VP-16体外杀伤小鼠结肠癌细胞ct26的作用

目的探讨刚地弓形虫RH株速殖子对小鼠结肠癌ct26细胞周期的影响及其协同细胞周期特异性抗癌药依托泊甙(VP-16)对ct26细胞的杀伤作用。方法建立弓形虫RH株速殖子与小鼠结肠癌ct26细胞不同比例的共培养模型(虫/细胞比例分别为2:1、4:1、8:1、16:1),流式细胞仪检测24h细胞周期改变;以虫细胞比例2:1预先感染ct26细胞24h,CCK-8比色法观察不同浓度VP-16对细胞增殖抑制的改变;荧光显微镜观察VP-16(40μg/mL)对感染虫体细胞的形态学改变。台盼蓝染色观察不同浓度VP-16分别作用弓形虫RH株速殖子悬液(1×106/mL)2h、4h、12h、24h的虫体存活度改变。琼脂糖凝胶电泳观察VP-16(20μg/mL)对虫体DNA影响。结果流式细胞仪检测发现各浓度弓形虫速殖子均可明显改变ct26细胞24h周期分布,使G0/G1期细胞百分比下降,G2/M期百分比显著升高(P0.01)。2×106个速殖子感染细胞24h可使S期和G2期细胞百分比共同增长14.92%。CCK-8比色法检测结果显示虫细胞比例2:1预先感染ct26细胞24h可有效增强各浓度VP-16对ct26细胞的杀伤作用;Hochest33258染色结合荧光显微镜观察到虫体胞质寄生形态及VP-16诱导感染细胞凋亡典型形态;10μg/mL及更高浓度VP-16作用弓形虫4h以上均可使虫体全部死亡;琼脂糖凝胶电泳发现VP-16(20μg/mL)可使弓形虫速殖子DNA呈现典型的云梯状条带。结论弓形虫RH株速殖子可使小鼠结肠癌ct26细胞发生G2/M期,可有效增强细胞周期特异性抗癌药VP-16的抗癌作用,且弓形虫增殖亦明显受抑并发生凋亡。     

激光对弓形虫速殖子的作用研究

本实验采用激光致弱弓形虫速殖子接种ICR小鼠腹腔,以期提高小鼠抗弓形虫感染的免疫保护力。结果表明：激光照射能够抑制虫体的繁殖和分裂能力。照射虫体免疫小且能诱导产生持续升高的IgG抗体。免疫民经弓形虫RH株攻击后,其发病和死亡时间延长,表现出部分的保护性免疫力。提示：激光对弓形虫速殖子的作用明显,可望在弓形虫病的治疗和预防中得到应用。     

兴奋毒作用下皮层神经细胞Ca^2＋变化机制

目的：检测谷氨酸兴奋毒损伤时,有胞外有Ca^2 或无Ca^2 ,,或有尼莫地平和Ca^2 条件下,神经细胞内[(Ca^2 )]i的变化,方法：用Fura-2/AM体外检测新生大鼠皮层神经细胞内游离钙离子浓度的方法。结果：发现谷氨酸兴奋毒可使皮层神经细胞[Ca^2 ]i上升并有剂量依赖性,胞外无Ca^2 或加入尼莫地平时神经细胞[Ca^2 ]i虽也上升,但幅度降低。三种条件下神经细胞在兴奋毒作用下[Ca^2=]i变化各不相同。结论：表明谷氨酸活化神经细胞膜上受体,使胞内钙库动员和细胞膜Ca^2 通道开放,导致[Ca^2_]i在短时间内总体表现为持续上升,存在兴奋毒损伤过程中有膦脂酶A2活化过度参与的信使基础。     

Nitric oxide donors modify free intracellular calcium levels in rat anterior pituitary cells

The effect of nitric oxide donors on intracellular calcium concentration [Ca2+]i was studied in anterior pituitary cells using ratiometric FURA 2 fluorescence measurements. Sodium nitroprusside (NP) induced a transient decrease in [Ca2+]i, after which [Ca2+]i returned to, or even increased over basal values. S-Nitroso glutathione (GSNO) induced a similar decrease. NP also inhibited high [Ca2+]i achieved by depolarization with 25 mM K+. The inhibitory effect of NP was partially blunted by pretreatment with methoxy-verapamil, and in calcium free buffer, and was not altered by thapsigargin. Interestingly, in calcium free buffer there was a significant stimulatory effect of NP, which was partially blunted by thapsigargin. We conclude that NO donors modify [Ca2+]i in anterior pituitary cells. The action is biphasic, with an initial decrease in [Ca2+]i probably related to a decrease of Ca2+ influx through VDCC, and an increase evidenced in calcium free buffer in which the inhibitory component is absent, and partially depends on thapsigargin sensitive calcium stores.     

Role of Ca<Superscript>2+</Superscript> in the Intracellular Signaling Pathway of Anti-IgM—Induced Apoptosis in the Human B-Cell Line,MBC-1, Established from Burkitt Lymphoma

The role of Ca2+ in the intracellular signal transduction process that causes antibody-induced apoptotic cell death in B-cells is not completely understood. We previously established a B-cell line (MBC-1) from a patient with Burkitt lymphoma at the leukemic stage that demonstrated the typical morphology and internucleosomal DNA fragmentation of apoptosis when treated with anti-immunoglobulin (Ig)M antibody. This antibody-induced cell death was partially inhibited by pretreatment with ethyleneglycol-bis-tetraacetic acid (EGTA) and actinomycin-D. FK506, an immunosupressive agent and calcineurin inhibitor, also partially rescued the anti-IgM antibody-induced death of MBC-1 cells. These results show that the calcium signaling pathway, which leads to a change in gene expression, plays an important role in anti-IgM-induced apoptosis in MBC-1 cells. Flow cytometric measurement of the cytosolic free Ca2+ concentration ([Ca2+]i) showed that nontoxic concentrations of 4-bromo-calcium ionophore A23187 (Ca2+ IP) increased [Ca2+]i more than did the anti-IgM antibody. A brief Ca2+ spike was observed on anti-IgM antibody treatment, but a gradual increase and decrease were observed when the cells were treated with Ca2+ IP at a nontoxic concentration of 1 microg/mL. These findings suggest that interpretations differ for the 2 patterns of calcium signaling and that the brief spiked elevation of Ca2+ produces distinct biological and cellular responses compared to the gradual increase and decrease of [Ca2+]i. Our results support the hypothesis that Ca2+ plays a significant role as a multifunctional second messenger providing specific information to the nucleus in anti-IgM antibody-induced apoptosis in MBC-1 cells.     

系统性红斑狼疮患者T细胞TCR/CD3复合物介导的信号传导研究

目的：证实系统性红斑狼疮（SLE）患者T细胞功能异常是否与其生物化学信号传导异常有关。方法：用CD3单抗与羊抗鼠二抗IgG相交联刺激T细胞并用Thapsigargin和依地酸（EGTA）干预后，分别用粘附细胞仪连续观察10min T细胞[Ca^2 ]i的变化，并评价[Ca^2 ]i反应与CD3分子和三磷酸肌醇（InsP3）生成量的相关性。结果：正常人和SLE患者T细胞[Ca^2 ]i反应的基准值相似（P＝0．105）；SLE患者高峰者，平台值T细胞的[Ca^2 ]i反应明显高于正常对照（P＜0．001，P＜0．001），加入Thapsigargin后二者[Ca^2 ]i反应差异无显著性，而加入EGTA后二者[Ca^2 ]i反应差异有显著性，二者的T细胞CD3阳性率和InsP3生成量差异无显著性（P＝0．665，P=0．537）。结论：SLE患者T细胞TCR／CD介导的信号传导途径存在异常，SLE患T细胞功能异常可能是因细胞内生物化学信号传导途径异常所致。     

Regulation of calcium current by intracellular calcium in smooth muscle cells of rabbit portal vein

Effects of concentrations of intracellular calcium, [Ca2+]i, on the voltage-dependent Ca2+ current (ICa) recorded from dispersed single smooth muscle cells of the rabbit portal vein were studied, using a whole cell voltage clamp method combined with an intracellular perfusion technique. Outward currents were minimized by replacement of Cs+ -rich solution in the pipette and 20 mM tetraethylammonium in the bath. The ICa was evoked by command pulses of above -30 mV, and the maximum amplitude was obtained at about 0 mV. This ICa was dose dependently inhibited by increases in the [Ca2+]i above 30 nM. The Kd value of the [Ca2+]i required to inhibit the ICa was about 100 nM. The Ba2+ current was also inhibited by increases in the [Ca2+]i. Conversely, perfusion of Ba2+ into the cell up to 100 microM did not suppress the ICa. Changes in the [Ca2+]i did not modify the steady-state inactivation curve. The inhibition of the ICa evoked by the test pulse is most prominent when the preceding influx of Ca2+ during the conditioning pulse was large, as estimated using a double pulse protocol. This inhibition was proportionally reduced by increases in the concentration of the Ca2+ chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Therefore, the Ca2+ -dependent inactivation of the Ca2+ channel may contribute toward regulating [Ca2+]i in smooth muscle cells of the rabbit portal vein.     

Distribution of nitric oxide synthase and secretory role of exogenous nitric oxide in the isolated rat pancreas.

BACKGROUND: Pancreatic production and in vivo effects of nitric oxide (NO) have been shown by several studies. In order to examine the direct actions of the NO donor sodium nitroprusside (SNP), this study used in vitro specimens of the rat pancreas where the distribution of neuronal nitric oxide synthase (NOS) and the secretory effects of SNP and the cyclic GMP (cGMP) analog 8-bromo cyclic GMP (8-Br cGMP) were investigated. METHODS: NO containing pancreatic nerves were visualized by NOS immunohistochemistry. Basal and stimulated amylase output from rat pancreatic segments was measured by an on-line fluorimetric method. Stimulation was achieved by either acetylcholine (ACh) or electrical field stimulation (EFS). Intracellular free calcium concentration ([Ca2+]i) was measured in dispersed pancreatic acinar cells. RESULTS: NOS containing nerves were demonstrated in the vicinity of pancreatic acini and blood vessels. SNP and 8-Br cGMP inhibited both basal and EFS evoked amylase output but failed to inhibit ACh induced amylase output. Basal [Ca2+]i was decreased by both SNP and 8-Br cGMP but neither SNP nor 8-Br cGMP influenced the ACh evoked increase in [Ca2+]i. CONCLUSION: NO is well distributed in the rat exocrine pancreas. Exogenous nitric oxide may have a dual action in the isolated rat pancreas: Inhibition of basal amylase secretion in acinar cells and inhibition of ACh release from intrinsic nerve terminals. Both effects seem to be calcium dependent and possibly mediated by cGMP.     

Angiotensin II receptor-mediated calcium influx in bovine adrenal glomerulosa cells.

The cytoplasmic calcium ([Ca2+]i) response to angiotensin II (AII) in bovine adrenal glomerulosa cells is characterized by an initial transient peak due to intracellular Ca2+ mobilization, followed by a sustained plateau phase that is dependent on Ca2+ entry from the extracellular fluid. In Fura-2 loaded cells, the calcium channel antagonists, nifedipine (1 microM) and verapamil (20 microM), only partially reduced the cytosolic calcium profile induced by AII. The dihydropyridine agonist, Bay K 8644, caused a moderate increase in [Ca2+]i when added in concentrations of 50-100 nM, but did not enhance the AII-induced rise in [Ca2+]i. These results indicate that most of the AII-stimulated Ca2+ influx is through channels that are insensitive to dihydropyridines and verapamil. In contrast, the inorganic Ca2+ channel blocker, LaCl3 (10 microM), inhibited the AII-induced plateau phase by more than 50%. The AII-induced Ca2+ signal was not affected by treatment with pertussis toxin (100-300 ng/ml for 12 h). The prior addition of specific AII-antagonists (DuP 753, a nonpeptide antagonist, and three peptide analogs, [Sar1,Thr8]AII, [Sar1,Ala8]AII, and [Sar1,Ile8]AII) completely inhibited the AII-induced Ca2+ signal. However, addition of up to 25,000 molar excess of these antagonists at intervals from 10 sec to 5 min after AII caused progressively less attenuation of the sustained Ca2+ signal. After 5 min, addition of antagonists did not inhibit the agonist-induced Ca2+ response for up to 20 min. The progressive loss of ability of the antagonists to inhibit the sustained elevation of [Ca2+]i could reflect prolonged activation of the receptor or of a subsequent process that maintains Ca2+ influx despite receptor blockade. It is possible that sequestration and/or endocytosis of the AII-receptor complex is accompanied by continued generation of intracellular signals that contribute to the maintenance of the [Ca2+]i response.     

1,25-Dihydroxyvitamin D3 enhances cytosolic free calcium in HL-60 cells

1,25-Dihydroxyvitamin D3 (1,25[OH]2D3) caused a rise in the concentration of intracellular free calcium ions ([Ca2+]i) in HL-60 cells. This effect of 1,25(OH)2D3 parallels its suppression of cell proliferation and its induction of cell differentiation into monocyte-like cells. The changes in [Ca2+]i are dose and time dependent. The concentration of 1,25(OH)2D3 (10(-7) M) that induced maximal differentiation also caused the maximal increase in intracellular Ca2+. The rise in cytoplasmic free Ca2+ concentration was not immediate and reached statistical significance only after 24 h. The [Ca2+]i reached its peak at 48 h (134 +/- 4 nM vs 101 +/- 3 nM in controls) and remained stable at this level. The increase in intracellular Ca2+ was found to be related to new protein synthesis, because it was inhibited in the presence of specific RNA and protein synthesis inhibitors. The rise in [Ca2+]i was not observed during incubation of HL-60 cells with 24,25-dihydroxyvitamin D3 (24,25[OH]2D3), a vitamin D metabolite that does not induce the differentiation of HL-60 cells. In contrast, 25-hydroxyvitamin D3 (25-OH-D3) and phorbol 12-myristate 13-acetate (TPA), both of which induce differentiation in this cell line, also increase [Ca2+]i. In conclusion, the present study emphasizes that a significant increase in intracellular free Ca2+ occurs in the effect of 1,25(OH)2D3 on HL-60 cells.     

Regulation of cytosolic pH in bovine parathyroid cells: effect of fluoride

In the present investigation, sodium fluoride (NaF) was employed to explore the role of guanine nucleotide-binding proteins (G-proteins), protein kinase-C, or cytosolic calcium [( Ca]i) in the regulation of cytosolic pH [( pH]i) in dispersed bovine parathyroid cells, using the pH-sensitive fluorescent dye BCECF. When cells acidified by nigericin in Na-free medium were resuspended in Na-containing buffer, [pH]i returned to basal levels. This recovery was blocked by continued removal of Na+ or the addition of amiloride. NaF (10 mM) increased [32P]phosphate incorporation into phosphatidylinositol bisphosphate, suggesting an increase in phosphatidylinositol bisphosphate turnover. NaF caused an initial acidification, followed by an alkaline recovery in a dose-dependent manner (1-10 mM). Amiloride blocked the NaF-induced alkaline recovery. The protein kinase-C activator phorbol 12-myristate 13-acetate (10(-7) M) caused cytosolic alkalinization, while the protein kinase-C inhibitor H7 (6 x 10(-5) M) significantly inhibited the NaF-induced alkaline recovery. Pertussis toxin (1 microgram/ml) did not affect the NaF-induced changes in [pH]i. Removal of extracellular Ca2+ with EGTA blocked the NaF-induced increase in [Ca]i and alkaline recovery. Ionomycin (5 x 10(-7) M) caused cytosolic alkalinization, but pretreatment with EGTA inhibited the ionomycin-induced cytosolic alkalinization. The present studies clearly demonstrated the presence of an amiloride-sensitive Na+/H+ exchanger in parathyroid cells. Our findings suggest that the NaF-induced cytosolic alkaline recovery was via two complementing pathways: 1) activation of protein kinase-C, followed by stimulation of a Na+/H+ exchanger, and 2) existence of extracellular calcium and/or an increase in [Ca]i.