The effect of exercise training in cold on shivering and nonshivering thermogenesis in adult and aged C57BL/6J mice

The behaviour of plasma membrane was studied in UV-treated cells to investigate its involvement in apoptosis. It was studied in HL60 cells, in which DNA oligonucleosomic cleavage occurs, and in Molt-4 cells, which are characterised by a different fragmentation pattern. During the early stages of apoptosis, a membrane lipid rearrangement occurs, which involves phosphatidylserine translocation from the inner to the outer leaflet. This molecular alteration was investigated by annexin V-FITC binding, analysed by flow cytometry and confocal microscopy. It was correlated with transmission electron microscopy, subdiploid peak appearance and DNA fragmentation. Our data indicate that the plasma membrane represents an early apoptotic target, even if its alterations are not detectable by ultrastructural analysis, which indicates its good preservation until late apoptotic stages. In addition, the study of apoptotic cells with absent or inactivated endonuclease demonstrates the independence of this membrane mechanism from nuclear activity.     

Lipid peroxidation in presence of ebselen

BACKGROUND: Apoptosis is a morphologically distinctive form of programmed cell death/cell suicide in which genomic DNA degradation/fragmentation and variegated dense chromatin aggregates are characteristic hallmarks that have never been demonstrated in mitotic cells. Perceptions of mutual exclusivity between apoptosis and mitosis imply that M-phase cells cannot be apoptotic. However, in the present study we show apoptotic morphologies in M-phase cells after an acute oxidative stress and endonuclease digestion. METHODS: Degradation of genomic DNA in human Chang liver cells (American Type Culture Collection, ATCC CCL13) was demonstrated by flow cytometric cell-by-cell evaluation of (a) propidium iodide intercalative binding to DNA and (b) terminal deoxynucleotidyl transferase (TdT)-mediated 3'OH nick end labeling (TUNEL) of fragmented DNA. Oxidative stress was imposed by a 30-min prepulse with 200 microM vanadyl(4), which produces hydroxyl free radicals (OH*), the most reactive of the free radical species. Oxidative stress in the cells was demonstrated by evaluating glutathione-S-transferase (GST)-mediated monochlorobimane-glutathione adduct fluorescence for glutathione content, the main reducing agent of a cell, and methylene blue redox metachromasia, which is a deep color when oxidized and colorless when reduced. Cells with DNA fragmentation were highlighted by TUNEL. Apoptotic morphologies were visualized by staining with Giemsa and neutral red dyes and by DNA-propidium iodide binding to chromatin. Direct endonuclease induction of apoptotic morphologies in permeabilized M-phase cells was produced by 1 hr incubation (37 degrees C) with 16 units/ml of micrococcal nuclease. RESULTS: The genomic DNA of proliferative cells, namely in G2/M phase of the cell cycle, was degraded by vanadyl(4) prepulsing and by micrococcal nuclease digestion, concomitantly with DNA fragmentation shown by TUNEL. Cytological profiles showed GSH depletion and M-phase cells with particularly high oxidative reactivity indicated by methylene blue redox metachromasia. DNA fragmentation in M-phase cells was highlighted by TUNEL. Characteristic apoptotic condensations, ranging from single-ball condensations to "pulverized" aggregates of a mitotic catastrophe, buddings, and "apoptotic bodies," were found in prophase, metaphase, anaphase, and telophase mitotic cells. The observed separation of condensed chromatin aggregates from the main chromosome mass in prophase and metaphase cells could explain micronuclei, linking it with apoptosis. Direct endonuclease digestion readily produced apoptotic morphologies in interphase and in M-phase cells. CONCLUSION: Apoptotic morphologies in M-phase cells can be induced indirectly via oxidative stress or directly via endonuclease activity, which has long been established as a pervading hallmark of apoptosis.     

Light-induced apoptosis: differential timing in the retina and pigment epithelium

Apoptosis is a genetically regulated form of cell death. Individual cells show condensed nuclear chromatin and cytoplasm, and biochemical analysis reveals fragmentation of the DNA. Ensuing cellular components, apoptotic bodies, are removed by macrophages or neighboring cells. Genes involved in the regulation of apoptosis as well as stimuli and signal transduction systems, are only beginning to be understood in the retina. Therefore, we developed a new in vivo model system for the investigation of events leading to apoptosis in the retina and the pigment epithelium. We induced apoptosis in retinal photoreceptors and the pigment epithelium of albino rats by exposure to 3000 lux of diffuse, cool white fluorescent light for short time periods of up to 120 minutes. Animals were killed at different time intervals during and after light exposure. The eyes were enucleated and the lower central retina was processed for light- and electron microscopy. DNA fragmentation was analysed in situ by TdT-mediated dUTP nick-end labeling (TUNEL) or by gel electrophoresis of total retinal DNA. We observed that the timing of apoptosis in the photoreceptors and pigment epithelium was remarkably different, the pigment epithelium showing a distinct delay of several hours before the onset of apoptosis. In photoreceptors, apoptosis was induced within 90 minutes of light exposure, with the morphological appearance of apoptosis preceding the fragmentation of DNA. In the pigment epithelium, the morphological appearance of apoptosis and DNA fragmentation were coincident. Different regulative mechanisms may lead to apoptotic cell death in the retinal photoreceptors and pigment epithelium. This in vivo model system will allow measurement of dose-responses, a potential spectral dependence and the molecular background of apoptotic mechanisms in the retina.     

A comparison of distinct modes of tumor cell death in Hodgkin's disease using morphology and in situ DNA fragmentation

The study examined the morphology and frequency of cell death occurring spontaneously in lymph nodes from patients with Hodgkin's disease. In addition to necrosis, which was infrequent and usually in patches, we document two cell types showing features of individual cell death: mummy cells end apoptotic cells. Mummy cells present no evidence of DNA fragmentation, but show electron microscopic features of "dark cells." Apoptotic Hodgkin-Reed-Sternberg cells are found frequently and are easier to demonstrate by in situ and labeling of fragmented DNA than by light microscopy only. In many cases phagocytosis of apoptotic cells is also documented. The significance of these findings to the limited number of Hodgkin-Reed-Sternberg cells in most cases of Hodgkin's disease is discussed.     

Serotype distribution of invasive group B streptococcal isolates in Maryland: implications for vaccine formulation. Maryland Emerging Infections Program

In human in-vitro fertilization (IVF)-embryo transfer, the in-vitro culture environment differs from in-vivo conditions in that the oxygen concentration is higher, and in such conditions the mouse embryos show a higher concentration of reactive oxygen species (ROS) in simple culture media. ROS are believed to cause damage to cell membranes and DNA fragmentation in somatic cells. This study was conducted to ascertain the level of H2O2 concentration within embryos and the morphological features of cell damage induced by H2O2. A total of 62 human oocytes and embryos (31 fragmented, 15 non-fragmented embryos, 16 unfertilized oocytes) was obtained from the IVF-embryo transfer programme. The relative intensity of H2O2 concentrations within embryos was measured using 2',7'-dichlorodihydrofluorescein diacetate by Quanti cell 500 fluorescence imaging and DNA fragmentation was observed with transmission electron microscopy and an in-situ apoptosis detection kit. The H2O2 concentrations were significantly higher in fragmented embryos (72.21 +/- 9.62, mean +/- SEM) compared to non-fragmented embryos (31.30 +/- 3.50, P < 0.05) and unfertilized oocytes (30.75 +/- 2.67, P < 0.05). Apoptosis was observed only in fragmented embryos, and was absent in non-fragmented embryos. Electron microscopic findings confirmed apoptotic bodies and cytoplasmic condensation in the fragmented blastomeres. We conclude that there is a direct relationship between increased H2O2 concentration and apoptosis, and that further studies should be undertaken to confirm these findings.     

Apoptosis-specific protein (ASP) identified in apoptotic Xenopus thymus tumor cells

A novel apoptosis-specific protein (ASP) has recently been identified in the cytoplasm of apoptotic mammalian cells. This paper investigates whether ASP is found in Xenopus thymus tumor-derived lymphoid cell lines undergoing apoptosis and also in apoptotic, nontransformed splenocytes. Cultured Xenopus tumor lymphoid cells induced to undergo apoptosis by serum deprivation or treatment with the calcium ionophore, ionomycin, displayed altered morphology typical of apoptotic cells, as judged by flow cytometric light-scatter characteristics and by fluorescence microscopy of acridine-orange-stained cells. Flow cytometry of permeabilized cells and fluorescence microscopy of acetone-fixed cytospins revealed that apoptotic Xenopus tumor cells, especially those displaying loss or condensation of DNA, displayed increased expression of epitopes recognized by a rabbit polyclonal antibody against ASP. Flow cytometry confirmed that ASP is also expressed in splenocytes induced to apoptose by culture in ionomycin or following concanavalin A stimulation. No increased expression of ASP was seen when lymphoid tumor cells or splenocytes were induced into necrosis by overdose with the antifungal agent amphotericin B. Western blotting with antibody against ASP identified the emergence of several protein bands in cell lysates from apoptotic, but not necrotic, Xenopus tumor cells. The new and simple methodology for identifying apoptotic cells described here is likely to be of value to those studying immune system development and associated programmed cell death in Xenopus.     

Serum deprivation and protein synthesis inhibition induce two different apoptotic processes in N18 neuroblastoma cells

N18 are murine neuroblastoma cells that underwent cell death upon serum deprivation or inhibition of protein synthesis by means of cycloheximide (CHX). In both cases, an ultrastructural morphology and an internucleosomal pattern of DNA fragmentation typical of apoptosis were found. However, electron microscopy revealed abundant lipid vesicles in the cytoplasm of CHX-treated cells that were not found in their serum-deprived counterparts. In addition, when both types of apoptotic cells were compared by means of flow cytometry and chromatin staining with propidium iodide, the former showed consistently less fluorescence than the latter. Therefore, in N18 cells, both apoptotic processes seemed to differ at a structural level. At a functional level, we found that apoptosis was blocked by the protease inhibitor TLCK in CHX-treated but not in serum-deprived cells. On the other hand, we generated N18 clones that overexpressed Bcl-2 protein. After a period of 48 h we found that identical levels of Bcl-2 protein were able to block apoptosis in serum-deprived but not in CHX-treated cells. In conclusion, two different biochemical pathways leading to apoptosis seem to coexist in N18 neuroblastoma cells.     

Induction of apoptosis by benzene metabolites in HL60 and CD34+ human bone marrow progenitor cells

Two cell types, HL60 human promyelocytic leukemia cells and CD34+ human bone marrow progenitor cells, were used as model systems to explore a possible role for apoptosis in the myelotoxicity of the phenolic metabolites of benzene. HL60 cells were treated with either phenol, catechol, hydroquinone, or 1,2,4-benzenetriol and then stained with Hoechst 33342 and propidium iodide and subjected to fluorescent microscopy. Cells with nuclear condensation and fragmentation were scored as apoptotic, and etoposide (40 microM) was used as a positive control. Catechol, 1,2,4-benzenetriol, and hydroquinone induced marked time- (0-24 hr) and concentration- (25-100 microM) dependent apoptosis, whereas phenol (750 microM) did not. Under these conditions, no significant necrosis was observed. The induction of apoptosis was confirmed by internucleosomal cleavage of DNA, assessed by agarose gel electrophoresis. CD34+ cells treated with etoposide (40 microM) or hydroquinone (50 microM) for 18 hr were stained and subjected to fluorescent microscopy as above. The percentage of cells exhibiting nuclear condensation and/or fragmentation as well as high intensity staining significantly increased in both cases. The induction of apoptosis was confirmed using a terminal deoxynucleotidyl transferase assay. These data show that apoptosis can be induced in both HL60 and CD34+ human bone marrow progenitor cells by benzene metabolites. The ability of phenolic metabolites of benzene to induce apoptosis in human bone marrow progenitor cells may contribute to benzene myelotoxicity.     

DNA fragmentation in granular cells of human cerebellum following global ischemia

Vulnerability of human cerebellum in two autopsy cases following global brain ischemia was examined histologically by using a specific in situ nick-end labeling method for DNA breaks. In both cases, DNA fragmentation was observed in approximately one-third of the granular cells in cerebellar cortex, whereas Purkinje cells were still alive and no DNA fragmentation was recognized in the nuclei. The present study suggests that some granular cells of cerebellar cortex are more vulnerable to transient ischemia than Purkinje cells and death of granular cells is induced by an apoptotic DNA fragmentation following global brain ischemia.     

Frequency-shaped amplification changes the neural representation of speech with noise-induced hearing loss

Apoptosis is a mode of active cell death. We have examined whether 2-chloroethylethyl sulfide (CEES), a sulfur vesicating agent, triggers apoptosis as a cytotoxic mechanism. Incubation of thymocytes with CEES, resulted in an induction of apoptotic features of cell death. Treatment of cells with 100 microM CEES for 5 h increased DNA fragmentation to approximately 40% of control. The fragmentation of DNA was visualized by agarose gel electrophoresis. It showed ladder pattern of DNA fragmentation, which indicates internucleosomal cleavage of DNA. Further evidence of apoptosis was observed in morphological changes of nuclei by using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. The percentage of TUNEL positive cells was dependent upon CEES concentrations. CEES induced the classical morphological features of apoptosis in nucleus. These features were accompanied by condensation of chromatin, which arranged in sharply declined clumps and fragmentation of nucleus. To study requirement for synthesis of new protein in CEES-induced apoptosis, we studied the effect of cycloheximide for apoptotic activity. This protein synthesis inhibitor did not suppress the CEES-induced apoptotic activity. Taken together, these results suggest that CEES-induced apoptosis as a cytotoxicmechanism and this process occurs independent of synthesis of new protein.     

Apoptosis in the chicken bursa of fabricius induced by X-irradiation

Immature B lymphocytes in the chicken bursa of Fabricius have previously been reported to undergo apoptosis by low doses of ionizing radiation. In the present study, newly hatched chickens were subjected to whole-body X-irradiation, and the bursa of Fabricius was examined at various postirradiation times by light and electron microscopy to obtain information on the change of ultrastructure of irradiated bursal cells as well as on the time course and dose-response for the induction of apoptosis. Histological examination by light microscopy showed that pyknotic cells started to increase in the bursa within a few hours after irradiation and the frequency of occurrence reached a maximum at 6 hr. An evident increase of the pyknotic cells in number was observed at a dose of as little as 1 Gy, and the frequency increased with increases in the dose, reaching over 90% at 15 Gy. Electron microscopy of the irradiated bursa revealed typical apoptotic morphology such as chromatin condensation, nuclear fragmentation, formation of apoptotic bodies and phagocytosis of pyknotic cells. Induction of apoptosis was also confirmed by the appearance of a typical DNA ladder pattern on agarose gel. Thus, the present results demonstrate that the chicken bursal cells are hypersensitive to X-irradiation with regard to induction of apoptosis, and that the apoptotic bursal cells exhibit most of the ultrastructural features known to be typical of apoptosis.     

Differential effects of indomethacin on the sheep ovary: prostaglandin biosynthesis, intracellular calcium, apoptosis, and ovulation

Cells of the apical wall of the dominant follicle and contiguous ovarian surface epithelium become apoptotic with the approach of ovulation in the sheep. It was hypothesized that indomethacin, an established inhibitor of prostaglandin biosynthesis and ovulation, would protect apical ovarian cells from programmed death. The anovulatory potencies of two systemic doses of indomethacin (200 and 800 mg) were tested in gonadotropin-stimulated ewes. A complete blockade of ovulation occurred at the higher dose of indomethacin. Ovulation was not inhibited by 200 mg indomethacin. Both doses of drug suppressed follicular prostaglandin production below pregonadotropin levels. Immunofluorescence detection of digoxigenin end-labeled (fragmented) DNA was used as a marker of apoptosis among ovarian surface epithelial and granulosa cells recovered from the optical hemisphere of preovulatory ovine follicles. Cellular DNA fragmentation was averted in animals given 800 mg indomethacin, whereas apoptosis ensued after 200 mg. A sustained increase in cytosolic calcium is generally a prerequisite to apoptotic DNA fragmentation and cell death. Indeed, intracellular calcium, detected by fluorescence of fura-2, was elevated in ovarian cells of animals destined to ovulate (controls, 200 mg indomethacin) in comparison to (safeguarded) cells of anovulatory ewes (800 mg indomethacin). These observations provide circumstantial evidence that apical ovarian cell degeneration by calcium-mediated apoptosis is a determinant of follicular instability and rupture, but that these events are unrelated to the gonadotropin-induced rise in prostanoid production characteristic of preovulatory follicles.     

Apoptosis induced in vivo during acute infection by porcine reproductive and respiratory syndrome virus

We studied apoptosis caused by porcine reproductive and respiratory syndrome virus (PRRSV) in vivo, focusing on the tissues that constitute the main targets for infection: lung and lymphoid tissues. Previous investigators have shown that the PRRSV glycoprotein p25, encoded by PRRSV open reading frame 5, induces apoptosis when expressed in COS-1 cells. Results of studies conducted in our laboratory indicate the simultaneous occurrence of PRRSV-induced alterations of spermatogenesis and apoptotic death of germinal epithelial cells in the testicle. In this study, the goal was to determine whether virus-induced apoptosis is a direct mechanism of cell death caused by PRRSV in infected pigs. Eight 3-week-old pigs were intranasally inoculated with PRRSV 16244B, a highly virulent field strain. Lung, tonsil, bronchial lymph node, spleen, and heart were assessed histologically at 4 and 7 days postinfection. To characterize PRRSV-infected cells and apoptotic cell death, we used immunohistochemical methods for detection of viral antigen, DNA electrophoresis for detection of DNA fragmentation, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-fluorescein nick end labeling method for in situ detection of DNA strand breaks, and electron microscopy for ultrastructural morphologic studies. PRRSV infection resulted in widespread apoptosis in the lungs and lymphoid tissues of infected pigs. Virus infection-induced apoptotic cells were more abundant than PRRSV-infected cells in all tissues. DNA laddering was detected in lung and lymphoid tissues. However, double-labeling experiments demonstrated that the majority of apoptotic cells did not colocalize with PRRSV-infected cells. Our findings suggest the presence of an indirect mechanism in the induction of apoptosis for PRRSV.     

Inhibition of apoptosis-associated DNA fragmentation activity in nonapoptotic cells: the role of DNA fragmentation factor-45 (DFF45/ICAD)

We have investigated the mechanism whereby nuclear DNA fragmentation activity emerging during early apoptosis is inhibited during normal cell life. In a cell-free system, cytosol fractions from diverse nonapoptotic human cell lines (Jurkat T-cell leukemia, HeLa carcinoma, SK-N-MC neuroblastoma, and WI-38 embryonic lung fibroblast) potently neutralized the nuclear DNA fragmentation activity of cytosol from apoptotic anti-Fas treated Jurkat cells. Recombinant human DNA fragmentation factor 45 kDa subunit (DFF45/ICAD), an inhibitor of the caspase-activated DNase DFF40/CAD, substituted for healthy cytosol in inhibiting DNA fragmentation. An antiserum against human DFF45 detected 44 and 34 kDa proteins (major and minor, respectively) in the cytosols but not in the nuclear or membrane fractions of various cultured human cells. Cytosols depleted of DFF45/ICAD by immunoadsorption had little or no inhibitor of nuclear DNA fragmentation activity and no caspase-activated DNA fragmentation activity. We conclude that immunoreactive DFF45/ICAD is the principal inhibitor of apoptotic DNase activity in the cytosol of healthy cells.     

Spermatogenic cell apoptosis induced by mitomycin C in the mouse testis

Spermatogenic cell degeneration in the mature mammalian testis occurs both spontaneously during normal spermatogenesis and in response to cytotoxic agents. Mitomycin C (MC) is an antibiotic that affects DNA synthesis. In the present study, we examined the induction of mouse spermatogenic cell apoptosis by MC, using TdT-mediated dUTP-biotin nick end labeling (TUNEL) to detect high levels of DNA fragmentation in situ, transmission electron microscopy (TEM) to observe nuclear chromatin condensation, and molecular methods to detect DNA ladders. This study shows that in the testis of MC-treated mice: (i) apoptotic cell death with fragmentation of nuclear DNA is induced by MC dose-dependently, (ii) apoptotic cell death is most commonly found in the spermatogonia and less frequently in spermatocytes, and (iii) apoptotic cell death induced by MC is not specific for the seminiferous stage of the tubules. The present study suggests that the spermatogenic cell apoptosis induced by MC might be involved in its testicular toxicity.     

Psammomatous melanotic schwannoma]

By histopathologic, electron microscopic, and immunochemical observation, the mechanism of cellular death was investigated in thymus, spleen, and liver of mice given intraperitoneally sublethal doses of T-2 toxin, a trichothecene mycotoxin. In the thymus and spleen of mice given 5.0 mg/kg body weight of T-2 toxin and killed 12 hours later, a massive cellular destruction characterized by chromatin condensation was evident, and electron microscopy analysis revealed the presence of apoptotic bodies. In the liver of mice given 2.5 mg/kg of T-2 toxin and killed 2 hours later, the induction of apoptotic cellular lesions was observed by electron microscopy, and Kupffer cells phagocytosed the apoptotic bodies. Such lesions were not observed in the mice killed 12 hours after receiving the toxin. In situ nick translation analysis (Tunel method) revealed DNA fragmentation in thymus, spleen, and liver shortly after administration of T-2 toxin. As previously observed in vitro, these findings indicated that T-2 toxin is a potent inducer of apoptotic cell death in thymus, spleen, and liver in vivo; especially in liver, apoptosis is induced rapidly as compared with the other tissues observed, and Kupffer cells play an important role for clearance of apoptosis.     

Distinct steps in DNA fragmentation pathway during camptothecin-induced apoptosis involved caspase-, benzyloxycarbonyl- and N-tosyl-L-phenylalanylchloromethyl ketone-sensitive activities

Monocytic-like leukemia U-937 cells rapidly undergo morphological changes and DNA fragmentation that is typical of apoptosis following treatment with DNA topoisomerase I inhibitor [20-S-camptothecin lactone (CPT)]. The tripeptide derivative benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone blocks Asp-Glu-Val-Asp-ase (DEVDase) activity and prevents the occurrence of high molecular weight and oligonucleosome-sized DNA fragments associated with apoptosis in CPT-treated cells. In contrast, N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) does not prevent DEVDase activity and high molecular weight DNA fragmentation but completely abrogates the appearance of oligonucleosome-sized DNA fragmentation. These results suggest that caspase 3-like activities are involved with high molecular weight DNA fragmentation pathway, whereas TPCK-sensitive activities are involved in oligonucleosome-sized DNA fragmentation pathway in CPT-treated cells. Electron micrographs reveal that caspase inhibition by benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone also abrogates the typical morphological changes associated with apoptosis, whereas TPCK does not delay these morphological changes that are typical of apoptosis. Caspase inhibition slows passage of the cells through G2 and causes a transient accumulation of these cells at the G0/G1 phase of the cell cycle following CPT treatment. In a cell-free system, when purified nuclei are incubated with apoptotic cytosolic extracts obtained from CPT-treated U-937 cells, TPCK causes a similar effect in abrogating the oligonucleosome-sized DNA fragmentation but does not affect DEVDase activity. Addition of either benzyloxycarbonyl-Val-Ala-Asp-free carboxyl group or acetyl-Asp-Glu-Val-Asp-aldehyde completely inhibits DEVDase activity in these extracts. However, acetyl-Asp-Glu-Val-Asp-aldehyde does not affect the occurrence of oligonucleosome-sized DNA fragmentation in the cell-free system, whereas the benzyloxycarbonyl derivatives benzyloxycarbonyl-Val-Ala-Asp-free carboxyl group, benzyloxycarbonyl-Val-Ala-free hydroxyl group, benzyloxycarbonyl-Val-free hydroxyl group, and benzyloxycarbonyl hydrazide abolish it markedly. Taken together, these observations show the pivotal role of DEVDase activity in triggering the apoptotic process and high molecular weight DNA fragmentation, whereas TPCK- and benzyloxycarbonyl-sensitive activities are involved in the oligonucleosome-sized DNA fragmentation pathway induced by CPT.     

Activin A and transforming growth factor-beta stimulate heart formation in axolotls but not rescue cardiac lethal mutants

The cytotoxicity of transforming growth factor beta 1 (TGF beta 1) was assessed in rat hepatocytes cultured under periportal (PP)-or pericentral (PC)-equivalent conditions. TGF beta 1 induced a 5-fold greater DNA fragmentation and LDH release in PC cultures than in PP cultures. At low exposure level (1 ng/ml TGF beta 1), albumin secretion and mitochondrial activity (rhodamine-123 uptake) were selectively reduced in PP cultures, whereas the incidence of apoptotic cells in PC cultures was about 10-fold higher than that in PP cultures. The time profiles of TGF beta 1-induced apoptotic and necrotic events and the concentration-response relationship differed in PC and PP cultures. In PC cultures the early appearance of cells with apoptotic nuclei was not associated with DNA fragmentation nor with an increase in LDH release or impaired mitochondrial function. At a high exposure level (5 ng/ml TGF beta 1), again cells with apoptotic nuclei were much more strongly induced in PC cultures but DNA fragmentation, LDH release, and impairment of mitochondrial activity all increased in an exposure-time dependent manner in both PP and PC cultures. At this exposure level 48 and 72% of the apoptotic cells detected in PC cultures after continuous exposure for 24 hr were induced within an exposure of 1 and 4 hr, respectively. Aurintricarboxylic acid (50 microM), an inhibitor of endonucleases, significantly inhibited the appearance of apoptotic cells and the progression in apoptosis. Clearly, TGF beta 1 preferentially induced apoptotic cell death in hepatocytes with PC-equivalent metabolism at low exposure levels. High exposure levels or prolonged exposure periods produced both apoptosis and necrosis.     

Explosive school-based measles outbreak: intense exposure may have resulted in high risk, even among revaccinees

Several studies have pointed out that L-selectin on CD34-positive cells plays a role in haematopoietic reconstitution after peripheral blood stem cell (PBSC) transplantation. Since it is known that a decrease in L-selectin expression in lymphocytes and granulocytes can be induced by a variety of stress situations, we have investigated in this study whether the freeze-thawing procedure, used in PBSC transplantation, would affect L-selectin expression on CD34+ stem cells. Flow cytometry was performed by labelling the cells with anti-CD34 (HPCA2 PE) and anti-CD62L (FMC46 FITC). The leucapheresis procedure itself caused a slight decrease of L-selectin expression on CD34 cells in 11 out of 12 cases (mean decrease of the percentage of positive cells 11.9; range 6-23). A much larger decrease was found upon freeze-thawing: a mean of 39% (range 4-78% in 27 cases) compared to fresh material. To determine if L-selectin expression might be up-regulated after cryopreservation, thawed transplant samples (n = 11) were incubated at 37 degrees C in RPMI with 10% FCS at 5% CO2. Already early in the course of incubation two CD34-positive populations appeared in the blast region, characterized by either a low or high forward scatter. Simultaneous viability staining with the DNA dye 7-Amino Actinomycin D and the DNA/RNA dye Syto16 revealed that the population with low forward scatter was apoptotic while the population with the high forward scatter was non-apoptotic. The latter population is considered to be most relevant for transplantation. In this population an increase of L-selectin expression after overnight incubation was observed in 8/11 samples up to values of 46-120% of the values of the fresh nonfrozen samples. In addition, the mean fluorescence intensity was significantly increased in 10/11 cases. Kinetic experiments with shorter incubation times revealed that only part of the leucapheresis samples (two out of 8) showed an increase of L-selectin expression within 4 h. In addition, a decrease of L-selectin expression was found upon CD34 purification from fresh leucapheresis material by magnetic isolation (decrease ranging from 59 to 92%, n = 5). In contrast to frozen samples, L-selectin reappearance was seen already within 4 h of incubation in all samples. Both the loss of L-selectin expression on CD34 cells occurring upon freeze-thawing, the emergence of apoptosis, as well as the recovery of L-selectin expression on non-apoptotic cells varies largely between individual leucapheresis samples, and therefore it is concluded that such processes should be considered when correlations with clinical outcome after transplantation are made.     

Mechanisms of induction of apoptotic DNA fragmentation

PURPOSE: Despite its common use as an indicator of apoptosis, little is known about the mechanisms controlling apoptotic DNA fragmentation in irradiated cells. This review discusses the pathways of chromatin fragmentation, and the role of both nucleases and chromatin structure in this process. DEFINITIONS: DNA fragmentation linked to apoptosis is a combination of cleavage events excising both large DNA fragments within the range 0.4-1.0 Mbp and 50 kbp followed by random cuts within internucleosomal regions (i.e. DNA laddering). The first two cleavage steps can be detected in virtually all apoptotic cells, but DNA laddering is not ubiquitously observed. Endonucleases that mediate this cleavage of chromatin may be classified by substrate specificity, mode of DNA cleavage and their cofactor requirements. CONCLUSIONS: Three major pathways of DNA fragmentation are proposed and discussed: (1) upregulation of endonucleases, (2) their intranuclear/intracellular redistribution and (3) primary changes of chromatin structure.