Life Sciences Commons^™

The Role Of Calcium In The Regulation Of Apoptosis, David J. Mcconkey

Scanning Microscopy

The recognition that apoptosis is regulated by an evolutionarily conserved set of polypeptides from the nematode Caenorhabditis elegans to humans suggests that a conserved set of biochemical mechanism(s) may also be involved in the response. Early evidence suggested that the endogenous endonuclease implicated in apoptosis in most model systems is Ca2+ -dependent, and subsequent work from a number of independent laboratories suggests that alterations in cytosolic Ca2+ homeostasis are one of the conserved biochemical pathways regulating the response. Molecular targets for Ca2+ are now being identified and include signal transduction intermediates, endonuclease(s) and proteases, and the enzymes involved in the …

Apoptosis And Red Blood Cell Echinocytosis: Common Features, Alexei B. Chukhlovin

Scanning Microscopy

Apoptosis of nucleated blood cells induced by oxidants and/or reactive oxygen species is accompanied by the typical membrane pathology. Meanwhile, red blood cell (RBC) membrane. is a popular object for studying appropriate cytotoxic effects. Scanning electron microscopy provides a reliable tool for detecting the oxidative changes in RBC shape and size. Transition of normal discoid erythrocytes to crenated forms (echinocytes) is often induced by the same factors which cause apoptosis of blood cells, e.g., ionizing radiation and other reactive oxygen intermediate-inducing agents, exogenous oxidants, in vitro aging conditions, cytosolic calcium increase, etc. Moreover, the biochemical membrane alterations in oxidant-induced echinocytosis …

Ionizing Radiation-Induced Apoptosis And Dna Repair In Murine Erythroleukemia Cells, R. Di Pietro, L. Centurione, E. Santavenere, M. A. Centurione, G. Sanitá Di Toppi, L. Zamai, R. Rana

Scanning Microscopy

A morphological study of DNA repair and apoptotic patterns in relationship with cell cycle events was performed on murine erythroleukemia cells. The presence and distribution of DNA replicon sites were evaluated through the BrdU-anti BrdU immunofluorescence and immunogold techniques in light and electron microscopy. Different patterns of labelling and percentages of BrdU positive cells were observed depending on irradiation dose (up to 60 Gy) and time in post-irradiation culture (up to 24 hours). An enlargement of the S phase of the cell cycle was evidenced 18 hours post-irradiation as determined by flow cytometry analysis. The high resolution approach showed that, …

Recognition And Phagocytosis Of Apoptotic Cells, Luciana Dini, Maria Teresa Ruzittu, Laura Falasca

Scanning Microscopy

Physiological elimination of unwanted cells within the organism occurs via cell death by apoptosis and phagocytosis of these cells represents a key event in the apoptotic process. Macrophages, which are the dedicated phagocytes, and other occasionally phagocytic cells ingest the apoptotic cells while they are still intact, thus preventing the leakage of potentially harmful materials from the dying cells. Although evidence has been presented that the elimination of apoptotic bodies from the tissue operates by means of specific recognition systems, the molecular mechanisms by which an apoptotic cell is recognized are poorly understood. Recent data indicate that phagocyte recognition of …

Different Approaches To The Study Of Apoptosis, E. Falcieri, L. Stuppia, A. Di Baldassarre, A. R. Mariani, C. Cinti, M. Columbaro, L. Zamai, M. Vitale

Scanning Microscopy

The morphological features of cell undergoing programmed cell death is well known and has been widely described in a number of experimental models with a variety of apoptotic triggering agents. Despite the similar cell behaviour, underlying molecular events seem variable and only partially understood. A multiple approach appears crucial to better clarify the phenomenon. The first technique, DNA gel electrophoresis, allows the identification of fragmented DNA and has been long considered the hallmark of apoptosis. Different patterns of DNA cleavage, which can be identified by conventional or "pulsed-field gel" electrophoresis, are presented and discussed. "In situ" labelling methods are also …

Thymocyte Proliferation And Apoptosis Induced By Ionizing Radiation, Vera V. Shaposhnikova, Yuri N. Korystov

Scanning Microscopy

Proliferation and apoptosis of rat and mouse thymocytes caused by ionizing radiation were studied. The percentage of proliferating cells was determined by the method of colchicine metaphases and the apoptosis was estimated as DNA fragmentation. In vitro irradiation with 0.05-0.2 Gy was found to stimulate thymocyte proliferation, the maximum was observed at 0.05 Gy for mouse thymocytes and at 0.1 Gy for rat thymocytes. These doses caused a slight decrease in DNA fragmentation, as compared to control. By raising the radiation dose, proliferation was reduced and DNA fragmentation was increased. The results obtained indicate that low radiation doses stimulate cell …

Human Skin Physiology Studied By Particle Probe Microanalysis, B. Forslind, M. Lindberg, K. G. Malmqvist, J. Pallon, G. M. Roomans, Y. Werner-Linde

Scanning Microscopy

Particle probe methods (electron probe and proton probe X-ray microanalysis) have been applied to investigate the distribution of elements and water over the different layers of the epidermis. For major elements, electron probe X-ray microanalysis (XRMA) provides the advantage of superior spatial resolution, but for trace element analysis the more sensitive proton probe (particle induced X-ray emission, PIXE) analysis has to be used. On a dry weight basis, the concentration of S is rather constant across the epidermis, whereas the concentrations of P, K, Cl and Na show gradients with high levels in stratum germinativum (basale) and stratum spinosum but …

Comparison Of Dna Fragmentation And Color Thresholding For Objective Quantitation Of Apoptotic Cells, D. R. Plymale, D. S. Ng Tang, C. D. Fermin, D. E. Lewis, D. S. Martin, R. F. Garry

Scanning Microscopy

Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4 + T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to γ-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with …

Cross-Linking Of Cell Surface Receptors As A Trigger Of Cell Apoptosis And Proliferation, Yuri N. Korystov

Scanning Microscopy

A hypothesis of the mechanism by which the protein cross-linking agents trigger apoptosis of lymphoid cells and proliferation of other cell types is proposed. It is assumed that both effects are triggered by aggregation of receptors on cell surface, which results from their cross-linking. This idea is substantiated by the example of one of these agents, ionizing radiation. As in the case of physiological agents, such as, antigens and growth factors, the aggregation of receptors induced by radiation activates receptor protein tyrosine kinases from which the signal is transduced to genes through protein kinase C. The hypothesis is consistent with …

Apoptosis Induced By Microtubule Disrupting Drugs In Normal Murine Thymocytes In Vitro, Vladimir Bumbaširević, Andjelija Škaro-Milić, Aleksandar Mirčić, Bogdan Djuričić

Scanning Microscopy

Disruption of cytoplasmic and spindle microtubules by colchicine or nocodazole increases mitotic index, but it also enhances apoptosis in isolated mouse thymocytes; the apoptotic index exceeds 20% after 4 hours of incubation with either drug (5% in controls). Apoptosis was confirmed by DNA fragmentation, and was blocked by calcium chelators and inhibitors of protein synthesis. The apoptotic effect of microtubule disrupting drugs (MOD) was directed to interphase thymocytes and was independent on MOD action on mitotic cells. However, cell death of mitotically arrested cells showed ultrastructural changes similar in many aspects to apoptosis.

Cell Death In The Embryonic Developing Limb, J. M. Hurle, M. A. Ros, V. Garcia-Martinez, D. Macias, Y. Ganan

Scanning Microscopy

In amniote vertebrates, the development of form and structure of the limb bud is accompanied by precise patterns of massive mesodermal cell death with morphological features of apoptosis. These areas of cell death appear to eliminate undifferentiated cells which are required only for a limited time period of limb development. Predictable skeletal and morphological anomalies of the limb occur when the pattern of cell death is modified in mutant species or under experimental conditions. Most evidence points to the occurrence of local triggering mechanisms to account for the establishment of the areas of cell death and the subsequent activation of …

Morphologic Changes Of Apoptosis Induced In Human Chronic Myelogenous Leukemia "Blast" Cells By Sc41661a (Searle), A Selective Inhibitor Of 5-Lipoxygenase, K. M. Anderson, T. M. Seed, J. Peng, A. Jajeh, J. Meng, J. E. Harris

Scanning Microscopy

Several inhibitors of the arachidonic acid-metabolizing enzyme, 5-lipoxygenase reduce proliferation of hematopoietic and non-hematopoietic cells and cell lines and some cells undergo limited differentiation. Cells were cultured from patients with chronic myelogenous leukemia in "blast" crisis with the selective inhibitor of 5-lipoxygenase,SC41661A[3-{3,5-bis(1,1-dimethyl)-4-hydroxyphenyl}hiol]-N-methyl-N-[2-(2-phridinyl-propanamide)]. Cells cultured for 3 to 5 days with 40 μM SC41661A exhibited reduced cellular numbers along with ultrastructural changes and DNA laddering characteristic of apoptosis. Similar culture conditions reduced proliferation of U937 monoblastoid cells. In U937 cells, the ultrastructural features of apoptosis were not observed at 72 hours, when DNA laddering was present and cell numbers were reduced, …

Ultrastructural Patterns Of Cell Damage And Death Following Gamma Radiation Exposure Of Murine Erythroleukemia Cells, R. Di Pietro, E. Falcieri, L. Centurione, M. A. Centurione, G. Mazzotti, R. Rana

Scanning Microscopy

Radiation causes damage to cell surface membranes, cytoplasmic organelles, and the nuclear process of DNA synthesis and repair, and this eventually results in different modes of cell death. In this study we examined murine erythroleukemia (MEL) cells, exposed to 15 and 60 Gy of 10 MeV photonic energy, and left in culture for up to 96 hours. Electron microscopical analysis was performed on conventionally embedded samples and freeze-fracture replicas, in order to detect ultrastructural patterns of cell damage and death. Of interest was the observation of chromatin condensates, nuclear membrane associations and nuclear pore redistribution during early apoptosis. Pronounced rearrangements …

Ultrastructural Features Of Apoptosis, Elisabetta Falcieri, Pietro Gobbi, Loris Zamai, Marco Vitale

Scanning Microscopy

Apoptosis is a gene-directed physiological and programmed process of cell deletion aimed at the regulation of tissue and organ development. It affects different cell types and is triggered by a variety of stimuli all inducing closely comparable structural changes. Despite the deeply different morphology and metabolism of the cell models and the various inducers and their initial effects, a convergence seems to take place in a common metabolic pathway that, in most cases, involves the activation of a Ca²⁺ dependent endonuclease. A growing body of data is now available on the molecular events that lead to DNA damage. DNA …

Sertoli Cell Death By Apoptosis In The Immature Rat Testis Following X-Irradiation, David J. Allan, Glenda C. Gobé, Brian V. Harmon

Scanning Microscopy

The importance of the morphological study of cell death has recently been emphasized by the recognition that the ultrastructural features of dying cells allow categorization of the death as either apoptosis or necrosis. This classification enables inferences to be drawn about the mechanism and biological significance of the death occurring in a particular set of circumstances.

In this study, Sertoli cell death induced in the immature testis of three and four day old rats by 5 Gy (500 rads) x-irradiation was described by light and transmission electron microscopy with the objective of categorizing the death as apoptosis or necrosis. The …

X-Ray-Induced Cell Death By Apoptosis In The Immature Rat Cerebellum, Brian V. Harmon, David J. Allan

Scanning Microscopy

The cells of the external granular layer (EGL) of the developing cerebellum are known to be particularly sensitive to radiation. In the past, changes induced in this layer by irradiation have been referred to by non-specific terms such as "pyknotic cells" and the mode of cell death has been assumed to be necrosis. However, in published light micrographs of these dying cells, the appearance is suggestive of apoptosis, a distinctive mode of cell death which occurs spontaneously in normal adult and embryonic tissues and can also be triggered by certain pathological stimuli.

This light and transmission electron microscopic study of …