Morphological Evidence Of Apoptosis Research Articles

Toxicity and apoptosis induced by the mycotoxins nivalenol, deoxynivalenol and fumonisin B 1 in a human erythroleukemia cell line

The toxicity of the mycotoxins nivalenol (NIV), deoxynivalenol (DON) and fumonisin B 1 (FB 1) were studied in the K562 human erythroleukemia cell line using the Trypan Blue, MTT and BrdU uptake analyses of cytotoxicity, cell metabolism and cell proliferation, respectively. Nuclear staining with propidium iodide and DNA analysis by flow cytometry were used to identify apoptosis and cell cycle distribution. By the MTT and BrdU tests, both NIV and DON were significantly more toxic than FB 1 by at least one order of magnitude, with ID 50s ranging from 0.5 μM for NIV to 70 μM for FB 1. The MTT test indicated that NIV was significantly (approximately four times) more toxic than DON. In contrast, the Trypan Blue test did not reveal any effects of mycotoxin exposure suggesting that, at the concentrations tested, NIV, DON and FB 1 did not induce cytotoxicity through plasma membrane damage. Cell cycle analysis suggested apoptotic cytotoxicity, revealing 100% cellular debris at the highest concentrations of NIV and DON and approximately 2.9 times more debris than control at the highest FB 1 concentration. Morphological evidence of apoptosis was related to the toxicity of the substances, such that the more toxic NIV and DON resulted in more late stage apoptotic events than FB 1. This study suggests that human blood cells are sensitive to mycotoxin exposure, that NIV is more toxic than DON which is more toxic than FB 1, and that DNA damage and apoptosis rather than plasma membrane damage and necrosis may be responsible for the observed cytotoxicity.

Absence of prostate apoptosis response-4 protein in substantia nigra of Parkinson's disease autopsies.

The morphological evidence for apoptosis of dopaminergic neurons in Parkinson's disease (PD) remains a conflicting issue. The present study examined autopsy material containing substantia nigra and putamen of PD ( n=7) and control subjects ( n=5) for the expression of prostate apoptosis response-4 (Par-4) protein, a protein expressed by apoptotic cells. Par-4 was not detected in the substantia nigra pars compacta. By contrast, duodenal enterocytes, which served as positive controls and are known to be apoptotic, profoundly expressed Par-4. The present study is in agreement with previous studies of the substantia nigra pars compacta in PD, which failed to detect molecules expressed by apoptotic cells. The absence of Par-4 immunoreactivity suggests that death of dopaminergic neurons in PD follows a degenerative pathway that circumvents the induction of Par-4.

Ultrastructural characterization of the erythroid cells in a novel case of congenital anemia

Congenital dyserythropoietic anemia type I (CDA-I) is a rare genetic disease that affects erythropoiesis. On the other hand, hemoglobin H (HbH) disease is a severe form of α-thalassemia. We herein present ultrastructural and immunocytochemical data concerning the first reported case of congenital anemia with clinical and molecular diagnosis of HbH disease complicated by CDA-I-specific dysplasies of the erythroid cells. Fine structure and transmission electron microscope immunolabeling analysis of the bone marrow and peripheral blood samples were consistent with a potential co-existence of the two defects in the same patient, producing a novel and diagnostically important dyserythropoietic profile. In the patient under investigation both nuclear and plasma membrane of the erythroid cells are almost equally defective. The unknown defect causes the concomitant precipitation of β- and α-globin chains (or hemoglobin), along with an unidentified protein(s). The unusual inclusions gain access to the euchromatin area and exhibit higher affinity for the plasma membrane than the classic inclusions of precipitated α- or β-globin chains seen in thalassemia. The affected erythroid precursors are presented with severe nuclear distortions, endonuclear globin loads, morphological evidence of apoptosis and increased erythrophagocytosis. Plasma membrane distortions and the rate of protein precipitation were aggravated with differentiation. Our findings provide additional evidence for a specific activation of a β-thalassemic-like mechanism in CDA-I, containing not only the hemoglobin biosynthesis as previously suggested, and interpret the prototypal hematological portrait, which is an HbH disease, modified and partially counterbalanced by the effect of CDA-I or an unidentified CDA-I-like disease. The reported data describe the complexity of the interactions between the CDA-I and the HbH disease, revealing essential pathogenic events of the novel anemia and, indirectly, of the CDA-I.

Susceptibility of sensory neurons to apoptosis following infection by bovine herpesvirus type 1.

Like other members of the alpha subfamily of herpesviruses, bovine herpesvirus type 1 (BHV-1) establishes latent infections in sensory neurons. BHV-1 induces apoptosis in lymphoid cells in vivo and in epithelial cell lines, but the ability of BHV-1 to induce apoptosis in sensory neurons remains unknown. In this report, the susceptibility of rabbit ganglionic neurons to infection by BHV-1 was examined in vitro and in vivo. Following infection of cultured neurons with BHV-1, hallmarks of apoptosis such as chromatin condensation, DNA fragmentation and membrane blebbing were detected. The appearance of these changes was preceded by active viral DNA replication as determined by in situ hybridization. When viral DNA replication was blocked by treatment of cultures with an inhibitor of eukaryotic DNA polymerases, apoptosis but not virus attachment to neurons or bICP0 gene expression was completely prevented. Taken together, these results demonstrate that sensory neurons are not intrinsically resistant to BHV-1-induced apoptosis and that viral DNA replication plays a role in triggering the apoptotic programme. Infection of rabbits with BHV-1 resulted in pathological changes in the trigeminal ganglia (TG) which included mononuclear cell infiltration and neuronophagia. Morphological evidence of apoptosis was not detected in neurons, even in cells with advanced cytophatology. Furthermore, whereas DNA fragmentation was common in infiltrating cells, it was very rare and sporadic in neurons. Therefore, mechanisms in the TG should exist to prevent neuronal apoptosis upon BHV-1 infection.

Lack of apoptosis in patients with progressive external ophthalmoplegia and mutated adenine nucleotide translocator-1 gene.

Adenine nucleotide translocator-1 (ANT-1), encoded by chromosome 4 (4q34-35 locus), is a component of the mitochondrial permeability transition pores that are involved in apoptotic mechanisms. We studied muscle biopsies from seven individuals with autosomal dominant progressive external ophthalmoplegia caused by ANT-1 mutations. We found no instance of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) positivity nor significant expression of apoptosis-related proteins. Furthermore, there was no morphological evidence of apoptosis at the ultrastructural level. Thus, degeneration of muscle in this disorder is nonapoptotic.

Increase in ceramide level alters the lysosomal targeting of cathepsin D prior to onset of apoptosis in HT-29 colon cancer cells.

Ceramide has been suggested as an important mediator of apoptosis. In HT-29 colorectal cancer cells increased ceramide levels, induced by exogenous N-acetylsphingosine (NAS, also known as C2-ceramide) or by 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol (PDMP), inhibited the transport and processing of cathepsin D (CD), a lysosomal protease implicated in apoptosis of tumour cells. C2-dihydroceramide (DH-C2), an inactive analogue of NAS, had no effect on CD transport and maturation. The treatment with either NAS or PDMP was revealed to be cytotoxic for HT-29 cells and led to cell death with classical features of apoptosis. Morphological signs of apoptosis and DNA fragmentation became apparent only between 24 and 48 h of incubation and poly(ADP ribose)-polymerase cleavage, a hallmark of caspase 3 activity, occurred no earlier than 8 h from incubation. Secretion of proCD was almost abolished and the formation of double-chain mature CD was reduced and delayed by NAS, whereas PDMP largely inhibited the lysosomal targeting and maturation of proCD. NAS- and PDMP-induced alteration of proCD transport and maturation were apparent already 2 h after incubation with the drugs, which is much earlier than when classical biochemical and morphological evidence of apoptosis could be detected. These data indicate that alteration of CD (and possibly of other glycoproteins) transport along the secretory pathway due to increased levels of cell-associated ceramide is an early event in cells undergoing apoptosis.

Alpha-MSH decreases apoptosis in ischaemic acute renal failure in rats: possible mechanism of this beneficial effect.

Apoptosis frequently occurs in acute renal injury but the molecular mechanisms responsible for this distinct form of cell death are largely unknown. Fas belongs to the tumour necrosis factor (TNF)/nerve growth factor superfamily and engagement by Fas ligand induces apoptosis in various epithelial cells. To investigate the role of apoptosis and associated mechanisms, we examined the occurrence of apoptosis and Fas and Fas ligand expression, and the therapeutic effect of alpha-melanocyte-stimulating hormone (alpha-MSH), a potent anti-inflammatory cytokine in an ischaemic acute renal failure (ARF) rat model. We also examined neutrophil infiltration together with intercellular adhesion molecule-1 (ICAM-1) expression because of their possible involvement in apoptosis due to their ability to release various inflammatory cytokines and reactive oxygen species. After unilateral nephrectomy in female Sprague-Dawley rats, the renal artery of the contralateral kidney was clamped for 40 min and reperfused. alpha-MSH or vehicle was injected intraperitoneally immediately after reperfusion and at 1, 6, or 24 h after reperfusion. The expression of Fas and Fas ligand was studied by western blot analysis and semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) method, and neutrophil infiltration by naphthol AS-D chloracetate staining. The degree of apoptosis, neutrophil infiltration, and Fas and Fas ligand, and ICAM-1 expression, as well as biochemical and histological data were compared between the alpha-MSH and the vehicle-treated groups. Intraperitoneally administered alpha-MSH significantly reduced renal injury, measured by blood urea nitrogen (BUN) and creatinine and by the degree of tubular necrosis (109.6+/-7.1/54.7+/-3.1 mg/dl for BUN, and 1.6+/-0.2/1.03+/-0.06 mg/dl for creatinine 24 h after ischaemia) (5.4+/-0.8/2.6+/-0.3 for injury score 24 h after ischaemia). Ischaemia caused an increase in Fas and Fas ligand expression and was accompanied by morphological evidence of apoptosis. alpha-MSH significantly reduced the degree of apoptosis, as well as Fas and Fas ligand expression (mean apoptotic cell number, 41.7+/-3.5/14.2+/-2.2 per x200 field at 24 h after ischaemia. Fas protein expression: sham, 1409+/-159 DI (densitometric index); vehicle/alpha-MSH, 2818+/-635/1306+/-321 DI at 24 h and 5542+/-799/2867+/-455 DI at 72 h after ischaemia. Fas ligand protein expression: sham, 1221+/-181 DI; vehicle/alpha-MSH, 2590+/-85/1279+/-169 DI at 4 h, 4376+/-268/2432+/-369 DI at 24 h and 5200+/-648/2253.7+/-1104 DI at 72 h after ischaemia). Neutrophil infiltration and ICAM-1 expression were also significantly reduced in alpha-MSH group (neutrophil infiltration: vehicle/ alpha-MSH, 5.05+/-1.8/1.59+/-0.4) (ICAM-1 expression, vehicle/alpha-MSH 0.46+/-0.21/0.29+/-0.19). These results suggest that apoptosis clearly contributes to tubular cell loss in ischaemia/reperfusion (I/R) injury possibly by neutrophil-mediated pathways or an increase in Fas-Fas ligand expression. The observed beneficial effect of alpha-MSH could be related to these mechanisms.

Bcl-2 Family Gene Expression during Severe Hyperoxia Induced Lung Injury

Exposure of the lung to severe hyperoxia induces terminal transferase dUTP end-labeling (TUNEL) indicative of DNA damage or apoptosis and increases expression of the tumor suppressor p53 and of members of the Bcl-2 gene family. Because cell survival and apoptosis are regulated, in part, by the relative abundance of proteins of the Bcl-2 family, we hypothesized that lung cells dying during exposure would show increased expression of pro-apoptotic members, such as Bax, whereas surviving cells would have increased expression of anti-apoptotic members, such as Bcl-XL. The hypothesis is tested in the current study by determining which Bcl-2 genes are regulated by hyperoxia, with specific focus on correlating expression of Bax and Bcl-XL with morphologic evidence of apoptosis or necrosis. Adult mice exposed to greater than 95% oxygen concentrations for 48 to 88 hours had increased whole-lung mRNA levels of Bax and Bcl-XL, no change in Bak, Bad, or Bcl-2, and decreased levels of Bcl-w and Bfl-1. In situ hybridization revealed that hyperoxia induced Bax and Bcl-XL mRNA in uniform and overlapping patterns of expression throughout terminal bronchioles and parenchyma, coinciding with TUNEL staining. Electron microscopy and DNA electrophoresis, however, suggested relatively little classical apoptosis. Unexpectedly, Western analysis demonstrated increased Bcl-XL, but not Bax, protein in response to hyperoxia. Bax and Bfl-1 were not altered by hyperoxia in p53 null mice; however, oxygen toxicity was not lessened by p53 deficiency. These findings suggest that oxygen-induced lung injury does not depend on the relative expression of these Bcl-2 members.

Morphological and biochemical assessment of DNA damage and apoptosis in Down syndrome and Alzheimer disease, and effect of postmortem tissue archival on TUNEL

We have previously shown that Alzheimer disease (AD) brain exhibits terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) for DNA damage and morphological evidence for apoptosis. Down syndrome (DS) is a neurodegenerative disorder that exhibits significant neuropathological parallels with AD. In accordance with these parallels and the need to clarify the mechanism of cell death in DS and AD, we investigated two principal issues in the present study. First, we investigated the hypothesis that TUNEL labeling for DNA damage and morphological evidence for apoptosis is also present in the DS brain. All DS cases employed had a neuropathological diagnosis of AD. Analysis of these cases showed that DS brain exhibits a significant increase in the number of TUNEL-labeled nuclei relative to controls matched for age, Postmortem Delay, and Archival Length, and that a subset of TUNEL-positive nuclei exhibits apoptotic morphologies. We also report that Archival Length in 10% formalin can significantly affect TUNEL labeling in postmortem human brain, and therefore, that Archival Length must be controlled for as a variable in this type of study. Second, we investigated whether biochemical evidence for the mechanism of cell death in DS and AD could be detected. To address this question we employed pulsed-field gel electrophoresis (PFGE) as a sensitive method to evaluate DNA integrity. Although apoptotic oligonucleosomal laddering has not previously been observed in AD, PFGE of DNA from control, DS and AD brain in the present study revealed evidence of high molecular weight DNA fragmentation indicative of apoptosis. This represents biochemical support for an apoptotic mechanism of cell death in DS and AD.

Neuronal and glial DNA fragmentation in Pick's disease.

Pick's disease (PD) is characterized by severe neuronal loss and gliosis in a frontotemporal lobar distribution, often associated with Pick bodies and ballooned neurons. Abnormal tau metabolism has been implicated in the pathogenesis of PD; however, the underlying mechanism of neuronal degeneration remains poorly understood. Evidence from other neurodegenerative diseases has suggested that DNA damage and apoptosis may play a major role in cellular degeneration and death. In the present study, an in situ nucleotidyl transferase assay (ISNTA) was used to identify DNA fragmentation in three cases of classical PD with Pick bodies and ballooned neurons, and two PD "variants", one with ballooned neurons only and the other without Pick bodies or ballooned neurons. In all cases large numbers of ISNTA-positive neurons were present in anatomic regions having obvious degenerative changes (neuronal atrophy and loss, gliosis, cytoplasmic inclusions) by conventional histology. There was no clear association between neuronal DNA fragmentation and the presence of structural abnormalities such as Pick bodies or ballooned cytoplasm. ISNTA-positive glia were present in both cortex and subcortical white matter. Morphologic evidence of apoptosis was not detected in either neurons or glial cells. We suggest that DNA fragmentation in PD and probably other neurodegenerative disorders most likely specifies a population of potentially vulnerable cells in which both cell death and repair mechanisms have been activated. It is likely that only a very small number of these vulnerable cells at a given time will proceed to cell death; however, it is uncertain whether this occurs by apoptosis or some other mechanism.

Extracellular phosphate ions cause apoptosis of terminally differentiated epiphyseal chondrocytes.

Epiphyseal chondrocytes end their life cycle through apoptosis. While this event provides a mechanism for the removal of terminally differentiated cells from cartilage, agents that promote this physiological process have not been defined. To address this issue, using a cell culture technique that models events that take place in the growth plate, we asked the following questions: Can agents that promote chondrocyte maturation and cartilage mineralization serve as specific triggers for cell death? Are chondrocytes susceptible to apoptogens at a singular developmental stage? Treatment of embryonic tibial chondrocytes with inorganic phosphate (Pi) induced death in a dose- and time-dependent manner. Within 48 hr, 3 mM Pi increased chondrocyte death by 30%; lower concentrations of Pi induced death after 48 hr. To ascertain if death was due to apoptosis, we evaluated Pi-induced death by a number of different methods and compared the results to those induced by the apoptogen, staurosporine. Analysis of the death process indicated that cartilage cells shared many of the common biological features of the apoptotic process. Thus, there was DNA fragmentation, terminal deoxynucleotidyl transferase (TUNEL) labeling, an increase in cells in the sub-G1 fraction of the cell cycle, and morphological evidence of apoptosis. To explore the specificity of the Pi effect, the experiment was repeated using embryonic sternal cephalic and caudal chondrocytes, cells that are at an earlier developmental stage than the terminally differentiated tibial cells. We noted that these cells remained vital despite a major increase in the medium Pi content. Results of this study suggest that Pi is a stage-specific inducer of apoptosis in maturing chondrocytes and that this role may be linked to chondrocyte maturation and mineralization of the extracellular matrix.

Continuing postischemic neuronal death in CA1: influence of ischemia duration and cytoprotective doses of NBQX and SNX-111 in rats.

Transient forebrain ischemia results in a 24- to 72-hour delayed loss of CA1 neurons. Previous work has not assessed whether insult durations can vary the degree and maturation rate of CA1 injury and whether there are different ultrastructural features of death after brief or severe ischemia. We also tested whether known cytoprotective drugs achieve permanent or transient neuroprotection. In the first experiment, ischemia was induced for 5, 15, or 30 minutes with the use of the 4-vessel occlusion rat model with 1- to 28-day survival. Others subjected to 5 or 15 minutes of ischemia and allowed to survive for 14 or 7 days, respectively, were examined with electron microscopy. Finally, we determined whether NBQX (30 mg/kg x3 at 0 or 6 hours after ischemia), an AMPA antagonist, and SNX-111 (5 mg/kg at 6 hours after ischemia), an N-type Ca2+ channel antagonist, provided enduring CA1 protection against 10 minutes of ischemia. CA1 damage was not detected at 24 hours. Thirty minutes of ischemia produced 47% and 84% CA1 damage at 2 and 3 days, respectively. A 15-minute occlusion yielded 11%, 74%, and 86% loss at 2, 3, and 7 days, respectively. Five minutes of ischemia produced an even slower progression with 24%, 52%, and 59% loss at 3, 7, and 14 days, respectively. Ultrastructural examination after 5 and 15 minutes of ischemia revealed necrosis with no morphological evidence of apoptosis. Both NBQX (P<0.021) and SNX-111 (P<0.001) significantly reduced CA1 death at 7 days (</=35%) but not at 28 days (>/=80%) compared with saline treatment ( approximately 79%). Brief forebrain ischemia results in a slower progression of CA1 loss than more severe insults. Nonetheless, neuronal injury had necrotic, not apoptotic, morphology. NBQX and SNX-111 only postponed CA1 injury.

P3A13 - Implication of lipid peroxidation in the cytotoxicity and genotoxicity of fumonisin B 1

The toxicity of the mycotoxins nivalenol (NIV), deoxynivalenol (DON) and fumonisin B1 (FB1) were studied in the K562 human erythroleukemia cell line using the Trypan Blue, MTT and BrdU uptake analyses of cytotoxicity, cell metabolism and cell proliferation, respectively. Nuclear staining with propidium iodide and DNA analysis by flow cytometry were used to identify apoptosis and cell cycle distribution. By the MTT and BrdU tests, both NIV and DON were significantly more toxic than FB1 by at least one order of magnitude, with ID50s ranging from 0.5 μM for NIV to 70 μM for FB1. The MTT test indicated that NIV was significantly (approximately four times) more toxic than DON. In contrast, the Trypan Blue test did not reveal any effects of mycotoxin exposure suggesting that, at the concentrations tested, NIV, DON and FB1 did not induce cytotoxicity through plasma membrane damage. Cell cycle analysis suggested apoptotic cytotoxicity, revealing 100% cellular debris at the highest concentrations of NIV and DON and approximately 2.9 times more debris than control at the highest FB1 concentration. Morphological evidence of apoptosis was related to the toxicity of the substances, such that the more toxic NIV and DON resulted in more late stage apoptotic events than FB1. This study suggests that human blood cells are sensitive to mycotoxin exposure, that NIV is more toxic than DON which is more toxic than FB1, and that DNA damage and apoptosis rather than plasma membrane damage and necrosis may be responsible for the observed cytotoxicity.

DNA fragmentation reduced by antioxidants following ischaemia‐reperfusion in the isolated perfused rat kidney

SUMMARY: The role of oxygen‐derived free radicals (OFR) in modifying structure and function after ischaemia‐reperfusion (IR) injury was studied in isolated perfused rat kidneys (IPRK). Control kidneys were studied after 20 min of ischaemia followed by 15 or 60 min of reperfusion. the xanthine oxidase inhibitor allopurinol and the hydroxyl radical scavenger dimethylthiourea (DMTU) were used to prevent OFR‐related damage. Morphological injury was assessed in cortex, inner and outer medulla and compared with indices of global renal function (inulin clearance, fractional sodium excretion and renal vascular resistance). Apoptosis was assessed using both morphological criteria and in situ end‐labelling (ISEL) to identify DNA fragmentation. Tubular damage, as evidenced by cellular blebbing, tubular cast formation, epithelial necrosis, and occasional apoptosis, was greatest in the straight proximal tubule and thick ascending limb (TAL) in the outer zone of the outer medulla. Pretreatment with allopurinol or DMTU did not significantly improve renal function. However, structural damage and luminal debris were diminished in allopurinol‐ and DMTU‐treated kidneys. These changes may lead to functional improvement after more prolonged reperfusion. In situ end‐labelling was more frequent in distal tubular epithelial cells after IR than either morphological evidence of apoptosis or necrosis. Decreased ISEL was observed after pretreatment with both allopurinol and DMTU. the data demonstrate that OFR produce DNA damage after IR, increasing ISEL. This probably represents reversible DNA damage rather than incipient apoptosis. Thus, antioxidants reduce or prevent DNA and cellular injury after IR and may reduce functional impairment after prolonged reperfusion.

The myelotoxicity of chloramphenicol: in vitro and in vivo studies: II: in vivo myelotoxicity in the B6C3F mouse

1. Chloramphenicol continues to be widely used in many parts of the world despite its known haematotoxicity. Until now, elucidation of the mechanisms involved and any attempt at amelioration of the toxic effects have been hampered by the lack of an animal model. 2. In this study neither acute nor chronic administration of chloramphenicol as its succinate ester in the drinking water produced anaemia in mice as assessed by changes in peripheral blood parameters. 3. Chloramphenicol could not be detected in the bone marrow when the antibiotic was administered either in the drinking water or by gavage, although it was detected in the serum. 4. In marrow taken from mice after chloramphenicol succinate administration and cultured in vitro, depression of the differentiation of immature committed erythroid progenitors occurred 15 min after administration of the antibiotic by gavage. However, recovery was beginning to occur at 48 h after administration of chloramphenicol succinate at 50 and 200 mg/kg and this was then followed by an 'overshoot' response at the higher dose. A toxic effect was therefore achieved in the bone marrow but this was probably masked in the peripheral blood by enhanced proliferation. 5. Morphological evidence of apoptosis was seen in erythroid and myeloid precursors in mice treated with 200 mg/kg. 6. The data suggest that the effect of chloramphenicol was at the differentiation stage of the committed marrow progenitor cells rather than at the replication stage of the stem cells and therefore this response appears to mimic the reversible bone marrow depression seen in the treated patient.

Multinucleated variant endothelial cells (MVECs) of human aorta: expression of tumor suppressor gene p53 and relationship to atherosclerosis and aging.

Multinucleated variant endothelial cells (MVECs) generally exist in atherosclerotic human aorta and even in nonatherosclerotic aorta. Because the number of nuclei is increased in every MVEC, and because DNA instability was suspected, a series of oncogene expressions was conducted to clarify the nature of nuclear abnormality. The tumor suppressor gene p53 was found to be specifically expressed in the multinuclei of MVECs, while double nuclei were sometimes positive, and mononuclear typical endothelial cells were always negative for p53. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) revealed extra bands in exons 5 and 7 of the p53 gene, but no additional band in exons 6 and 8. In a BCL family, BCL-2 was coexpressed in one or two nuclei in the perinuclear space of the multinuclei of MVECs, whereas MCL-1, BCL-XS/L, and BAX were all negative, indicating that the BCL-2 coding gene is expressed only in the corresponding one or two nuclei of the multinuclei. Another oncogene, c-MET (hepatocyte growth factor receptor), was universally expressed in either type of endothelial cells, but other oncogenes, k-RAS and c-ERBB2, were not expressed in either type. MVECs were derived from human aorta and therefore non-tumorous somatic cells. No morphologic evidence of apoptosis was found. Although it is unclear that the extra bands came from the MVECs or just from ECs associated with atherosclerosis, combined immunocytological studies and PCR analysis suggest that MVECs express mutant type p53.

Morphological evidence of apoptosis in chickens infected with infectious bursal disease virus

Two-week-old specific pathogen-free chickens were infected with the infectious bursal disease virus by the ocular route. Immediately, and at 4 and 8 days after infection, groups of three chickens were killed and tissue samples were collected. Under electron microscopical examination, typical apoptotic cells were seen in the bursa of Fabricius (BF) and in the spleen. Tissue sections stained with haematoxylin and eosin were subjected to image analysis to quantify cellular depletion in the follicles of the BF. Unstained sections were treated with a terminal deoxy-transferase-based kit to detect apoptotic cells. The numbers of apoptotic cells at different stages of infection were counted by image analysis. The results revealed a rapid depletion of cells in the BF and a simultaneous increase in apoptotic cells.

Morphological evidence of apoptosis and the prevalence of apoptotic versus mitotic cells in the membrana granulosa of ovarian follicles during spontaneous and induced atresia in ewes.

Apoptosis is a process by which granulosa cells are thought to be deleted during ovarian follicular atresia. The aims of the present studies, using sheep as the experimental model, were to determine 1) whether morphological changes in cells composing the membrana granulosa during the process of atresia conformed with the general criteria of apoptotic cell death as assessed using tissue sections stained with hematoxylin and eosin; 2) whether cells classified as apoptotic on the basis of their morphology contained fragmented DNA using an in situ 3' end-labeling technique; and 3) the degree of apoptosis and mitosis within the granulosa cell populations of large antral follicles (> or = 3 mm in diameter) during both spontaneous and experimentally induced atresia using stereological methods. The results showed that most degenerate granulosa cells in follicles undergoing atresia display the morphological characteristics of apoptosis, suggesting that this is the most common pathway of cell deletion. Typical features were cells containing nuclei with marginated chromatin; cells with a single small densely staining nucleus (pyknotic appearance); cells with multiple smaller, densely staining nuclear fragments; and densely staining membrane-bound bodies (apoptotic bodies) either singly or in clusters. Cells with morphological features more typical of oncosis or necrosis were sometimes observed, but mainly during the later stages of atresia. All cells classified as apoptotic on the basis of morphological criteria contained fragmented DNA as measured by 3' end-labeling. Apoptotic bodies and/or cells were found in all follicles examined, including those classified as healthy. The overall prevalence of apoptotic cells plus apoptotic bodies expressed as a percentage of the total granulosa cell number per follicle varied from 0.02% to 0.20% in healthy follicles, varied from 0.21% to 2.00% in follicles in early (primary) atresia, and was > 2.0% in follicles in later (secondary) atresia. Percentages of mitotic cells in healthy follicles were > 0.5% in all but one of those examined and were < 1.0% in all follicles classified as atretic. Both morphological and 3' end-labeling results indicated that apoptotic cells were widely disseminated throughout the membrana granulosa, including the cell layer adjacent to the basement membrane. Collectively, these observations indicate that during early atresia, apoptosis occurs randomly and is not limited to specific areas within follicles. Our finding that apoptotic cell death and mitosis occur simultaneously within the same follicle is consistent with the notion that atresia is determined by a dynamic equilibrium between cell division, differentiation, and death.

P53 and Bax activation in 6-hydroxydopamine-induced apoptosis in PC12 cells

p53, Bax and Bcl-x L proteins have been implicated in apoptotic neuronal cell death. We have investigated whether those proteins are involved in 6-OHDA-induced PC12 cell death. After a 24-h exposure to the neurotoxin (100 μM), morphological evidence for apoptosis was observed in PC12 cells. Up-regulation of p53 and Bax proteins was demonstrated 4 and 6 h, respectively, after 6-OHDA treatment; in contrast, no change in Bcl-x L levels was found. These findings suggest that p53 and Bax could be relevant markers of neuronal apoptosis as previously described in kainic acid- or ischemia-induced neuronal cell death and may participate to neuronal degeneration in Parkinson's disease. © 1997 Elsevier Science B.V. All rights reserved.

Apoptosis in the pancreas of genetically diabetic rats with a disrupted cholecystokinin (CCK-A) receptor gene.

In a previous study, we reported that the pancreatic wet weight in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, cholecystokinin-A (CCK-A) receptor-defective because of a congenital gene abnormality, was significantly lower than in control rats (Long-Evans Tokushima Otsuka; LETO) from 3 weeks of age. In this study we examined apoptosis of pancreatic acinar cells in OLETF rats at 5 to 6 weeks of age in comparison with that in LETO rats. We present here direct morphologic evidence of apoptosis in OLETF rats, using a 3'-OH nick end-labeling method for detecting cells with DNA strand breaks and electron microscopy. Nick end-labeling revealed a small number of positively labeled acinar cells in OLETF rats. On electron microscopic examination, small numbers of apoptotic cells were seen in the lobules in OLETF rats but not in LETO rats. These results suggest that apoptosis plays an important role in the destruction of acinar cells of OLETF rats and induces atrophy of the pancreas.