Heterochromatin Condensation Research Articles

Nitric oxide and reactive oxygen species do not elicit hypersensitive cell death but induce apoptosis in the adjacent cells during the defense response of oat.

Nitric oxide (NO) acts as a signaling molecule in many cellular responses in plants and animals. Oat plants (Avena sativa L.) evoke the hypersensitive response (HR), which shares morphological and biochemical features with mammalian apoptosis, such as DNA laddering and heterochromatin condensation, in response to the avirulent crown rust fungus (Puccinia coronata f. sp. avenae). We examined the role of NO and reactive oxygen species (ROS) in the initiation of hypersensitive cell death, which is induced by direct contact with the pathogen, and apoptotic cell death in the adjacent cells. Cytofluorimetric analysis using the fluorescent NO probe DAF and the H2O2 probe DCF demonstrated that NO and H2O2 were generated simultaneously in primary leaves at an early stage of the defense response. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) markedly enhanced H2O2 accumulation detected by 3,3-diaminobenzidine staining and DCF, whereas treatment with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) strongly suppressed it. Superoxide dismutase (SOD) increased NO accumulation, suggesting that endogenous NO may modulate the level of H2O2 by interacting with O2- in the HR lesion. Cytological observation showed that administration of cPTIO, SNAP, or SOD had no effect on elicitation of hypersensitive cell death, but clearly reduced heterochromatin condensation in the nearby cells and DNA laddering. These findings indicate that NO and ROS are not essential mediators for the initiation of hypersensitive cell death. However, NO and O2- but not H2O2 are required for the onset of apoptotic cell death in the adjacent cells, where excess NO may exert its anti-apoptotic function by regulating cellular redox state.

Dying to Make Platelets

Two groups, de Botton et al. and Clarke et al. , showed that platelet formation occurs through a variation of apoptosis involving compartmentalized caspase activation. Megakaryocytes, the platelet precursor cells, undergo a morphological reorganization in which they extend processes called proplatelets, which develop nodes that are eventually released as mature platelets. De Botton et al. found that caspase inhibitors blocked thrombopoietin-stimulated proplatelet formation in cultured human megakaryocytes, as did overexpression of Bcl-2 (an antiapoptotic protein). The authors used Western analysis to demonstrate proteolytic maturation of caspase-3 and caspase-9 and cleavage of caspase substrates during megakaryocyte maturation. Confocal microscopy analysis of immunofluorescently labeled cells indicated that punctate, localized caspase-3 activation occurred before proplatelet formation. Furthermore, early localized caspase-3 activation was associated with mitochondrial release of cytochrome c but not DNA fragmentation or cell death. Clarke et al. found that caspase inhibitors blocked platelet production and proplatelet formation in a human megakaryoblastic cell line that spontaneously generated functional platelets. Fluorescence analysis indicated that the cell bodies of proplatelet-bearing cells contained activated caspases but the proplatelets did not. Cells developing proplatelets showed heterochromatin condensation consistent with early apoptosis. Fas ligation stimulated proplatelet formation, platelet production, and nuclear condensation, and augmented platelet production by primary cultures of mouse megakaryocytes and human bone core explants. The authors used a fluorescent mitochondrial dye to show that proplatelet mitochondria maintained their membrane potentials in cells showing nuclear condensation. Dying platelets cultured in the absence of survival factors, however, showed collapsed mitochondrial membrane potentials and released cytochrome c. Western analysis together with caspase-activating treatments suggested that platelets lacked caspase-9. S. de Botton, S. Sabri, E. Daugas, Y. Zermati, J. E. Guidotti, O. Hermine, G. Kroemer, W. Vainchenker, N. Debili, Platelet formation is the consequence of caspase activation within megakaryocytes. Blood 100 , 1310-1317 (2002). [Online Journal] M. C. H. Clarke, J. Savill, D. B. Jones, B. S. Noble, S. B. Brown, Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death. J. Cell Biol . 160 , 577-587 (2003). [Abstract] [Full Text]

Dying to Make Platelets

Two groups, de Botton et al. and Clarke et al., showed that platelet formation occurs through a variation of apoptosis involving compartmentalized caspase activation. Megakaryocytes, the platelet precursor cells, undergo a morphological reorganization in which they extend processes called proplatelets, which develop nodes that are eventually released as mature platelets. De Botton et al. found that caspase inhibitors blocked thrombopoietin-stimulated proplatelet formation in cultured human megakaryocytes, as did overexpression of Bcl-2 (an antiapoptotic protein). The authors used Western analysis to demonstrate proteolytic maturation of caspase-3 and caspase-9 and cleavage of caspase substrates during megakaryocyte maturation. Confocal microscopy analysis of immunofluorescently labeled cells indicated that punctate, localized caspase-3 activation occurred before proplatelet formation. Furthermore, early localized caspase-3 activation was associated with mitochondrial release of cytochrome c but not DNA fragmentation or cell death. Clarke et al. found that caspase inhibitors blocked platelet production and proplatelet formation in a human megakaryoblastic cell line that spontaneously generated functional platelets. Fluorescence analysis indicated that the cell bodies of proplatelet-bearing cells contained activated caspases but the proplatelets did not. Cells developing proplatelets showed heterochromatin condensation consistent with early apoptosis. Fas ligation stimulated proplatelet formation, platelet production, and nuclear condensation, and augmented platelet production by primary cultures of mouse megakaryocytes and human bone core explants. The authors used a fluorescent mitochondrial dye to show that proplatelet mitochondria maintained their membrane potentials in cells showing nuclear condensation. Dying platelets cultured in the absence of survival factors, however, showed collapsed mitochondrial membrane potentials and released cytochrome c. Western analysis together with caspase-activating treatments suggested that platelets lacked caspase-9.S. de Botton, S. Sabri, E. Daugas, Y. Zermati, J. E. Guidotti, O. Hermine, G. Kroemer, W. Vainchenker, N. Debili, Platelet formation is the consequence of caspase activation within megakaryocytes. Blood 100, 1310-1317 (2002). [Online Journal]M. C. H. Clarke, J. Savill, D. B. Jones, B. S. Noble, S. B. Brown, Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death. J. Cell Biol. 160, 577-587 (2003). [Abstract] [Full Text]

Novel actin-related proteins in vertebrates: similarities of structure and expression pattern to Arp6 localized on Drosophila heterochromatin

Actin-related proteins (Arps), which share a basal structure with actin isoforms but possess different functions, have been identified in a wide variety of organisms. The Arps are classified into subfamilies based on the relatedness of their sequences and functions. Recently, several Arp subfamilies have been shown to be localized in the nucleus and included in protein complexes involved in the organization of chromatin structure, for example, in chromatin remodeling and histone acetyltransferase complexes. A member of the Arp6 subfamily in Drosophila, dArp6, is localized on centric heterochromatin together with heterochromatin protein 1 (HP1). We have identified the first examples of the Arp6 subfamily in vertebrates, novel human and chicken Arps, hArp6 and gArp6, respectively. They are closely related to each other (98% similar) and show apparent similarity to dArp6 (70%). In addition, the hArp6 gene possesses evolutionarily conserved exon/intron structures compared with genes for members of the Arp6 subfamily in invertebrates. Like Drosophila dArp6, gArp6 is expressed abundantly in the early developmental stages, when heterochromatin condensation and nuclear maturation occur. The finding of a conserved Arp6 subfamily in vertebrates will contribute to the understanding of molecular mechanisms of heterochromatin organization.

Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana.

The genome of the model plant Arabidopsis thaliana has been sequenced by an international collaboration, The Arabidopsis Genome Initiative. Here we report the complete sequence of chromosome 5. This chromosome is 26 megabases long; it is the second largest Arabidopsis chromosome and represents 21% of the sequenced regions of the genome. The sequence of chromosomes 2 and 4 have been reported previously and that of chromosomes 1 and 3, together with an analysis of the complete genome sequence, are reported in this issue. Analysis of the sequence of chromosome 5 yields further insights into centromere structure and the sequence determinants of heterochromatin condensation. The 5,874 genes encoded on chromosome 5 reveal several new functions in plants, and the patterns of gene organization provide insights into the mechanisms and extent of genome evolution in plants.

The Drosophila mus101 gene, which links DNA repair, replication and condensation of heterochromatin in mitosis, encodes a protein with seven BRCA1 C-terminus domains.

The mutagen-sensitive-101 (mus101) gene of Drosophila melanogaster was first identified 25 years ago through mutations conferring larval hypersensitivity to DNA-damaging agents. Other alleles of mus101 causing different phenotypes were later isolated: a female sterile allele results in a defect in a tissue-specific form of DNA synthesis (chorion gene amplification) and lethal alleles cause mitotic chromosome instability that can be observed genetically and cytologically. The latter phenotype presents as a striking failure of mitotic chromosomes of larval neuroblasts to undergo condensation of pericentric heterochromatic regions, as we show for a newly described mutant carrying lethal allele mus101(lcd). To gain further insight into the function of the Mus101 protein we have molecularly cloned the gene using a positional cloning strategy. We report here that mus101 encodes a member of the BRCT (BRCA1 C terminus) domain superfamily of proteins implicated in DNA repair and cell cycle checkpoint control. Mus101, which contains seven BRCT domains distributed throughout its length, is most similar to human TopBP1, a protein identified through its in vitro association with DNA topoisomerase IIbeta. Mus101 also shares sequence similarity with the fission yeast Rad4/Cut5 protein required for repair, replication, and checkpoint control, suggesting that the two proteins may be functional homologs.

Cloning and characterization of an extremely conserved satellite DNA family from the root-knot nematode Meloidogyne arenaria.

A new satellite DNA family, named pMaE, has been cloned from the genome of the phytoparasitic nematode, Meloidogyne arenaria (Nematoda: Tylenchida). It is represented as tandemly repeated sequences with a monomeric unit of 172 bp. The monomers are present at approximately 15700 copies per haploid genome, and represent about 5.3% of the total genomic DNA. Twenty-seven independent monomers have been cloned and sequenced. The deduced consensus sequence is 70.9% A + T rich, with frequent stretches of A and (or) T. Several direct or inverted sub-repeats are present in the sequence, which may allow the formation of a dyad structure, suggesting some potential role of this repetitive sequence in heterochromatin condensation. The monomers are very homogeneous in sequence, showing on average 1.8% divergence from their consensus sequence. Moreover, Southern blot experiments and sequence analysis of homologous monomers from the genome of geographically distinct M. arenaria populations have shown that this satellite DNA is uniformly distributed and highly conserved within the species. Therefore, it is hypothesized that this unusually low level of variability, either within the genome of a given population or between populations, could be achieved as the result of some highly effective homogenization mechanism acting upon the nematode genome.

Myelokathexis, a congenital disorder of severe neutropenia characterized by accelerated apoptosis and defective expression ofbcl-x in neutrophil precursors

Myelokathexis is a congenital disorder that causes severe chronic leukopenia and neutropenia. Characteristic findings include degenerative changes and hypersegmentation of mature neutrophils and hyperplasia of bone marrow myeloid cells. The associated neutropenia can be partially corrected by treatment with granulocyte colony-stimulating factor (G-CSF) or granulocyte–macrophage colony-stimulating factor (GM-CSF). These features led us to propose that accelerated apoptosis of neutrophil precursors might account for the neutropenic phenotype. Blood and bone marrow aspirates were obtained from 4 patients (2 unrelated families) with myelokathexis before G-CSF therapy and from 2 of the affected persons after G-CSF therapy (1 μg/kg per day subcutaneously for 3 weeks). Bone marrow was fractionated using immunomagnetic bead cell sorting into CD34+, CD33+/CD34−, and CD15+/CD34−/CD33− cell populations. Examination of these cells by flow cytometry and electron microscopy revealed abundant apoptosis in the CD15+ neutrophil precursor population, characterized by enhanced annexin-V binding, extensive membrane blebbing, condensation of heterochromatin, and cell fragmentation. Colony-forming assays demonstrated significant reduction in a proportion of bone marrow myeloid-committed progenitor cells. Immunohistochemical analysis revealed a selective decrease inbcl-x, but not bcl-2, expression in the CD15+/CD34−/CD33− cell population compared with similar subpopulations of control bone marrow-derived myeloid precursors. After G-CSF therapy, apoptotic features of patients' bone marrow cells were substantially reduced, and the absolute neutrophil counts (ANC) and expression ofbcl-x in CD15+/CD34−/CD33−cells increased. The authors concluded that myelokathexis is a disease characterized by the accelerated apoptosis of granulocytes and the depressed expression of bcl-x in bone marrow-derived granulocyte precursor cells. These abnormalities are partially corrected by the in vivo administration of G-CSF. (Blood. 2000;95:320-327)

Myelokathexis, a congenital disorder of severe neutropenia characterized by accelerated apoptosis and defective expression ofbcl-x in neutrophil precursors

AbstractMyelokathexis is a congenital disorder that causes severe chronic leukopenia and neutropenia. Characteristic findings include degenerative changes and hypersegmentation of mature neutrophils and hyperplasia of bone marrow myeloid cells. The associated neutropenia can be partially corrected by treatment with granulocyte colony-stimulating factor (G-CSF) or granulocyte–macrophage colony-stimulating factor (GM-CSF). These features led us to propose that accelerated apoptosis of neutrophil precursors might account for the neutropenic phenotype. Blood and bone marrow aspirates were obtained from 4 patients (2 unrelated families) with myelokathexis before G-CSF therapy and from 2 of the affected persons after G-CSF therapy (1 μg/kg per day subcutaneously for 3 weeks). Bone marrow was fractionated using immunomagnetic bead cell sorting into CD34+, CD33+/CD34−, and CD15+/CD34−/CD33− cell populations. Examination of these cells by flow cytometry and electron microscopy revealed abundant apoptosis in the CD15+ neutrophil precursor population, characterized by enhanced annexin-V binding, extensive membrane blebbing, condensation of heterochromatin, and cell fragmentation. Colony-forming assays demonstrated significant reduction in a proportion of bone marrow myeloid-committed progenitor cells. Immunohistochemical analysis revealed a selective decrease inbcl-x, but not bcl-2, expression in the CD15+/CD34−/CD33− cell population compared with similar subpopulations of control bone marrow-derived myeloid precursors. After G-CSF therapy, apoptotic features of patients' bone marrow cells were substantially reduced, and the absolute neutrophil counts (ANC) and expression ofbcl-x in CD15+/CD34−/CD33−cells increased. The authors concluded that myelokathexis is a disease characterized by the accelerated apoptosis of granulocytes and the depressed expression of bcl-x in bone marrow-derived granulocyte precursor cells. These abnormalities are partially corrected by the in vivo administration of G-CSF. (Blood. 2000;95:320-327)

Ultrastructural and biochemical observations on the early changes in apoptotic epithelial cells of the rat prostate induced by castration.

The present study describes the sequential ultrastructural changes in the apoptotic cells of the rat ventral and dorsal prostates during the early period of 1-3 days postcastration. The major morphological changes include: (1) condensation of heterochromatin along the nuclear envelope and fragmentation into crescent-shaped micronuclei; (2) formation of membrane-bound cytoplasmic spherical bodies, which contain various organelles and micronuclei, within the apoptotic cells; (3) formation of non-membrane-bound autolytic vacuoles by autolysis of cytoplasm; (4) focal rupture of outer mitochondrial membrane; and (5) phagocytosis of the fragmented cytoplasmic spherical bodies and apoptotic cells by macrophages. The occurrence of both cytoplasmic apoptotic bodies and autolytic vacuoles in apoptotic cells suggests that the cytoplasm of the apoptotic cells could be destroyed by different means. The responsiveness of different prostatic lobes to androgen withdrawal and the time course of the transitory apoptotic activity in different lobes were analyzed by counting the indices of the TUNEL-labeled apoptotic cells against the postcastration periods. The results showed that the ventral lobe responded more rapidly to castration than the lateral and dorsal lobes. The dorsal lobe was the slowest in response to castration among the three lobes. Analysis of protease activities by zymography has identified two Ca(2+)-independent proteases of apparent MW 20 and 24 kDa (expressed in both ventral and dorsolateral lobes), and one Ca(2+)-dependent protease of MW 66.5 kDa (expressed only in the dorsolateral lobe) which became activated at day 3 postcastration. Their expression patterns were different from that of CPP-3 in the castrated prostates, suggesting that the activated proteases were enzymes other than CPP-3. The association of their highest activities with the maximum apoptotic activity at day 3 postcastration and also their loss of activity at day 15 suggest that these protease activities might be related to apoptosis or glandular involution.

Genomic alterations in the interspecific hybrid Helianthus annuus×Helianthus tuberosus

The genome of a Helianthus annuus (2n=34) ×Helianthus tuberosus (2n=102) hybrid was studied at cytological, biochemical and molecular levels and compared to those of the parental species. Cytophotometric analyses showed that the hybrid has a 4C DNA content higher than expected and with a larger variability than in the parents. This high variability is probably not related to chromosome-number variations since the hybrid always had 2n=68 chromosomes. Moreover, hybrid interphase nuclei showed lower heterochromatin condensation than the parental ones. Thermal denaturation of genomic DNAs indicated that quantitative variation of some DNA families occurred in the hybrids compared to parents. Finally, molecular analyses of DNAs restricted with different enzymes, after Southern blotting and hybridization with HR probes, showed restriction patterns in the hybrid different from those observed in parents. These results indicate that interspecific hybridization between H. annuus and H. tuberosus may determine quantitative variation of some DNA families and differential DNA methylations that probably modify the nuclear structure. These phenomena are probable responses to a “genomic shock” following the interspecific cross.

The influence of 5-azacytidine on the condensation of the short arm of rye chromosome 1R in Triticum aestivum L. root tip meristematic nuclei.

This paper describes the effects of 5-azacytidine on the condensation state of rye (Secale cereale L.) chromatin introduced into the wheat genome (Triticum aestivum L. cv. Beaver). The wheat cultivar Beaver carries a translocation between the short arm of rye chromosome 1R (1RS) and the long arm of wheat chromosome 1B (1BL/1RS). 1RS can be detected using genomic in situ hybridisation and carries a ribosomal DNA (rDNA) locus that can be simultaneously detected using multiple labelling strategies. The rDNA locus divides 1RS into a distal region that is gene rich and a proximal region that is gene poor and highly methylated. 1RS also carries a large block of subtelomeric heterochromatin. The drug, which acts to inhibit DNA methylation in plants, has three pronounced effects on interphase nuclei. (1) It induces aberrant condensation of the rye subtelomeric heterochromatin and in many cases induces sister chromatid separation in the subtelomeric heterochromatin of G2 nuclei. (2) Nuclei trisomic for 1RS are observed at low frequency in treated material and are probably a consequence of aberrant sister chromatid separation or condensation. (3) The drug alters normal condensation of 1RS euchromatin. However, contrary to expectation the effect is not simply to induce decondensation. The proximal region of the arm actually condenses at low levels of drug administration while the distal region remains unaltered or increases its decondensation state. Increasing the concentration of 5-azacytidine induces a biphasic response and at the highest concentration used all regions of the arm show signs of decondensation. Thus the influence of the drug on chromatin condensation depends on the genomic structure.

Ultrastructure of rat lung following inhalation of ricin aerosol.

Ricin is one of a group of structurally related plant lectins and is extracted from the seeds of the Castor Oil plant, Ricinus communis. Groups of rats were exposed to ricin aerosol by inhalation, total LCt1-11.21 mg.min.m-3 (an approximate LCt30 exposure) and examined, using transmission electron microscopy, at intervals up to 48 h after exposure. The first signs of change in ultrastructure were seen at between 6 and 12 h post exposure in alveolar macrophages and took the form of apoptotic changes primarily in the nucleus. These included heterochromatin condensation at the nuclear periphery and crenulation of the nuclear membrane. There then followed a sequence of changes in the cells of the alveolar wall and blood/air barrier culminating in intra-alveolar oedema at 12 and 15 h after exposure. Damage was first observed in the capillary endothelium and type I epithelial cell changes were evident from 12 h post exposure onward. These changes appeared to be necrotic rather than apoptotic in nature and suggest that mechanisms other than a direct effect of ricin may be involved. Associated with these changes were mixed inflammatory cell infiltrates in the interstitium, isolated type II pneumocyte necrosis and evidence of microvascular microthrombosis. By 48 h after exposure, the intra-alveolar oedema appeared less marked with prominent hyperplasia of type II pneumocytes. The identification that apoptosis of alveolar macrophages plays a significant part in the mechanism of toxicity following exposure to ricin raises the possibility of developing new therapeutic strategies against poisoning by ricin.

Histopathological changes in Yucatan minipig skin following challenge with sulphur mustard. A sequential study of the first 24 hours following challenge.

Sulphur mustard (HD) or "mustard gas' is a potent vesicant chemical warfare agent whose biological effects in man have been well documented. The histopathological features of the developing cutaneous HD lesion in female Yucatan minipigs up to 24 hours post exposure are reported. Following challenge with HD vapour at a concentration of 12.0 mumol/cm2 a sequence of ultrastructural changes to keratinocytes occurred which were initially seen in the stratum basale. Condensation of nuclear heterochromatin and loss of euchromatin was accompanied by cytoplasmic swelling and culminated in focal epidermal necrosis which was evident at 24 h after the challenge. The melanocyte appeared to be the cell type most sensitive to HD challenge with a loss of cytoplasmic electron density in areas of the cytoplasm immediately surrounding the melanosomes, chromatin condensation, nuclear membrane blebbing with mitochondrial and generalized cytoplasmic swelling. Isolated complete cell necrosis and disruption were noted as early as 2 h following the challenge with generalized necrosis being seen from 12 h onwards. From 12 h onwards, areas of basement membrane degeneration were also observed. These appeared similar in ultrastructure to that observed in other animal model systems but did not progress to the large blisters so typical of the human lesion. Damage to the upper dermis took the form of an inflammatory response typified by vascular endothelial swelling and vacuolation, dermal oedema and inflammatory cell (mainly neutrophil) infiltration. There was some evidence of transitory epidermal damage outside the prescribed wound area. This finding may be of clinical importance when surgical treatments are being considered.

Topoisomerase II alpha is associated with the mammalian centromere in a cell cycle- and species-specific manner and is required for proper centromere/kinetochore structure.

A study of the distribution of Topoisomerase II alpha (Topo II) in cells of six tissue culture cell lines, human (HeLa), mouse (L929), rat, Indian muntjac, rat kangaroo (PTK-2), and wallaby revealed the following features: (1) There is a cell cycle association of a specific population of Topo II with the centromere. (2) The centromere is distinguished from the remainder of the chromosome by the intensity of its Topo II reactivity. (3) The first appearance of a detectable population of Topo II at the centromere varies between species but is correlated with the onset of centromeric heterochromatin condensation. (4) Detectable centromeric Topo II declines at the completion of cell division. (5) The distribution pattern of Topo II within the centromere is species- and stage-specific and is conserved only within the kinetochore domain. In addition, we report that the Topo II inhibitor ICRF-193 can prevent the normal accumulation of Topo II at the centromere. This results in the disruption of chromatin condensation sub-adjacent to the kinetochore as well as the perturbation of kinetochore structure. Taken together, our studies indicate that the distribution of Topo II at the centromere is unlike that reported for the remainder of the chromosome and is essential for proper formation of centromere/kinetochore structure.

Nuclear and mitochondrial changes of co-cultivated spinal cord, spinal ganglia and muscle fibers following treatment with various doses of zidovudine.

Long-term zidovudine (also termed azidothymidine, AZT) treatment of AIDS patients may cause severe myopathy characterized by conspicuous mitochondrial and nuclear changes. The mitochondrial changes are attributed to an inhibitory effect of AZT on the mitochondrial gamma-polymerase in a variety of cells. Inhibition of the nuclear alpha-polymerase is another well-known side effect of AZT, whereas the (nuclear) beta-polymerase appears to be rather insensitive. The nuclear changes seen in AIDS patients are usually considered secondary to the human immunodeficiency virus infection. To eliminate the influence of the virus on the nuclei, we studied the effect of AZT on non-infected, organotypic co-cultures of spinal ganglia, spinal cord, and skeletal muscle from fetal rats. We noted significant changes not only in the mitochondria but also in the nuclei of spinal ganglia, spinal cord, and muscle cells, which depended more on the duration of AZT application (1, 3, 5, and 8 days) than on the concentration (0.1, 1, 10, 100 and 1000 microM). The alterations of the mitochondria consisted mainly of swelling, loss of cristae and, finally, disappearance. The nuclei showed nucleolar segregation, marginal condensation of heterochromatin, formation of interchromatin and perichromatin granules, nuclear protrusions and pseudoinclusions and, finally, disintegration. The changes were not as pleomorphic as those seen in biopsy specimens from AIDS patients who had received long-term treatment with AZT. However, this difference can easily be attributed to the short duration of drug application in tissue culture compared to the long-term medication in patients.

Morphology of ricin and abrin exposed endothelial cells is consistent with apoptotic cell death.

Cultures of bovine pulmonary endothelial (BPE) cells were exposed to LC70 doses of ricin or abrin (15.5 and 4.5 pM respectively) over a period of up to 40 h. The viability of the cultures (as determined by the neutral red (NR) dye retention assay) declined after 6 h exposure to the toxins. From 15 h onwards, cellular material in toxin exposed cultures became detached from the substratum of the culture vessels. Hoffman modulation contrast photomicrography showed that this process was due to ricin and abrin exposed cells collapsing into membrane bound vesicles which retained the NR dye, became detached and floated into the medium. These apoptotic-like structural changes were further investigated by transmission electron microscopy (TEM) and by agarose gel electrophoresis of DNA from control and exposed cultures. Many of the characteristic changes associated with apoptotic cell death were seen using TEM, including heterochromatin condensation at the nuclear periphery, crenulation of the nuclear membrane and progressive degeneration of residual nuclear and cytoplasmic structures. The plasma membrane of many cells remained intact, and contained nuclear and cytoplasmic debris. Agarose gel electrophoresis of DNA extracted from toxin-treated cells revealed oligonucleosome sized DNA fragments, characteristic of apoptosis, from adherent cells at 7 h and both adherent and floating populations when harvested from 15 h; DNA from unexposed control cells did not show this fragmentation. The identification of apoptosis as being a significant additional mechanism of toxicity following exposure to ricin and abrin holotoxins raises the possibility of developing new therapeutic strategies against poisoning by these phytotoxins.

F-actin disorganization in apoptotic cell death of cultured rat renal proximal tubular cells.

The mechanism of nephrotoxin-induced apoptosis was studied in rat renal proximal tubular cells (PTC) exposed to the nephrotoxin S-(1,2-dichlorovinyl)-L-cysteine (DCVC). After a 6-h incubation, DCVC caused a condensation of heterochromatin and a fragmentation of the nucleus in 84 and 16% of the cells, respectively, which is indicative of apoptosis. This was confirmed biochemically by agarose gel electrophoresis demonstrating the formation of DNA fragments with multiples of 200 bp. The antioxidant N,N'-diphenyl-p-phenylenediamine prevented neither the fragmentation of the nucleus nor the formation of DNA fragments, but it did prevent lactate dehydrogenase release and bleb formation by DCVC. Apoptosis induced by DCVC was closely associated with F-actin disorganization: every cell with a fragmented nucleus displayed completely disorganized F-actin, while cells with a normal nucleus still possessed at least some intact F-actin also induced apoptosis in PTC. Similarly, dithiothreitol, which damages F-actin in PTC, caused apoptosis of PTC. These data suggest a causal relationship between F-actin disorganization and apoptosis of PTC.

The thromboxane mimetic, U-46619, induces the aggregation of fish thrombocytes.

Fish blood contains nucleated cells termed thrombocytes which are thought to be functionally analogous to mammalian platelets. The present study was undertaken to assess the aggregatory response of peripheral blood thrombocytes from the rainbow trout, Oncorhynchus mykiss. Incubation of Percoll density gradient purified thrombocytes with the thromboxane A2 mimetic, U-46619 (0.03-10 microM), alone or in the presence of human or trout fibrinogen, elicited a dose-dependent aggregatory response. A greater amount of aggregation was observed in the presence of fish, rather than human, fibrinogen (e.g. c 55% compared with 15% maximal aggregation respectively). This response was inhibited by pre-incubation of thrombocytes with the specific thromboxane A2 receptor antagonist, GR32191 (0.01-10 microM) with an IC50 value of 5.7 microM. Ultrastructural and aggregometry studies of thrombocyte aggregation at various time periods (1-12 min) after incubation with U-46619 (0.5 microM) revealed clear differences between the amount and dynamics of thrombocyte clumping in the presence of human, compared with trout, fibrinogen (final concentration 400 micrograms ml-1). Thrombocytes rapidly underwent shape change and aggregation after only 1 min incubation without the initial involvement of any other cell types. The maximum degree of aggregation was achieved after 4-8 min with larger aggregates formed in the presence of trout compared than human fibrinogen. Ultrastructurally, the thrombocytes in these aggregates displayed a number of changes compared with non-stimulated cells, including increased pseudopodial activity, a more pronounced canalicular system and condensation of the nuclear heterochromatin. After 12 min incubation the clumps of thrombocytes showed progressive disaggregatory behaviour, with some cells reverting back to their normal in vivo appearance. Neutrophilic granulocytes, present as contaminants in the thrombocyte suspensions, were occasionally found attached to the thrombocyte aggregates, perhaps suggesting a specific interaction similar to that occurring in mammals. Finally, and also of some significance, was the finding that trout thrombocytes exhibit phagocytic activity in vitro towards cell debris and contaminating bacteria, indicating that this cell type may possess both haemostatic and immunological capacities.

Characterization of a renal epithelial cell model of apoptosis using okadaic acid and the NRK-52E cell line.

Apoptotic cell death plays an important role in the pathogenesis of renal tubular epithelial damage and repair following tubular injury. Presently, the cellular factors involved in regulating apoptotic pathways in the kidney are unknown. To address the possibility that protein phosphorylation may regulate apoptosis in kidney cells, okadaic acid (OKA), a specific inhibitor of protein phosphatases 1A and 2A, was tested for its morphologic and biochemical effects on normal rat kidney epithelial cells (NRK-52E) in culture. As revealed by the DNA-specific stain DAPI, nuclei of cells treated with 1.0 microM okadaic acid contained irregular clumps of dense chromatin. Additional morphologic alterations typical of apoptosis were apparent within 2 hr after treatment with 1.0 microM OKA, including marked cellular rounding, cytoplasmic condensation, and cytoplasmic blebs. Ultrastructurally, 1.0 microM OKA caused cytoplasmic bleb formation, cellular fragmentation, condensation of heterochromatin into clumps, and segregation of nucleoli. At this stage, the cytoplasmic fragments and blebs contained many normal mitochondria. The attached, rounded cells also effectively excluded propidium iodide, demonstrating maintenance of membrane integrity despite pronounced morphologic alterations. A 2-fold increase in intracellular free Ca2+ was apparent 90 min after treatment with 1.0 microM okadaic acid. Transverse alternating field electrophoresis revealed the appearance of large DNA fragments of approximately 300-kbp. The appearance of these 300-kbp fragments correlated temporally with the observed elevation in intracellular calcium and the onset of morphologic alterations. However, preloading cells with EGTA-AM, an intracellular calcium chelator, obliterated the calcium elevation and had no effect on OKA-induced morphology, DNA fragmentation, or cell death. Detectable internucleosomal fragmentation occurred much later than the onset of morphologic changes (24-hr treatment time) and did not correlate with elevations in cellular calcium. These studies support the hypothesis that during apoptosis, chromatin condensation reflects chromatin cleavage at nuclease-sensitive sites between hexameric rosettes. These results also suggest that morphologic and nuclear alterations in the pathway of OKA-induced apoptosis occur independent of observed increases in intracellular calcium.