一种特殊的长寿细胞:毛竹茎秆纤维细胞

利用显微和细胞化学方法,对毛竹(Phyllostachys edulis)茎秆纤维次生壁形成过程中超微结构变化以及ATP酶、Ca2 -ATPase和酸性磷酸酶的超微细胞化学定位进行了研究.研究发现,次生壁形成早期,细胞核具有双层核膜,染色质凝聚,可见大量的线粒体、粗面内质网和高尔基体等细胞器存在于纤维细胞中;随后,双层核膜消失,细胞器将逐渐解体,多泡体开始出现在纤维细胞的细胞质;随着年龄的增加,纤维细胞壁逐渐增厚,并出现多层结构现象,而运输小泡、细胞膜、胞间连丝和凝聚的染色质将持续存在.在次生壁形成的整个过程中,ATP酶、Ca2 -ATPase和酸性磷酸酶在运输小泡、细胞膜、质膜内陷、胞间连丝和凝聚的染色质中将持续存在.结果表明,毛竹茎秆纤维细胞是一种不同于木本双子叶植物的长寿细胞,纤维原生质体中ATP酶和酸性磷酸酶的持续存在与次生壁的持续增厚密切相关.     

毛竹茎秆纤维细胞发育过程中ATP酶的超微细胞化学定位研究

采用磷酸铅沉淀技术,对毛竹茎秆纤维细胞发育过程中的ATP酶进行了超微细胞化学定位研究.在初生壁形成时期,大量的ATP酶的活性产物沉积在质膜、质膜内陷、运输小泡、胞间连丝等膜体系以及细胞核和各种细胞器上;在次生壁形成的初期,ATP酶在多泡小体和裂解的液泡膜上出现,凝聚并边缘化的染色质上仍然具有ATP酶活性;随着次生壁的逐渐加厚,在前四年中持续存在具有ATP酶活性的质膜内陷结构,以后消失;而在六年生纤维细胞的质膜、运输小泡、纹孔、胞间连丝和凝聚化的染色质上仍然发现有明显的ATP酶分布,并发现在染色质上ATP酶活性会随着凝聚程度的加深而增强.结果表明,ATP酶在毛竹茎秆纤维细胞壁的整个形成过程中发挥重要作用,而纤维细胞的次生壁形成过程是一个由核基因控制的主动的PCD过程;并证实毛竹茎秆纤维细胞的发育有别于其它木本植物纤维细胞的发育过程,这种纤维细胞是一种典型的长寿细胞.     

毛竹茎秆纤维发育过程的超微结构观察

利用透射和扫描电镜观察了毛竹（Phyllostachys edulis (Carr.) H. De Lehaie）茎秆纤维发育过程中的超微结构变化。在纤维细胞初生壁形成期,细胞质中线粒体、内质网、高尔基体等细胞器数量有明显的增加,出现大量的由内质网与高尔基体分泌形成的运输小泡,周质微管平行分布于质膜内侧,出现环状片层结构,并在细胞壁与质膜之间出现壁旁体结构。随着次生壁的逐渐形成,细胞质中细胞器逐渐地解体并出现多泡小体;纤维细胞核出现染色质凝聚并边缘化,但在8 年生的纤维中可以持续存在;在纤维次生壁形成的整个阶段都存在与周围细胞相联系的胞间连丝和运输小泡;次生壁 在前4 年加厚明显,以后加厚程度减缓,但可以持续很长一段时间,并随着加厚出现宽窄交替的多层结构。结果表明,线粒体、内质网、高尔基体和壁旁体等细胞器与周质微管一起参与了初生壁和次生壁早期的形成;纤维细胞次生壁的形成过程就是一个漫长的程序性细胞死亡（PCD）,而PCD 的产物与胞间连丝一起参与了次生壁的形成与加厚;染色质凝聚并边缘化的细胞核与胞间连丝的持续存在,证明毛竹茎秆纤维细胞是一种典型的长寿细胞。     

毛竹茎秆纤维发育过程的超微结构观察

利用透射和扫描电镜观察了毛竹(Phyllostachys edulis(Carr.)H.De Lehaie)茎秆纤维发育过程中的超微结构变化.在纤维细胞初生壁形成期,细胞质中线粒体、内质网、高尔基体等细胞器数量有明显的增加,出现大量的由内质网与高尔基体分泌形成的运输小泡,周质微管平行分布于质膜内侧,出现环状片层结构,并在细胞壁与质膜之间出现壁旁体结构.随着次生壁的逐渐形成,细胞质中细胞器逐渐地解体并出现多泡小体;纤维细胞核出现染色质凝聚并边缘化,但在8年生的纤维中可以持续存在;在纤维次生壁形成的整个阶段都存在与周围细胞相联系的胞间连丝和运输小泡;次生壁在前4年加厚明显,以后加厚程度减缓,但可以持续很长一段时间,并随着加厚出现宽窄交替的多层结构.结果表明,线粒体、内质网、高尔基体和壁旁体等细胞器与周质微管一起参与了初生壁和次生壁早期的形成;纤维细胞次生壁的形成过程就是一个漫长的程序性细胞死亡(PCD),而PCD的产物与胞间连丝一起参与了次生壁的形成与加厚;染色质凝聚并边缘化的细胞核与胞间连丝的持续存在,证明毛竹茎秆纤维细胞是一种典型的长寿细胞.     

毛竹茎秆纤维细胞发育过程中的细胞程序性死亡

利用TUNEL检测、细胞学及细胞化学方法,对毛竹茎秆纤维细胞发育过程中的细胞程序性死亡进行了研究。在次生壁形成的早期,纤维细胞出现染色质凝聚、细胞器膨胀、液泡膜解体和细胞质泡状化等典型的细胞程序性死亡形态学特征;TUNEL检测反应呈阳性,显示此时的纤维细胞核DNA发生了片段化。此时,在纤维细胞裂解的液泡膜、降解的细胞质和凝聚的染色质上具有ATPase活性。纤维细胞质的Ca^2＋水平会随着次生壁的形成而逐渐升高,随后Ca^2＋聚集成块状。在初生壁形成后期,纤维细胞染色质上的酸性磷酸酶（APase）活性增强。随着纤维次生壁的持续增厚,ATPase、酸性磷酸酶和Ca^2＋将在裂解的细胞质和凝聚的染色质上持续存在多年。结果表明,毛竹茎秆纤维细胞的次生壁形成过程是一个主动自溶的细胞程序性死亡过程。初生壁形成后期染色质上酸性磷酸酶活性增强及次生壁形成期胞质Ca^2＋的聚集,与纤维细胞的程序性死亡密切相关。ATPase,Ca^2＋和APase参与了纤维细胞程序性死亡过程中原生质体的降解。     

毛竹茎秆纤维细胞发育过程中的细胞程序性死亡(英文)

利用TUNEL检测、细胞学及细胞化学方法,对毛竹茎秆纤维细胞发育过程中的细胞程序性死亡进行了研究。在次生壁形成的早期,纤维细胞出现染色质凝聚、细胞器膨胀、液泡膜解体和细胞质泡状化等典型的细胞程序性死亡形态学特征;TUNEL检测反应呈阳性,显示此时的纤维细胞核DNA发生了片段化。此时,在纤维细胞裂解的液泡膜、降解的细胞质和凝聚的染色质上具有ATPase活性。纤维细胞质的Ca2+水平会随着次生壁的形成而逐渐升高,随后Ca2+聚集成块状。在初生壁形成后期,纤维细胞染色质上的酸性磷酸酶(APase)活性增强。随着纤维次生壁的持续增厚,ATPase、酸性磷酸酶和Ca2+将在裂解的细胞质和凝聚的染色质上持续存在多年。结果表明,毛竹茎秆纤维细胞的次生壁形成过程是一个主动自溶的细胞程序性死亡过程。初生壁形成后期染色质上酸性磷酸酶活性增强及次生壁形成期胞质Ca2+的聚集,与纤维细胞的程序性死亡密切相关。ATPase,Ca2+和APase参与了纤维细胞程序性死亡过程中原生质体的降解。     

百合花粉母细胞腺苷三磷酸酶反应产物的X-射线微区分析

百合早前期花粉母细胞经腺苷三磷酸酶反应处理后,一部分经过锇酸后固定和铀染色,一部分不经锇酸后固定和铀染色,其余的经锇酸固定,但不经铀染色,在此三种情况下,细胞质膜和染色质中都出现有致密的,电子不通透的沉淀。进一步的X-射线微区分析表明这些沉淀物中含有一定量的铅。X-射线微区分析结果也表明核膜和胞间连丝通道内部的酶反应沉淀中也含有铅,并且质膜、染色质和胞间连丝通道中酶反应沉淀中的铅较为丰富,细胞融合期染色质酶反应沉淀物中的铅含量较高,进入粗线期后,酶反应沉淀物中铅的含量下降。本研究结果表明百合早前期花粉母细胞的质膜、染色质、核膜及胞间连丝通道内部的确具有ATP酶活性;ATP酶在细胞融合过程中可能起重要作用。     

百合花粉母细胞间染色质穿壁运动前次生胞间连丝形成的研究

百合花粉母细胞间染色质穿壁运动前(细线期到偶线期)的花药,用一般电镜制片法和铅沉淀法对酸性磷酸酶活性的细胞化学反应产物的定位实验,其结果总结如下:(1)形成次生胞间连丝通道水解作用所需的酶可能是由“类溶酶体”小泡或由内质网腔直接分泌的;(2)次生胞间连丝通道的水解作用,可在细胞壁的两边细胞同时开始,先形成半胞间连丝,然后贯穿??在一起;或从一侧开始,一直穿孔到另一边,最后两者都能形成胞间连丝;(3)用铅沉淀法进行的酸性磷酸酶细胞化学的定位实验表明:在质膜、内质网、类溶酶体小泡中的酶活性反应产物沉积的部位与一般电镜法制备的切片上看到的电子致密度物质的分布情况完全一致,(4)用X-射线微区能谱分析的结果表明:沉淀物中含有铅元素,确实是磷酸铅。因此我们推测所谓“类溶酶体”以及内质网所分泌的水解酶,可能具有果胶酶、纤维素酶和半纤维素酶的性质,它们都能降解、穿孔各自的细胞壁形成胞间连丝。     

高温胁迫下外源钙对菊花叶片光合机构与活性氧清除酶系统的影响

以切花菊品种‘神马’为材料,研究了高温胁迫下外源Ca²⁺对菊花光合系统及活性氧清除酶系统的影响,探讨了Ca²⁺可能的作用机制.结果表明：高温胁迫下外源Ca²⁺抑制了净光合速率(P_n）与实际量子效率（Φ_PSⅡ）的大幅度降低,24 h后,二者分别比对照高出31.11％与21.88％,而初始荧光（F_o）比对照降低了13.19％;Ca²⁺明显激活了高温胁迫下叶片超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）的活性,活性氧得到及时清除,24 h后,膜脂过氧化产物丙二醛（MDA）的积累量与相对电导率（REC）分别比对照降低29.20％与35.81％,缓解了短期高温胁迫对菊花光合系统的破坏.     

毛竹茎纤维次生壁形成过程的超微结构观察

利用透射电镜观察了毛竹（Ｐｈｙｌｌｏｓｔａｃｈｙｓ ｐｕｂｅｓｃｅｎｓ Ｍａｚｅｌ）茎纤维发育过程中次生壁的形成过程。纤维发育早期,细胞具有较大的细胞核和核仁;细胞质浓稠,具有核糖体、线粒体和高尔基体等细胞器。随着纤维次生壁的形成,细胞壁加厚,细胞质变得稀薄,内质网和高尔基体的数量明显增加,并且两者共同参与了运输小泡的形成;在质膜内侧可观察到大量周质微管分布。随着次生壁的进一步加厚及木质化,细胞壁     

Mechanism and role of high-potassium-induced reduction of intracellular Ca2+ concentration in rat osteoclasts

Osteoclasts are multinucleated, bone-resorbing cells that show structural and functional differences between the resorbing and nonresorbing (motile) states during the bone resorption cycle. In the present study, we measured intracellular Ca²⁺ concentration ([Ca²⁺]_i) in nonresorbing vs. resorbing rat osteoclasts. Basal [Ca²⁺]_i in osteoclasts possessing pseudopodia (nonresorbing/motile state) was around 110 nM and significantly higher than that in actin ring-forming osteoclasts (resorbing state, around 50 nM). In nonresorbing/motile osteoclasts, exposure to high K⁺ reduced [Ca²⁺]_i, whereas high K⁺ increased [Ca²⁺]_i in resorbing state osteoclasts. In nonresorbing/motile cells, membrane depolarization and hyperpolarization applied by the patch-clamp technique decreased and increased [Ca²⁺]_i, respectively. Removal of extracellular Ca²⁺ or application of 300 µM La³⁺ reduced [Ca²⁺]_i to 50 nM in nonresorbing/motile osteoclasts, and high-K⁺-induced reduction of [Ca²⁺]_i could not be observed under these conditions. Neither inhibition of intracellular Ca²⁺ stores or plasma membrane Ca²⁺ pumps nor blocking of L- and N-type Ca²⁺ channels significantly reduced [Ca²⁺]_i. Exposure to high K⁺ inhibited the motility of nonresorbing osteoclasts and reduced the number of actin rings and pit formation in resorbing osteoclasts. These results indicate that in nonresorbing/motile osteoclasts, a La³⁺-sensitive Ca²⁺ entry pathway is continuously active under resting conditions, keeping [Ca²⁺]_i high. Changes in membrane potential regulate osteoclastic motility by controlling the net amount of Ca²⁺ entry in a "reversed" voltage-dependent manner, i.e., depolarization decreases and hyperpolarization increases [Ca²⁺]_i. membrane depolarization; resorbing and motile activities; bone resorbing cycle     

Effect of Cytoplasmic Ca2+ on the Membrane Potential and Membrane Resistance of Chara Plasmalemma

Effects of cytoplasmic Ca²⁺ on the electrical properties ofthe plasma membrane were investigated in tonoplast-free cellsof Chara australis that had been internally perfused with media,containing either 1 mM ATP to fuel the electrogenic pump orhexokinase and glucose to deplete the ATP and stop the pump. In the presence of ATP, cytoplasmic Ca²⁺ up to 2.5?10^–5M did not affect the membrane potential (about -190 mV), butmembrane resistance decreased uniformly with increasing [Ca²⁺]_i.In the absence of ATP, the membrane potential, which was onlyabout -110 mV, was depolarized further by raising [Ca²⁺]_i from1.4?10^–6 to 2.5?10^–5 M. Membrane resistance, whichwas nearly the twofold that of ATP-provided cells, decreasedmarkedly with an increase in [Ca²⁺]_i from zero to 1.38?10^–6M, but showed no change for further increases. Internodal cellsof Nitellopsis obtusa were more sensitive to intracellular Ca²⁺with respect to membrane potential than were those of Charaaustralis, reconfirming the results obtained by Mimura and Tazawa(1983). The effect of cytoplasmic Ca²⁺ on the ATP-dependent H⁺ effluxwas measured. No marked difference in H⁺ effluxes was detectedbetween zero and 2.5?10^–5 M [Ca²⁺]_i; but, at 10^–4M the ATP-dependent H⁺ efflux was almost zero. Ca²⁺ efflux experimentswere done to investigate dependencies on [Ca²⁺]_i and [ATP]_i.The efflux was about 1 pmol cm^–2 s^–1 at all [Ca²⁺]_iconcentrations tested (1.38?10^–6, 2.5?10^–5, 10^–4M).This value is much higher than the influx reported by Hayamaet al. (1979), and this efflux was independent of [ATP]_i. Thepossibility of a Ca²⁺-extruding pump is discussed. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, 5300 Bonn, F.R.G. (Received September 22, 1984; Accepted February 19, 1985)     

Calcineurin-independent regulation of plasma membrane Ca2+ ATPase-4 in the vascular smooth muscle cell cycle

Calcineurin mediates repression of plasma membrane Ca²⁺-ATPase-4 (PMCA4) expression in neurons, whereas c-Myb is known to repress PMCA1 expression in vascular smooth muscle cells (VSMC). Here, we describe a novel mouse VSMC line (MOVAS) in which ⁴⁵Ca efflux rates decreased 50%, fura 2-AM-based intracellular Ca²⁺ concentrations ([Ca²⁺]_i) increased twofold, and real-time RT-PCR and Western blot revealed a 40% decrease in PMCA4 expression levels from G₀ to G₁/S in the cell cycle, where PMCA4 constituted 20% of total PMCA protein. Although calcineurin activity increased fivefold as MOVAS progressed from G₀ to G₁/S, inhibition of this increase with either BAPTA or retroviral transduction with peptide inhibitors of calcineurin (CAIN), or its downstream target nuclear factor of activated T cells (NFAT) (VIVIT), had no effect on the repression of PMCA4 mRNA expression at G₁/S. By contrast, Ca²⁺-independent activity of the calmodulin-dependent protein kinase-II (CaMK-II) increased eightfold as MOVAS progressed from G₀ to G₁/S, and treatment with an inhibitor of CaMK-II (KN-93) or transduction of a c-Myb-neutralizing antibody significantly alleviated the G₁/S-associated repression of PMCA4. These data show that G₁/S-specific PMCA4 repression in proliferating VSMC is brought about by c-Myb and CaMK-II and that calcineurin may regulate cell cycle-associated [Ca²⁺]_i through alternate targets. calcineurin; c-Myb; plasma membrane Ca²⁺-ATPase-4; cell cycle     

Calcitonin gene-related peptide elevates calcium and polarizes membrane potential in MG-63 cells by both cAMP-independent and -dependent mechanisms

Published data suggest that the neuropeptide calcitonin gene-related peptide (CGRP) can stimulate osteoblastic bone formation; however, interest has focused on activation of cAMP-dependent signaling pathways in osteogenic cells without full consideration of the importance of cAMP-independent signaling. We have now examined the effects of CGRP on intracellular Ca²⁺ concentration ([Ca²⁺]_int) and membrane potential (E_m) in preosteoblastic human MG-63 cells by single-cell fluorescent confocal analysis using fluo 4-AM-fura red-AM and bis(1,3-dibarbituric acid)-trimethine oxanol [DiBAC₄(3)] bis-oxonol assays. CGRP produced a two-stage change in [Ca²⁺]_int: a rapid transient peak and a secondary sustained increase. Both responses were dose dependent with an EC₅₀ of 0.30 nM, and the maximal effect (initially 3-fold over basal levels) was observed at 20 nM. The initial phase was sensitive to inhibition of Ca²⁺ mobilization with thapsigargin, whereas the secondary phase was eliminated only by blocking transmembrane Ca²⁺ influx with verapamil or inhibiting cAMP-dependent signaling with the Rp isomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS). These data suggest that CGRP initially stimulates Ca²⁺ discharge from intracellular stores by a cAMP-independent mechanism and subsequently stimulates Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channels by a cAMP-dependent mechanism. In addition, CGRP dose-dependently polarized cellular E_m, with maximal effect at 20 nM and an EC₅₀ of 0.30 nM. This effect was attenuated with charybdotoxin (–20%) or glyburide (glibenclamide; –80%), suggesting that E_m hyperpolarization is induced by both Ca²⁺-activated and ATP-sensitive K⁺ channels. Thus CGRP signals strongly by both cAMP-dependent and cAMP-independent signaling pathways in preosteoblastic human MG-63 cells. osteoblastic cells; calcium; membrane potential; potassium channels; adenosine 3',5'-cyclic monophosphate     

An Excel-based model of Ca2+ diffusion and fura 2 measurements in a spherical cell

We wrote a program that runs as a Microsoft Excel spreadsheet to calculate the diffusion of Ca²⁺ in a spherical cell in the presence of a fixed Ca²⁺ buffer and two diffusible Ca²⁺ buffers, one of which is considered to be a fluorescent Ca²⁺ indicator. We modeled Ca²⁺ diffusion during and after Ca²⁺ influx across the plasma membrane with parameters chosen to approximate amphibian sympathetic neurons, mammalian adrenal chromaffin cells, and rat dorsal root ganglion neurons. In each of these cell types, the model predicts that spatially averaged intracellular Ca²⁺ activity ([Ca²⁺]_avg) rises to a high peak and starts to decline promptly on the termination of Ca²⁺ influx. We compared [Ca²⁺]_avg with predictions of ratiometric Ca²⁺ measurements analyzed in two ways. Method 1 sums the fluorescence at each of the two excitation or emission wavelengths over the N compartments of the model, calculates the ratio of the summed signals, and converts this ratio to Ca²⁺ ([Ca²⁺]_avg,M1). Method 2 sums the measured number of moles of Ca²⁺ in each of the N compartments and divides by the volume of the cell ([Ca²⁺]_avg,M2). [Ca²⁺]_avg,M1 peaks well after the termination of Ca²⁺ influx at a value substantially less than [Ca²⁺]_avg because the summed signals do not reflect the averaged free Ca²⁺ if the signals come from compartments containing gradients in free Ca²⁺ spanning nonlinear regions of the relationship between free Ca²⁺ and the fluorescence signals. In contrast, [Ca²⁺]_avg,M2 follows [Ca²⁺]_avg closely. intracellular calcium; kinetic model; diffusion coefficient; fura 2ff; furaptra