Sclerostin Immunoreactivity Increases in Cortical Bone Osteocytes and Decreases in Articular Cartilage Chondrocytes in Aging Mice

Sclerostin is a 24-kDa secreted glycoprotein that has been identified as a negative modulator of new bone formation and may play a major role in age-related decline in skeletal function. Although serum levels of sclerostin markedly increase with age, relatively little is known about whether cells in the skeleton change their expression of sclerostin with aging. Using immunohistochemistry and confocal microscopy, we explored sclerostin immunoreactivity (sclerostin-IR) in the femurs of 4-, 9-, and 24-month-old adult C3H/HeJ male mice. In the femur, the only two cell types that expressed detectable levels of sclerostin-IR were bone osteocytes and articular cartilage chondrocytes. At three different sites along the diaphysis of the femur, only a subset of osteocytes expressed sclerostin-IR and the percentage of osteocytes that expressed sclerostin-IR increased from approximately 36% to 48% in 4- vs. 24-month-old mice. In marked contrast, in the same femurs, there were ~40% fewer hypertrophic chondrocytes of articular cartilage that expressed sclerostin-IR when comparing 24- vs. 4-month-old mice. Understanding the mechanism(s) that drive these divergent changes in sclerostin-IR may provide insight into understanding and treating the age-related decline of the skeleton.     

Induction of clusterin Expression by Neuronal Cell Death in Zebrafish

Clusterin, a protein associated with multiple functions, is expressed in a wide variety of mammalian tissues. Although clusterin is known to be involved in neurodegenerative diseases, ageing, and tumorigenesis, a detailed analysis of the consequences of gain- or loss-of-function approaches has yet to be performed to understand the underlying mechanisms of clusterin functions. Since clusterin levels change in neurological diseases, it is likely that clusterin contributes to cell death and degeneration in general. Zebrafish was investigated as a model system to study human diseases. During development, zebrafish clusterin was expressed in the notochord and nervous system. Embryonic overexpression of clusterin by mRNA microinjection did not affect axis formation, whereas its knock-down by anti-sense morpholino treatment resulted in neuronal cell death. To analyze the function of clusterin in neurodegeneration, a transgenic zebrafish was investigated, in which nitroreductase expression is regulated under the control of a neuron-specific huC promoter which is active between the stages of early neuronal precursors and mature neurons. Nitroreductase turns metronidazole into a cytotoxic agent that induces cell death within 12 h. After metronidazole treatment, transgenic zebrafish showed neuron-specific cell death. Interestingly, we also observed a dramatic induction of clusterin expression in the brain and spinal cord in these fish, suggesting a direct or indirect role of clusterin in neuronal cell death and thus, more generally, in neurodegeneration.     

Staurosporine Induces Programmed Cell Death in Embryonic Neurons and Activation of the Ceramide Pathway

Abstract: We activated the death pathway in embryonic chick cerebral hemisphere neuron (E7CH) cultures with staurosporine (0.1–1.0 µ M ) and observed the morphological changes, DNA laddering patterns, and DNA fragmentation (determined by Hoechst 33258 dye) associated with apoptosis. N -Acylsphingosine (C2-ceramide), a soluble ceramide analogue, was also able to induce apoptosis in these cells with the same characteristics and in the same time frame. We then observed that staurosporine was effective in inducing hydrolysis of sphingomyelin to ceramide as measured by a threefold increase in ceramide mass and increased incorporation of [³H]-palmitate into ceramide, concurrent with activating the cell death program. Furthermore, the coaddition of a specific ceramidase inhibitor, oleoylethanolamine (15 µ M ), enhanced the formation of ceramide as well as the degree of DNA fragmentation and cell death. Exogenous addition of sphingomyelinase activated the death pathway whereas ceramide glycanase did not, and inhibitors of sphingomyelin or protein synthesis failed to block this type of killing. Our data suggest that the formation of ceramide from sphingomyelin is a key event in staurosporine-induced and potentially all programmed cell death.     

环境胁迫诱导的植物细胞程序性死亡

在最近的10年中,兴起了对植物细胞程序性死亡的研究。大量的证据表明,在各种环境胁迫因子诱导植物细胞PCD过程中,活性氧、乙烯、Ca2+、水杨酸、NO等成为重要的信号分子。像动物细胞凋亡一样,在植物PCD中也存在一条依赖于天冬氨酸特异性半胱氨酸蛋白酶(Caspases)活性的信号传导途径,其中,线粒体处于PCD调控的中心位置。 Abstract:Programmed cell death (PCD) research in higher plants has blossomed in the past ten years.Many evidences suggested that reactive oxygen species,ethylene,Ca2+,salicylic acid,nitric oxide etc.are important signal molecules during environmental stress-induced PCD in plants.Like apoptosis in animals,there also exists a Caspase-dependent PCD signal transduction pathway,in which mitochondrion plays a role of central depot.     

Hypoxia-induced Cell Death and Activation of Pro- and Anti-apoptotic Proteins in Developing Chick Optic Lobe

Exposure of the CNS to hypoxia is associated with cell death. Our aim was to establish a temporal correlation between cellular and molecular alterations induced by an acute hypoxia evaluated at different post-hypoxia (p-h) times and at two stages of chick optic lobe development: embryonic days (ED) 12 and 18. TUNEL assays at ED12 disclosed a significant increase (300%) in pyknotic cells at 6 h p-h, while at ED18 no morphological changes were observed in hypoxic versus controls. At ED12 there was a significant increase (48%) in Bcl-2 levels at the end of the hypoxic treatment, followed by a significant increase of active caspase-9 (49%) and active caspase-3 (58%) at 30 and 60 min p-h, respectively, while at ED18 no significant changes were observed. These findings indicate that prenatal hypoxia produces an equilibrated imbalance in both pro- and anti-apoptotic proteins that culminates in a process of cell death, present at earlier stages of development.     

死亡信号传递: 叶绿体与线粒体间信号交流调控植物程序性细胞死亡

程序性细胞死亡(PCD)是生物体受遗传调控的自主细胞死亡现象, 在植物生长发育和抵抗环境胁迫中起重要作用。PCD的发生可受线粒体中活性氧(ROS)诱导。中国科学院遗传与发育生物学研究所李家洋研究组早期的研究发现了1个拟南芥(Arabidopsis thaliana)细胞死亡突变体mod1, 并暗示植物细胞中存在叶绿体与线粒体之间的信号交流调控PCD, 但其中的具体作用机制尚不清楚。最近, 他们通过大规模筛选mod1突变体的抑制突变体, 克隆了3个新的抑制基因plNAD- MDH、DiT1和mMDH1。此3个基因分别编码质体定位的NAD依赖的苹果酸脱氢酶、叶绿体被膜定位的二羧酸转运蛋白1和线粒体定位的苹果酸脱氢酶1, 突变后都可抑制mod1中ROS的积累及PCD的发生。通过对这些基因进行深入的功能分析, 他们论证了苹果酸从叶绿体到线粒体的转运对线粒体中ROS的产生及随后PCD的诱导起重要作用。该研究拓展了我们对植物细胞中细胞器间交流的认识, 为我们深入理解植物PCD发生机制提供了新线索, 是该领域的一项突破性进展。     

Activation of Caspase-3 in Developmental Models of Programmed Cell Death in Neurons of the Substantia Nigra

Programmed cell death has been proposed to play a role in the death of neurons in acute and chronic degenerative neurologic disease. There is now evidence that the caspases, a family of cysteine proteases, mediate programmed cell death in various cells. In neurons, caspase-3 (CPP32/Yama/apopain), in particular, has been proposed to play a role. We examined the expression of caspase-3 in three models of programmed cell death affecting neurons of the substantia nigra in the rat: natural developmental neuron death and induced developmental death following either striatal target injury with quinolinic acid or dopamine terminal lesion with intrastriatal injection of 6-hydroxydopamine. Using an antibody to the large (p17) subunit of activated caspase-3, we have found that activated enzyme is expressed in apoptotic profiles in all models. Increased p17 immunostaining correlated with increased enzyme activity. The subcellular distribution of activated caspase-3 differed among the models: In natural cell death and the target injury model, it was strictly nuclear, whereas in the toxin model, it was also cytoplasmic. We conclude that p17 immunostaining is a useful marker for programmed cell death in neurons of the substantia nigra.     

咪喹莫特调节Th1、Th2细胞相关趋化因子表达抑制兔耳瘢痕增生

目的:免疫因素在增生性瘢痕的发生中起重要作用,本实验研究免疫抑制剂咪喹莫特对兔耳增生性瘢痕组织中辅助性T淋巴(Th)细胞亚群Th1、Th2细胞相关趋化因子CXCL10、CXCL12、CCL2、CCL3、CCL5、CCL7、CCL13表达的影响,探讨咪喹莫特抑制兔耳瘢痕增生的作用机制。方法:选取16只新西兰大耳白兔,雌雄不限。建立兔耳增生性瘢痕模型,每只兔耳腹侧做四个直径为1 cm的圆形创面,每个相距1.5 cm,双侧对称,右耳为咪喹莫特组涂抹5%咪喹莫特软膏,左耳为空白对照组涂抹等量凡士林软膏,待术后14天上皮化均完全后开始涂抹,一日一次,持续一个月。分别于术后第21、28、35、42、49、56、63天同一时间空气栓塞法随机处死2只兔子,收集所有瘢痕标本。另外处死2只健康兔并采集兔耳正常皮肤组织。所有标本行HE染色及Masson三色法染色,观察形态学差异;测量并计算瘢痕增生指数(Scar elevation index,SEI);行Real-time PCR检测CXCL10、CXCL12、CCL2、CCL3、CCL5、CCL7、CCL13的表达。结果:HE及Masson染色可见咪喹莫特组胶原沉积较空白对照组明显减少,SEI显示空白对照组于术后第28天增生程度达到高峰,其增生程度明显高于咪喹莫特组(P0.05);Real-time PCR结果可见咪喹莫特组Th2细胞相关趋化因子CCL2、CCL3、CCL5、CCL7及CCL13表达在各时间点较空白对照组明显降低,Th1细胞相关趋化因子CXCL10、CXCL12的表达在各时间点较空白对照组明显增高(P0.05)。结论:咪喹莫特可通过调节Th1、Th2细胞相关趋化因子的表达来发挥抑制兔耳瘢痕增生的作用。     

An Early and Robust Activation of Caspases Heads Cells for a Regulated Form of Necrotic-like Cell Death

Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as “apoptosis-necrosis continuum.” To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death.     

Intracellular features of type II procollagen and chondroitin sulfate proteoglycan synthesis in chondrocytes

The intracellular compartments of chondrocytes involved in the synthesis and processing of type II procollagen and chondroitin sulfate proteoglycan (CSPG) monomer were investigated using simultaneous double immunofluorescence and lectin localization reactions. Type II procollagen was distributed in vesicles throughout the cytoplasm, whereas intracellular precursors of CSPG monomer were accumulated in the perinuclear cytoplasm. In this study, cytoplasmic vesicles that stained intensely with antibodies directed against CSPG monomer but did not react with type II collagen antibodies, also were observed. A monoclonal antibody, 5-D-4, that recognizes keratan sulfate determinants was used to identify the Golgi complex (the site of keratan sulfate chain elongation). Staining with 5-D-4 was restricted to the perinuclear cytoplasm. The vesicles outside the perinuclear cytoplasm that stained intensely with antibodies to CSPG monomer did not react with 5-D-4. Fluorescent lectins were used to characterize further subcellular compartments. Concanavalin A, which reacts with mannose-rich oligosaccharides, did not stain the perinuclear region, but it did stain vesicles throughout the rest of the cytoplasm. Because mannose oligosaccharides are added cotranslationally, the stained vesicles throughout the cytoplasm presumably correspond to the rough endoplasmic reticulum. Wheat germ agglutinin, which recognizes N-acetyl-D-glucosamine and sialic acid (carbohydrates added in the Golgi), stained exclusively the perinuclear cytoplasm. By several criteria (staining with the monoclonal antibody 5-D-4 and with wheat germ agglutinin), the perinuclear cytoplasm seems to correspond to the Golgi complex. The cytoplasmic vesicles that react with anti-CSPG monomer and not with anti-type II collagen contain precursors of CSPG monomer not yet modified by Golgi-mediated oligosaccharide additions (because they are not stained with wheat germ agglutinin or with the anti-keratan sulfate antibody); these vesicles may have a unique function in the processing of CSPG.     

铝诱导植物程序性细胞死亡信号转导的研究进展

铝是制约酸性土壤上作物生产的主要因素。铝诱导氧化胁迫产生大量活性氧/一氧化氮,引起胞质钙超载,通过线粒体信号转导途径激发相关凋亡基因,从而引起细胞主动死亡,以减轻铝对植物的进一步毒害。本文综述了铝诱导程序性细胞死亡的信号分子、相关基因以及信号转导途径,对未来的研究方向提出了展望,为深入研究植物铝毒害机理和耐铝机制提供参考。     

Mitochondrial Implication in Accidental and Programmed Cell Death: Apoptosis and Necrosis

Both physiological cell death (apoptosis) and at least some cases of accidental cell death (necrosis) involve a two-step-process. At a first level, numerous physiological or pathological stimuli can trigger mitochondrial permeability transition which constitutes a rate-limiting event and initiates the common phase of the death process. Mitochondrial permeability transition (FT) involves the formation of proteaceous, regulated pores, probably by apposition of inner and outer mitochondrial membrane proteins which cooperate to form the mitochondrial PT pore complex. Inhibition of PT by pharmacological intervention on mitochondrial structures or mitochondrial expression of the apoptosis-inhibitory oncoprotein Bcl-2 thus can prevent cell death. At a second level, the consequences of mitochondrial dysfunction (collapse of the mitochondrial transmembrane potential, uncoupling of the respiratory chain, hyperproduction of superoxide anions, disruption of mitochondrial biogenesis, outflow of matrix calcium and glutathione, and release of soluble intermembrane proteins) can entail a bioenergetic catastrophe culminating in the disruption of plasma membrane integrity (necrosis) and/or the activation and action of apoptogenic proteases with secondary endonuclease activation and consequent oligonucleosomal DNA fragmentation (apoptosis). The acquisition of the biochemical and ultrastructural features of apoptosis critically relies on the liberation of apoptogenic proteases or protease activators from the mitochondrial intermembrane space. This scenario applies to very different models of cell death. The notion that mitochondrial events control cell death has major implications for the development of death-inhibitory drugs.     

植物细胞程序性死亡分子机制和信号转导

细胞程序化死亡(PCD)在植物的发育、抗病及植物与环境互作等过程中发挥着极其重要的作用.总结了植物细胞凋亡发生的特征及其检测技术,概括了植细胞凋亡的分子机制,综述了植物细胞凋亡相关激素种类及其信号转导机制,并对植物细胞凋亡存在的问题进行分析和展望.     

Underlying the Mechanisms of Doxorubicin-Induced Acute Cardiotoxicity: Oxidative Stress and Cell Death

Cancer is a destructive disease that causes high levels of morbidity and mortality. Doxorubicin (DOX) is a highly efficient antineoplastic chemotherapeutic drug, but its use places survivors at risk for cardiotoxicity. Many studies have demonstrated that multiple factors are involved in DOX-induced acute cardiotoxicity. Among them, oxidative stress and cell death predominate. In this review, we provide a comprehensive overview of the mechanisms underlying the source and effect of free radicals and dependent cell death pathways induced by DOX. Hence, we attempt to explain the cellular mechanisms of oxidative stress and cell death that elicit acute cardiotoxicity and provide new insights for researchers to discover potential therapeutic strategies to prevent or reverse doxorubicin-induced cardiotoxicity.     

Activation of Cysteine Proteases in Cowpea Plants during the Hypersensitive Response-A Form of Programmed Cell Death

There is increasing evidence that the hypersensitive response during plant–pathogen interactions is a form of programmed cell death. In an attempt to understand the biochemical nature of this form of programmed cell death in the cowpea–cowpea rust fungus system, proteolytic activity in extracts of fungus-infected and uninfected cowpea plants was investigated, using exogenously added poly(ADP-ribose) polymerase as a marker. Unlike the proteolytic cleavage pattern of endogenous poly(ADP-ribose) polymerase in apoptotic animal cells, exogenously added poly(ADP-ribose) polymerase in extracts of fungus-infected plants was proteolytically cleaved into fragments of molecular masses 77, 52, 47, and 45 kDa.In vitroandin vivoprotease inhibitor experiments revealed the activation of cysteine proteases, and possibly a regulatory role, during the hypersensitive response.     

白藜芦醇以Caspase依赖和非依赖两种方式导致Jurkat细胞死亡

应用形态学观察、流式细胞仪检测、Western印迹和DNA凝胶电泳等方法研究白藜芦醇对Jurkat细胞的作用。发现白藜芦醇处理组中细胞有皱缩、出泡、染色质边集等现象,但染色质浓缩呈散在团块状且不致密。细胞质结构疏松,线粒体肿胀,脊消失。少见凋亡小体。在白藜芦醇处理组,Western印迹可检测到弱的17kDacaspase-3条带,DNA凝胶电泳可以检测到梯状DNA和弥散条带;流式细胞仪在白藜芦醇处理组检测到大量PI单阳性细胞和少量膜联蛋白V单阳性细胞。Z-VAD-FMK干预后可以发现细胞死亡率降低,同时该组梯状DNA消失,但是大分子量弥散DNA条带依然可以检测到。结果表明白藜芦醇可以通过caspase依赖和非依赖途径导致Jurkat细胞死亡。此分子机制的明确将为白藜芦醇应用于临床白血病的治疗打下理论基础。     

壳寡糖诱导水稻过敏性细胞死亡及抗病性的提高

作为真菌细胞壁的主要成分之一的壳寡糖(Oligo-GlcNAc)能够诱导水稻悬浮细胞和幼叶细胞发生过敏性死亡,并伴有H2O2的积累.以1 μg*mL-1壳寡糖处理水稻悬浮细胞12 h后细胞明显死亡;诱导水稻幼叶细胞出现明显的死亡所需壳寡糖浓度为5 μg*mL-1.以壳寡糖处理的水稻抗稻瘟病性也明显增强.