低温胁迫诱导玉米根尖细胞凋亡的形态和生化证据

近来体外培养细胞体系研究的结果表明,一定条件的低温胁迫可导致植物细胞凋亡。本文利用ＤＮＡ梯状图谱（ＤＮＡｌａｄｄｅｒｉｎｇ）以及染色体涂片的ＴＵＮＥＬ（ｔｅｒｍｉｎｄａｌ ｄｅｏｘｙｎｕｃｌｅｏｔｉｄｙｌ ｔｒａｎｓｆｅｒａｓｅ－ｍｅｄｉａｔｅｄ ｄＵＴＰｎｉｃｋ ｅｎｄ－ｌａｂｅｌｉｎｇ）原位检测,从原位以及生化水平和对低温胁迫下的玉米根尖细胞死亡作了研究,形态和生化方面的语气表明,同体外一样,     

盐胁迫诱导的植物细胞凋亡--植物抗盐的可能生理机制

近来的研究表明,一定条件的盐胁迫可导致植物细胞程序性死亡。本文利用DNALaddering、石蜡切片原位检测以及染色体涂片原位检测,从组织、细胞以及DNA等多个方面对盐胁迫下的玉米、水稻和烟草根尖细胞死亡作了研究,形态特别是生化方面的证据表明盐胁迫诱导的植物细胞凋亡可能在植物界具有一定的普遍性。但各个物种之间有一定差异。本实验结果对盐胁迫下的植物生理机制提供了新的研究思路。同时,我们还对基于染色体制片和石蜡切片的原位检测方法进行了比较和讨论。我们认为,基于染色体制片的原位标记技术适合于定性和定量检测单个细胞的凋亡,具有一些石蜡切片所不可及的优点。     

药物诱导的玉米根尖细胞凋亡

同时应用ＤＮＡ Ｌａｄｄｅｒｉｎｇ、ＤＮＡ Ｇｅｌ Ｂｌｏｔ以及基于染色体涂片 原位末端标记技术,从染色体、细胞核和ＤＮＡ不同水平对细胞毒素类药和的放线菌Ｄ、放线菌酮和秋水仙碱诱导的玉米（Ｚｅａ ｍａｙｓ Ｌ．）根尖分生组织细胞死亡作了检测。结果表明：同动物中一样,这些药物诱导的玉米根尖分生组织细胞死亡也具有ＤＮＡ Ｌａｄｄｅｒ、染色质和细胞核浓缩等典型的调亡特征,说明这些细胞毒素类药物能够诱导植     

渗透胁迫诱导的小麦叶片细胞程序性死亡

用20％PEG6000（－0.63MPa）溶液对小麦（Triticum aestivum)根系进行渗透胁迫,在DNA琼脂糖凝胶电泳图谱上观察到明显的梯状DNA条带,表明PEG处理诱发了DNA核小体间的断裂,从而表现出典型的细胞程序性死亡的发生特征;末端脱氧核糖核酸转移酶介导的3′-OH末端标记法（terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end labeling,TUNEL)检测出现阳性结果。这些结果表明在PEG诱导小麦叶片死亡过程中,有一个阶段存在明显的细胞程序性死亡过程。结果同时表明,在小麦叶片处于程序性死亡时期,蛋白质含量、叶绿素含量的下降和电解质泄漏率的增加都比处于坏死时期的慢。     

离子胁迫诱导洋葱鳞茎内表皮细胞凋亡

通过不同浓度离子胁迫诱导剂(NaCl、CaCl2)对洋葱鳞茎内表皮细胞进行不同时间的处理,发现0.1M、0.5M的NaCl和CaCl2处理2小时即可诱导出细胞凋亡现象,随处理时间延长直至10小时,细胞核凋亡的形态学变化和凋亡小体更加明显,基因组DNA降解更加梯状条带化。本实验对离子诱导的洋葱鳞茎内表皮细胞凋亡现象做了较系统的描述,为植物细胞凋亡的研究及细胞凋亡实验教学提供了经济、快捷、有效的诱导方法。     

药物诱导的玉米根尖凋亡细胞表面电特性研究

细胞凋亡过程中细胞表面膜的电位很可能会发生改变。本文首次报导：应用细胞电泳技术（cell electrophoresis)对细胞毒素类药物放线菌酮（cycloheximide)、放线菌素D（actinomycin D）和秋水仙碱（colchicine)等诱导的植物凋亡的细胞与正常细胞之间电泳迁移率（EPM）的差异进行了比较,对引起的膜电位变化进行了定量分析。实验以玉米根尖分生组织为材料,制备原生质体,经过适当剂量的药物处理（Fig.1-B),在尽量减少细胞膜被破坏的情况下（Fig.2),观察到：三种细胞毒素类药物的作用有所不同。被诱导的植物凋亡细胞的膜表面Zeta电位绝对值比正常细胞的高（Fig.1-A)。本研究提示细胞电泳可对凋亡细胞表面膜电位的变化进行定量分析,为细胞凋亡的检测在方法上提供了新思路。     

低温对紫外照射诱导人乳腺癌细胞凋亡的影响

采用Ｈｏｅｃｈｓｔ ３３２５８荧光染色技术,透射电镜,流式细胞术研究低温对紫外照射诱导体外培养的人乳腺癌细胞凋亡的影响。结果显示：４℃低温处理可增强紫外照射诱导人乳腺癌细胞凋亡,且低温增强细胞凋亡具有时间效应和剂量效应关系。细胞经４℃处理０,１２,２４ｈ并照射６ｍｉｎ,在培养６ｈ后各实验组细胞的调亡率开始升高,与培养０ｈ时相比差异显著（Ｐ〈０．０１）,培养１２￣１８ｈ时达到高峰（１５．４６－６４．     

TUNEL法检测Cu~(2+)胁迫诱导的中国树花共生藻细胞的死亡

为了探讨铜离子胁迫对中国树花共生藻细胞毒害作用的机制及细胞死亡的原因,通过徒手切片,采用两种TUNEL检测法(Promega和Roche试剂盒)对2mmol/L和4mmol/L Cu~(2+)胁迫24h的中国树花地衣体共生藻细胞凋亡情况进行观察。结果表明:(1)伊文思蓝染色法检测结果显示,中国树花共生藻细胞活力在2和4mmol/L Cu~(2+)胁迫下显著降低,其细胞的死亡率随着Cu~(2+)浓度的提高和处理时间的延长而明显增加;(2)在2和4mmol/L Cu~(2+)胁迫条件下,中国树花共生藻细胞的TUNEL阳性细胞率在Promega试剂盒中检测结果分别为50.30%和31.21%,而在Roche试剂盒中共生藻细胞TUNEL阳性标记核分别为53.17%和36.88%,两种TUNEL检测法结果类似。(3)与伊文思蓝染色法检测的Cu~(2+)胁迫中国树花共生藻细胞活力相比,发现较低浓度的Cu~(2+)(2mmol/L)胁迫对地衣共生藻细胞的毒害作用可诱导启动细胞凋亡程序,而较高浓度的Cu~(2+)(4mmol/L)胁迫对地衣共生藻产生较严重的毒害则导致大部分细胞的坏死,只有极少数细胞出现细胞凋亡。(4)两种试剂盒均可用于较低浓度Cu~(2+)胁迫引起的地衣体共生藻细胞凋亡的检测;直接将徒手切片材料用于TUNEL细胞凋亡原位检测中也得到了较理想的结果,避免了制备石蜡切片的繁琐步骤,缩短实验时间,简化了实验流程。     

硝基苯对蚕豆根尖细胞凋亡的影响

细胞凋亡(apoptosis)是一自然生理过程。是由一个主动由基因决定的自动结束生命的过程。由于细胞凋亡受到严格的由遗传机制决定的程序性调控,所以也常常被称为细胞程序性死亡(PCD,programmed cell death)。植物在正常发育时也会发生细胞程序性死亡,     

羟自由基诱导烟草细胞凋亡

用适当浓度组合的硫酸亚铁和过氧化氢反应以产生羟自由基来处理烟草悬浮细胞,首次观察到了细胞中染色质凝缩、边缘化、细胞核解体等典型的细胞凋亡的形态学特征;在ＤＮＡ 琼脂糖凝胶电泳图谱上观察到因核ＤＮＡ 有规则地在核小体间被降解而产生的阶梯状条带;末端脱氧核糖核酸转移酶介导的３’ＯＨ 末端标记法（ｔｅｒｍｉｎａｌ ｄｅｏｘｙｎｕｃｌｅｏｔｉｄｙｌ ｔｒａｎｓｆｅｒａｓｅ （ＴｄＴ）ｍｅｄｉａｔｅｄ ｄＵＴＰ ｎｉｃｋｅｎｄｌａｂｅｌｉｎｇ,ＴＵＮＥＬ） 检测结果为阳性。这些实验表明,羟自由基确能诱导烟草细胞凋亡。     

植物细胞凋亡的ELISA检测(英文)

本文利用ＴＵＮＥＬ方法在单个细胞水平上对苯胺灵诱导的玉米根尖细胞死亡进行了检测。结果表明,一定浓度的苯胺灵能诱导玉米根尖细胞发生主动性的细胞凋亡,具有细胞凋亡典型的形态和生化特征即细胞核浓缩并形成核碎片、染色质边缘化及ＤＮＡ特异片段化等（Ｆｉｇ．１）。首次利用ＥＬＩＳＡ方法在群体水平上对这种细胞凋亡进行了进一步检测。结果表明：动物细胞研究常用的ＥＬＩＳＡ方法同样也适合用于植物细胞凋亡的检测。在凋亡前期,随着凋亡过程的进行,细胞质中的ＯＤ值逐渐上井（Ｆｉｇ．２）。剂量实验表明,０．２ｍｇ／ｍＬ苯胺灵最适合于诱导细胞凋亡（Ｆｉｇ．３）。     

药物诱导的玉米根尖凋亡细胞表面电特性研究(英文)

细胞凋亡过程中细胞表面膜的电位很可能会发生改变。本文首次报导：应用细胞电泳技术（ｃｅｌｌ ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ）对细胞毒素类药物放线菌酮（ｃｙｃｌｏｈｅｘｉｍｉｄｅ）、放线菌素 Ｄ（ａｃｔｉｎｏｍｙｃｉｎ Ｄ）和秋水仙碱（ｃｏｌｃｈｉｃｉｎｅ）等诱导的植物凋亡细胞与正常细胞之间电泳迁移率（ＥＰＭ）的差异进行了比较,对引起的膜电位变化进行了定量分析。实验以玉米根尖分生组织为材料,制备原生质体,经过适当剂量的药物处理（Ｆｉｇ．１－Ｂ）,在尽量减少细胞膜被破坏的情况下（Ｆｉｇ．２）,观察到：三种细胞毒素类药物的作用有所不同,被诱导的植物凋亡细胞的膜表面Ｚｅｔａ电位绝对值比正常细胞的高（Ｆｉｇ．１－Ａ）。本研究提示细胞电泳可对凋亡细胞表面膜电位的变化进行定量分析,为细胞凋亡的检测在方法上提供了新思路。     

Cold stress induced high molecular weight membrane polypeptides are responsible for cold tolerance in Rhizobium DDSS69

Cold stress induces a lag phase in the growth cycle of Rhizobium DDSS69. Two cold sensitive mutants of DDSS69 were generated through Tn5 tagged mutagenesis. These mutants do not grow below 15 degrees C but show a growth curve comparable with the wild type grown at 5 degrees C. There is a rapid induction of two high molecular weight membrane polypeptides of 135 and 119 kDa within 15 min of exposure to 5 degrees C in DDSS69. PAGE membrane protein profiles of stressed and non-stressed cells reveal differential regulation of genes. At 15 degrees C both mutants lack the high molecular weight polypeptides, suggesting a role in alleviation of cold stress.     

Ultrastructural Distribution of DNA within Plant Meristematic Cell Nucleoli during Activation and the Subsequent Inactivation by a Cold Stress

We have investigated the precise location of DNA within the meristematic cell nucleolus ofZea maysroot cells andPisum sativumcotyledonary buds, in the course of their activation and induced inactivation following a subsequent treatment at low temperature. For this purpose, we combined the acetylation method, providing an excellent distinction between the various nucleolar components, with thein situterminal deoxynucleotidyl transferase-immunogold technique, a highly sensitive method for detecting DNA at the ultrastructural level. In addition to the presence of DNA in the condensed chromatin associated with the nucleolus, we demonstrated that a significant label was detected in the nucleolus of quiescent cells in both plant models. Evident labels were also found in the dense fibrillar component of actived nucleoli. Whereas in inactivated nucleoli no significant label was observed within the dense fibrillar component, an intense label was seen over the large heterogeneous fibrillar centres only during inactivation. The granular component was never significantly labelled. These results appear to indicate that the DNA present in the dense fibrillar component of activated nucleoli withdraws from this structure during its inactivation and becomes incorporated in the large fibrillar centres. These observations suggest that in plant cells inactivation of rRNA genes is clearly accompanied by changes in the conformation of ribosomal chromatin.     

Apoptosis induced by progesterone in human ovarian cancer cell line SNU-840

Progesterone has been used as an ingredient of anticancer drug for patients with ovarian carcinoma. However, the mechanism of anticancer effects by progesterone has not been understood. In this study, the effects of progesterone on ovarian cancer cells, SNU-840, were investigated. After the incubation with progesterone, the viability of the cells was evaluated by MTT assay. As a result, 45% of the cells were viable after 48 h of incubation with 100 microM progesterone. In addition, [(3)H]thymidine incorporation assay showed that the proliferation of the cells was completely inhibited by progesterone after 48 h of incubation at 100 microM concentration. Colorimetric TUNEL assay revealed the fragmentation of the chromosomal DNA, suggesting that the process of the cell death was apoptosis. The level of the p53 mRNA was determined by northern blotting assay, since many apoptosis processes are mediated by up-regulation of the p53 expression. The level of the p53 mRNA reached its maximum at 12 h and decreased after 24 h of incubation with progesterone. In conclusion, progesterone inhibits the proliferation and elicits apoptosis of SNU-840 cells. Also, it up-regulates the p53 mRNA transiently.     

Salt Stress Induced Nuclear and DNA Degradation in Meristematic Cells of Barley Roots

Root growth of barley (Hordeum vulgare L., cv. Akashinriki)was inhibited by 200 raM NaCl, when 1 mM CaCl₂ was present inthe hydroponic culture solution. Increasing the CaCl₂ up to10 mM partially prevented this inhibition. However, inhibitionalso occurred with 100 mM NaCl in the presence of 0.1 mM CaCl₂.The nuclei of meristematic cells in roots in which growth hadbeen inhibited by salt stress were studied after staining withDAPI (4',6-diamino-2-phenylindol). Nuclear deformation of thecells occurred with 12 h of salt stress with 500 mM NaCl, andwas followed by degradation. The nuclear degradation was alsoobserved when the roots were exposed to more than 300 mM NaClfor 24 h. Biochemical analysis revealed that nuclear degradationwas accompanied by apoptosis-like DNA fragmentation. The intracellularmechanisms of nuclear degradation in cells after salt stressare discussed. ¹Emertius professor, Okayama University.     

Cold stress and cold adaptation

1. 1. Results from more than half a century of investigation of human adaptation to cold have been so varied that some observers have doubted whether man can adapt to cold at all.

2. 2. This paper considers what challenges to the thermoregulatory system humans experience when living and working in a cold environment (specifically the Antarctic and Subantarctic), what kinds of adaptation have been shown to develop, and what factors might have contributed to the diversity of opinion.