Microwave irradiation of paraffin-embedded tissue sensitizes the TUNEL method for in situ detection of apoptotic cells

Apoptosis is a morphologically distinct form of programmed cell death that plays an important role in the growth regulation of a variety of tissues and also in the elimination of self-reacting immunocompetent cells. Several techniques for the qualitative and quantitative detection of this process have been established; recently, an in situ nick end-labelling technique based on the detection of DNA fragmentation, which is a molecular characteristic of apoptotic cell death, was described. Applying this method to paraffin sections of human tissues, sensitivity was observed to be inconsistently low with regard to the expected number of apoptotic cells. In the present study we show that irradiation of the tissue sections in 10 mM citrate buffer, pH 6.0, by microwaves at 750 W considerably enhances the sensitivity of this nick end-labelling technique.     

Effects of salmon GnRH and chicken GnRH-II on testicular apoptosis in goldfish (Carassius auratus)

Apoptosis is a form of cell death, characterized by morphological and biochemical changes. Apoptosis occurs in the normal testis and in response to different agents. In this study, we investigated the effect of gonadotropin-releasing hormone (GnRH) in testicular apoptosis in the goldfish. GnRH is a decapeptide that is expressed in different tissues, including gonads in mammalian and non-mammalian species. While GnRH is considered to be a paracrine mediator of ovarian follicular atresia, the role of GnRH in the testis is less clear. In the present study, treatments with native salmon GnRH and chicken GnRH-II increased DNA fragmentation (a hallmark of apoptosis) in the mature goldfish testis. On the other hand, gonadotropin hormone was found to act as survival factor, by decreasing spontaneous and GnRH-induced DNA fragmentation in the goldfish testis. The results demonstrate that GnRH plays an important paracrine role in the control of apoptosis in the goldfish testis.     

Extracellular superoxide dismutase (EC-SOD) binds to type i collagen and protects against oxidative fragmentation

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is mainly found in the extracellular matrix of tissues. EC-SOD participates in the detoxification of reactive oxygen species by catalyzing the dismutation of superoxide radicals. The tissue distribution of the enzyme is particularly important because of the reactive nature of its substrate, and it is likely essential that EC-SOD is positioned at the site of superoxide production to prevent adventitious oxidation. EC-SOD contains a C-terminal heparin-binding region thought to be important for modulating its distribution in the extracellular matrix. This paper demonstrates that, in addition to binding heparin, EC-SOD specifically binds to type I collagen with a dissociation constant (K(d)) of 200 nm. The heparin-binding region was found to mediate the interaction with collagen. Notably, the bound EC-SOD significantly protects type I collagen from oxidative fragmentation. This expands the known repertoire of EC-SOD binding partners and may play an important physiological role in preventing oxidative fragmentation of collagen during oxidative stress.     

Multiple feedback loops are key to a robust dynamic performance of tryptophan regulation in Escherichia coli

Internucleosomal DNA fragmentation is an apoptotic event that depends on the activity of different nucleases. Among them, the DNA fragmentation factor B, better known as caspase-activated DNase (CAD), is mainly responsible for this DNA fragmentation in dying cells. CAD is an endonuclease that is chaperoned and inhibited by inhibitor of CAD (ICAD). Activation of CAD needs the cleavage of ICAD by activated caspase-3. During the characterization of the staurosporine-induced apoptotic process in human neuroblastoma cell lines, we have found three novel splice variants of CAD. In all three messengers, the open reading frame is truncated after the second exon of the CAD gene. This truncated open reading frame codifies the CAD protein amino terminal part corresponding to the cell death-inducing DFF45-like effector-N (CIDE-N) domain. We have detected these splicing variants in human tissues and in peripheral white blood cells from 10 unrelated individuals, and their products have been showed to be expressed in certain mouse tissues. We demonstrate that these truncated forms of CAD are soluble proteins that interact with ICAD. We also provided evidences that these CIDE-N forms of CAD promote apoptosis in a caspase-dependent manner.     

Combining intensity correlation analysis and MALDI imaging to study the distribution of flavonols and dihydrochalcones in Golden Delicious apples

Mass spectrometry-based imaging techniques applied to small molecules complement the growing research field of metabolomics and can be used to interpret many important biological processes occurring in plants. In untargeted imaging applications, chemical identification is a critical step since it cannot take advantage of separative techniques applied to neutral molecules (e.g. liquid chromatography). The use of high resolution spectrometers is of great help, but fragmentation experiments are often necessary. In many cases, the information on ion fragmentation is embedded in the data sets, because analytes break up during ionization, but the extraction of this information is not easy considering the complexity of the imaging data files. Here an approach is proposed for applying conventional untargeted MALDI (matrix-assisted laser desorption ionization) profiling and advanced data analysis to perform imaging of metabolites in apple tissues. The pipeline, based on intensity correlation analysis, is used to extract fragmentation information from untargeted, high resolution, wide range mass spectra and to reconstruct compound-specific images which can be used for interpretation purposes. The proposed approach was used to investigate the distribution of glycosylated flavonols and dihydrochalcones in Golden Delicious apples. The results indicate that the method is effective, showing a high potential for ascertaining detailed metabolite localization.     

Coordinate involvement of cysteine protease and nuclease in the executive phase of plant apoptosis

We have developed an oat cell-free apoptosis system to investigate the execution mechanisms of plant apoptosis. Cell extracts derived from oat tissues undergoing toxin (victorin)-induced apoptosis caused nuclear collapse and internucleosomal DNA fragmentation in isolated nuclei. Pharmacological studies revealed that cysteine protease, which is E-64-sensitive but insensitive to caspase-specific inhibitors, is a crucial component in the morphological change of isolated nuclei, and that nuclease and the cysteine protease act cooperatively to induce the apoptotic DNA laddering. Interestingly, this finding is contrasted with those in well-studied animal cell-free systems in which an apoptotic endonuclease is solely responsible for the DNA fragmentation.     

Teratogen-induced cell death in postimplantation mouse embryos: differential tissue sensitivity and hallmarks of apoptosis

Teratogen-induced cell death is a common event in the pathogenesis associated with tissues destined to be malformed. Although the importance of this cell death is recognized, little information is available concerning the biochemistry of teratogen-induced cell death. We show that three teratogens, hyperthermia, cyclophosphamide and sodium arsenite induce an increase in cell death in day 9.0 mouse embryos with concurrent induction of DNA fragmentation, activation of caspase-3 and the cleavage of poly (ADP-ribose) polymerase (PARP). Teratogen-induced cell death is also selective, i. e., some cells within a tissue die while others survive. In addition, cells within some tissues die when exposed to teratogens while cells in other tissues are relatively resistant to teratogen-induced cell death. An example of the latter selectivity is seen in the cells of the developing heart, which are resistant to the cytotoxic potential of many teratogens. We show that the absence of cell death in the heart is accompanied by the complete lack of DNA fragmentation, activtion of caspase-3 and the cleavage of PARP.     

BAF as a caspase-dependent mediator of nuclear apoptosis in Drosophila

BAF is a double-stranded DNA binding protein required for proper nuclear morphology and function in Drosophila development. Imaginal discs of Drosophila baf-null mutants were found to exist only in younger larvae as small degenerative tissues. Immunohistochemical analyses showed diffuse lamin distribution, DNA fragmentation, and activation of caspase drICE in these tissues, suggesting that apoptotic events can be induced by the loss of baf. We therefore investigated the fate of BAF after induction of the pro-apoptotic hid transgene, and found that the loss of DNA binding forms of BAF preceded that of non-DNA binding forms of BAF. Furthermore, the DNA binding forms of BAF disappeared from nuclei before DNA fragmentation and NPC clustering were detected, showing that the loss of BAF occurs at the initial stages of nuclear apoptosis. This BAF loss was not detected before drICE activation and was inhibited by Ac-DEVD-CHO caspase inhibitors. In summary, BAF disappears at an early stage due to caspase activity when apoptosis is induced by hid, and its depletion in mutants is sufficient in itself to induce cell death, suggesting it is an apoptotic mediator.     

DNA fragmentation in apoptosis

Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis.Elegant biochemical work identified the DNA fragmentation factor(DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro Genetic studies in mice support the importence of DFF in DNA fragmentation and possibly in apoptosis in vivo.Recent work also suggests the existence of additional endonucleases for DNA degradation.Understanding the roles of individual endonucleases in apoptosis,and how they might coordinate to degrade DNA in different tissues during normal development and homeostasis,as well as in various diseased states,will be a major research focus in the near future.     

Autodigestion of chromatin in some radiosensitive and radioresistant mouse cells. Role of proteolysis and endonucleolysis

Evidence is presented indicating that mouse thymus, spleen, kidney, lung and heart contain a protease activity with relatively high specificity for histones. It is suggested that degradation of chromatin occurring in irradiated lymphoid tissues is produced by the action of alkaline endonuclease in association with this histone protease. The autodigestion of chromatin was assessed by determining the release of soluble chromatin from cells suspended in sucrose media of low ionic strength. It was found that the protease inhibitors, phenylmethylsulphonyl fluoride and especially NaHSO3, were also capable of depressing the activity of alkaline endonuclease, the fragmentation of chromatin, and the release of soluble chromatin. The results suggest that the release of histones from irradiated lymphoid tissues cannot be considered as a determinant step in the fragmentation of DNA in chromatin.     

Quantity and quality of suitable matrices matter in reducing the negative effect of fragmentation

The negative effect of fragmentation is one of the main concerns in the study of biodiversity loss in landscape ecology. The use of the matrix has been considered an important factor because it can change a population's relationship with the configuration of the landscape. A systematic way to assess the effect of matrix quality in fragmented landscapes could lead to a better understanding of how matrices can suppress the negative effect of fragmentation. We built a computational individual-based model capable of simulating bi-dimensional landscapes with three types of land cover (habitat, suitable matrix and hostile matrix) and individuals that inhabit those landscapes. We explored in which situations suitable matrix proportions and the degree of usability of this suitable matrix mitigate the negative effect of fragmentation per se. We observed that (i) an increase in the general matrix quality (increases in the suitable matrix proportion and/or usability) can suppress the fragmentation effect in 47% of the simulated scenarios; (ii) the less usable the matrix is, the more of it is needed to suppress the fragmentation effect; (iii) there is a level of usability below which increasing the suitable matrix proportion does cause the fragmentation effect to cease. These results point toward landscape management decisions that consider the similarity of the matrix to the native habitat under management. We suggest that an index to measure the usability of elements of the matrix could be an important tool for using computational models in landscape management more efficiently.     

The geometry of habitat fragmentation: Effects of species distribution patterns on extinction risk due to habitat conversion

Land‐use changes, which cause loss, degradation, and fragmentation of natural habitats, are important anthropogenic drivers of biodiversity change. However, there is an ongoing debate about how fragmentation per se affects biodiversity in a given amount of habitat. Here, we illustrate why it is important to distinguish two different aspects of fragmentation to resolve this debate: (a) geometric fragmentation effects, which exclusively arise from the spatial distributions of species and habitat fragments, and (b) demographic fragmentation effects due to reduced fragment sizes, and/or changes in fragment isolation, edge effects, or species interactions. While most empirical studies are primarily interested in quantifying demographic fragmentation effects, geometric effects are typically invoked as post hoc explanations of biodiversity responses to fragmentation per se. Here, we present an approach to quantify geometric fragmentation effects on species survival and extinction probabilities. We illustrate this approach using spatial simulations where we systematically varied the initial abundances and distribution patterns (i.e., random, aggregated, or regular) of species as well as habitat amount and fragmentation per se. As expected, we found no geometric fragmentation effects when species were randomly distributed. However, when species were aggregated, we found positive effects of fragmentation per se on survival probability for a large range of scenarios. For regular species distributions, we found weakly negative geometric effects. These findings are independent of the ecological mechanisms which generate nonrandom species distributions. Our study helps to reconcile seemingly contradictory results of previous fragmentation studies. Since intraspecific aggregation is a ubiquitous pattern in nature, our findings imply widespread positive geometric fragmentation effects. This expectation is supported by many studies that find positive effects of fragmentation per se on species occurrences and diversity after controlling for habitat amount. We outline how to disentangle geometric and demographic fragmentation effects, which is critical for predicting the response of biodiversity to landscape change.     

Role of mitochondrial remodeling in programmed cell death in Drosophila melanogaster

The role of mitochondria in Drosophila programmed cell death remains unclear, although certain gene products that regulate cell death seem to be evolutionarily conserved. We find that developmental programmed cell death stimuli in vivo and multiple apoptotic stimuli ex vivo induce dramatic mitochondrial fragmentation upstream of effector caspase activation, phosphatidylserine exposure, and nuclear condensation in Drosophila cells. Unlike genotoxic stress, a lipid cell death mediator induced an increase in mitochondrial contiguity prior to fragmentation of the mitochondria. Using genetic mutants and RNAi-mediated knockdown of drp-1, we find that Drp-1 not only regulates mitochondrial fission in normal cells, but mediates mitochondrial fragmentation during programmed cell death. Mitochondria in drp-1 mutants fail to fragment, resulting in hyperplasia of tissues in vivo and protection of cells from multiple apoptotic stimuli ex vivo. Thus, mitochondrial remodeling is capable of modifying the propensity of cells to undergo death in Drosophila.     

ABCA1-dependent but apoA-I-independent cholesterol efflux mediated by fatty acid-bile acid conjugates (FABACs)

PCD (programmed cell death) in plants presents important morphological and biochemical differences compared with apoptosis in animal cells. This raises the question of whether PCD arose independently or from a common ancestor in plants and animals. In the present study we describe a cell-free system, using wheat grain nucellar cells undergoing PCD, to analyse nucleus dismantling, the final stage of PCD. We have identified a Ca2+/Mg2+ nuclease and a serine protease localized to the nucleus of dying nucellar cells. Nuclear extracts from nucellar cells undergoing PCD triggered DNA fragmentation and other apoptotic morphology in nuclei from different plant tissues. Inhibition of the serine protease did not affect DNA laddering. Furthermore, we show that the nuclear extracts from plant cells triggered DNA fragmentation and apoptotic morphology in nuclei from human cells. The inhibition of the nucleolytic activity with Zn2+ or EDTA blocked the morphological changes of the nucleus. Moreover, nuclear extracts from apoptotic human cells triggered DNA fragmentation and apoptotic morphology in nuclei from plant cells. These results show that degradation of the nucleus is morphologically and biochemically similar in plant and animal cells. The implication of this finding on the origin of PCD in plants and animals is discussed.     

Spatial pattern of habitat quality modulates population persistence in fragmented landscapes

Habitat quality is one of the important factors determining population dynamics and persistence, yet few studies have examined the effects of spatial heterogeneity in within-patch habitat quality. In this paper, we use a spatially explicit agent-based model to investigate how habitat fragmentation and spatial pattern of within-patch habitat quality affect population dynamics and long-term persistence. We simulate three levels of habitat fragmentation (ranges from continuous to highly fragmented) and three types of spatial patterns in habitat quality within patches (i.e., negatively autocorrelated, randomly distributed, and positively autocorrelated). Hypothetical species differ in their niche specialization. The results demonstrate explicitly that the spatial pattern of within-patch habitat quality plays an important role in modulating the effects of habitat fragmentation on populations. Populations become less variable in size, and experience lower probability of extinction in landscapes with positively autocorrelated within-patch habitat quality. Specifically, specialized species are more vulnerable to habitat fragmentation, but this vulnerability is greatly mitigated by positively autocorrelated habitat quality within patches, in other words, exhibiting higher resistance to habitat fragmentation. The findings of this study suggest that managing habitat quality in existing habitat remnants is important to preserve species in habitats undergoing fragmentation, particularly for those with specialized habitat requirements.     

陕北黄土高原景观破碎化的时空动态研究

景观破碎化受到自然环境背景及社会经济活动的影响,并限制区域发展.为揭示景观破碎化的时空变化特征,整合了MODIS(中分辨率成像光谱仪)影像逐旬数据与多时段景观类型数据,并对其进行了分析.比较1987～2002年景观破碎化的年际差异表明,陕北黄土高原的景观破碎化过程较缓慢,但具有区域差异,且与景观类型组成息息相关.结果表明,林地的破碎化水平变化显著,而农、草地的破碎化水平变化相对缓慢.年际变化分析有助于说明区域景观破碎化的趋势,而年内季节变化则能帮助认识景观破碎化过程的周期规律.     

Apoptosis of Leishmania (Leishmania) chagasi amastigotes in hamsters infected with visceral leishmaniasis

Apoptosis in amastigotes from hamsters infected with visceral leishmaniasis was absent 30-day post-infection but appeared 90-day post-infection in the liver and spleen, as analysed using the TUNEL method. Necrosis was not present in these tissues and the nuclei of macrophages harbouring apoptotic amastigotes were preserved. Amastigote DNA fragmentation was demonstrated using agarose gel electrophoresis. DNA fragmentation was evident 90-day post-infection, coinciding with the occurrence of apoptosis of amastigotes in the tissues. Apoptosis of Leishmania amastigotes in vivo may constitute a mechanism that regulates growth of the parasite population during infection.     

EndoG links Bnip3-induced mitochondrial damage and caspase-independent DNA fragmentation in ischemic cardiomyocytes

Mitochondrial dysfunction, caspase activation and caspase-dependent DNA fragmentation are involved in cell damage in many tissues. However, differentiated cardiomyocytes repress the expression of the canonical apoptotic pathway and their death during ischemia is caspase-independent. The atypical BH3-only protein Bnip3 is involved in the process leading to caspase-independent DNA fragmentation in cardiomyocytes. However, the pathway by which DNA degradation ensues following Bnip3 activation is not resolved. To identify the mechanism involved, we analyzed the interdependence of Bnip3, Nix and EndoG in mitochondrial damage and DNA fragmentation during experimental ischemia in neonatal rat ventricular cardiomyocytes. Our results show that the expression of EndoG and Bnip3 increases in the heart throughout development, while the caspase-dependent machinery is silenced. TUNEL-positive DNA damage, which depends on caspase activity in other cells, is caspase-independent in ischemic cardiomyocytes and ischemia-induced DNA high and low molecular weight fragmentation is blocked by repressing EndoG expression. Ischemia-induced EndoG translocation and DNA degradation are prevented by silencing the expression of Bnip3, but not Nix, or by overexpressing Bcl-x(L). These data establish a link between Bnip3 and EndoG-dependent, TUNEL-positive, DNA fragmentation in ischemic cardiomyocytes in the absence of caspases, defining an alternative cell death pathway in postmitotic cells.     

Apoptosis in rat placenta is zone-dependent and stimulated by glucocorticoids

Apoptosis, or physiological cell death, is elevated in the placenta of human pregnancies complicated by fetal growth retardation, suggesting that placental apoptosis may be a key factor in the overall control of feto-placental growth. The present study used DNA internucleosomal fragmentation analysis to characterize apoptosis in the two morphologically and functionally distinct regions of the rat placenta, the basal and labyrinth zones, during the last week of pregnancy (Days 16, 22, and 23). In addition, because glucocorticoids are potent inhibitors of feto-placental growth and can stimulate apoptosis in other tissues, we examined whether dexamethasone treatment in vivo induces placental apoptosis. DNA fragmentation was clearly evident in both placental zones at each stage of pregnancy, with higher levels evident in the basal zone compared with the labyrinth zone on Days 22 and 23. TUNEL analysis, which identifies dying cells in situ, demonstrated positive staining of cells in the basal zone, particularly giant trophoblast cells. Dexamethasone treatment increased DNA fragmentation in the basal zone but not the labyrinth zone. Similarly, maternal treatment with carbenoxolone, which can enhance local concentrations of endogenous glucocorticoid by inhibition of 11 beta-hydroxysteroid dehydrogenase, also increased DNA fragmentation in the basal zone but not in the labyrinth zone. These effects of dexamethasone and carbenoxolone on placental apoptosis were associated with reduced placental and fetal weights. In conclusion, this study shows that apoptosis occurs in both zones of the rat placenta, particularly in the basal zone near term, and is elevated after increased glucocorticoid exposure in vivo. These data support the hypothesis that placental apoptosis is an important player in the regulation of feto-placental growth, and establish the rat as a useful model to study the endocrine control of placental apoptosis.