Ca, Mg-dependent endonuclease activity in different subpopulations of spleen cells from normal and erythroleukemic mice

We have detected Ca²⁺,Mg²⁺-dependent endonuclease activity in spleen cells of normal, Friend erythroleukemic, and phenylhydrazine-treated mice. When nuclei were isolated and incubated in the presence of Ca²⁺ and Mg²⁺ ions, the activity resulted in the production of 3′-OH termini in the cellular DNA and the release of chromatin due to internucleosomal DNA fragmentation. This enzyme activity was chromatin-bound and could be extracted from chromatin in an active form in 0.35 M KC1. The majority of endonuclease activity from erythroleukemic spleens was present in nuclei of precursor erythroid cells of low buoyant density (1.025–1.05 g/ml). Uninfected normal splenic tissue contained an endonuclease activity which was almost entirely confined to a B-lymphocyte population of high buoyant density (>1.07 g/ml). Erythroid cell-enriched spleens from phenylhydrazine-treated mice exhibited a distribution of endonuclease activity in cells at low and high densities reflecting a mixture of erythroid and lymphoid cells. Cloned erythroleukemic cell lines propagated in vitro lacked cells of low density and showed no detectable endonuclease activity. However, nuclei from these cell lines were susceptible to exogenously added endonuclease extracted from erythroleukemic spleen cells. These same cell lines propagated as subcutaneous tumors contained endonuclease activity and a morphologically-similar low-density cell population which accounted for the endonuclease activity in these tumors. Nuclei from cloned lymphoid cell lines, representing different B-lymphocyte phenotypes, showed differences in the presence of endonuclease activity. Among the cell lines tested, only those expressing late B-cell markers showed detectable endonuclease activity.     

Under-replication of intron+ rDNA cistrons in polyploid nurse cell nuclei of Calliphora erythrocephala

We have examined the possibility that the intron-containing (intron⁺) rDNA cistrons of Dipteran flies are active in the germ-line derived polyploid nurse cell nuclei of the ovarian follicles. Using the organism, Calliphora erythrocephala, we describe here a procedure which yields very pure nurse cell nuclei and compare the intron-free (intron^–) and intron⁺ rDNA cistron contents of nurse cell nuclei prepared by this procedure to those of 2–18 h embryo nuclei and 3 day pupal nuclei. DNA from three preparations of each nuclear type was examined and the intron^– and intron⁺ cistron contents quantitated using a Southern transfer procedure. The number of intron^– and intron⁺ rDNA cistrons per haploid genome in the presumed diploid 2–18 h embryo DNA was first established, and then the intron^– and intron⁺ cistron contents of nurse cell nuclear DNA and 3 day pupal DNA were determined relative to these values.The intron^– cistron content of nurse cell nuclear DNA was indistinguishable from that of embryonic DNA but the intron⁺ cistrons showed an 8-fold under-replication relative to the presumed diploid DNA. A slight under-representation of the intron^– cistrons and 3-fold under-replication of the intron⁺ cistrons were demonstrated for 3 day pupal DNA. These findings strongly suggest that intron⁺ rDNA cistrons are non-functional in nurse cell nuclei and substantiate the generality of this implication for the whole organism during early pupal life.     

Influence of cis-dichlorodiamineplatinum on glioma cell morphology and cell cycle kinetics in tissue culture

Summary C6 glioma cells (CCL 107) were cultured for three days and then treated withcis-dichlorodiamineplatinum (cis-DDP) at doses of 0.2–10 µg/ml medium. Changes in DNA synthesis and DNA content, as well as morphology of cells and chromatin distribution, were examined from the first post-treatment day onwards. The number of cells labelled with [³H]thymidine, detected autoradiographically, decreased after treatment with 0.2–10 µg/ml by approximately one half on post-treatment day 1 and diminished further by the third day after treatment. The labelled cells were entirely absent only after treatment with 10µg/ml, 7 days post-treatment. Mitoses decreased from 1.4–0.6% by post-treatment day 1 and completely disappeared by day 3 (1 µg/ml). Feulgen cytophotometry and propidium iodide cytofluorimetry revealed accumulation of cells in the S-phase, especially the latter part (0.5 and 1.0 µg/ml, post-treatment day 1) and subsequently also in G2 phase (post-treatment day 3). Incomplete cyto- and karyokinesis in some cycling cells was indicated by an increased number of binucleate cells and nuclei of higher ploidy classes. Labelled cells with intermediate DNA values were, on average, labelled less intensively, as was revealed by simultaneous measurements of DNA content and [³H]thymidine incorporation. Some cells displayed reduction in grain density over heterochromatin clumps. This would be in agreement with the late S-phase block of DNA replication. After post-treatment day 3 the density of cells in cultures was substantially lower. This was due to slowed transversing through the cell cycle and cell death occurring after post-treatment day 1 with higher doses or after day 2 with lower doses (up to 1 µg/ml). The size of the nuclei of surviving cells enlarged initially (post-treatment day 1) and later (day 7) giant cells with long, branched fibres similar to those of reactive astrocytes occurred. Texture analysis of Feulgen-stained nuclei revealed that the chromatin of cells treated withcis-DDP became less evenly distributed. This might be due either to the direct influence ofcis-DDP on the DNA molecule, or mediated by changes in cytoskeleton and cAMP levels described earlier.     

Programmed Myocyte Cell Death Affects the Viable Myocardium after Infarction in Rats

To determine whether apoptotic and necrotic myocyte cell death occur acutely and chronically after infarction, the formation of DNA strand breaks and the localization of myosin monoclonal antibody labeling were analyzed in the surviving myocardium from 20 min to 1 month. DNA strand breaks in myocyte nuclei were detected as early as 3 h following coronary artery occlusion and were still present at 1 month. This cellular process was characterized biochemically by internucleosomal DNA fragmentation which produced DNA laddering on agarose gel electrophoresis. Quantitatively, 155 myocyte nuclei per 10⁶cells exhibited DNA strand breaks in the portion adjacent to the infarcted tissue at 3–12 h. This parameter increased to 704 at 1–2 days and subsequently decreased to 364 at 7 days, 188 at 14 days, and 204 at 1 month. In the remote myocardium, the number of myocyte nuclei with DNA strand breaks was 84 per 10⁶at 3–12 h and remained essentially constant up to 1 month. Programmed myocyte cell death was accompanied by a decrease in the expression of bcl-2 and an increase in the expression of bax. The changes in the expression of these genes were present at 1 and 7 days after coronary artery occlusion. In conclusion, the mechanical load produced by myocardial infarction and ventricular failure may affect the regulation of bcl-2 and bax in the viable myocytes, triggering programmed cell death and the remodeling of the ventricular wall.     

Antibody against single-stranded DNA detects both programmed cell death and drug-induced apoptosis

Cyclophosphamide induced fragmented nuclei in mouse thymic epithelial cells. Agarose gel electrophoresis showed the fragmentation of the DNA extracted from mouse thymus exposed to cyclophosphamide. The cell death induced by cyclophosphamide was considered to be apoptotic. Polyclonal antibody against single-stranded DNA was used immunohistochemically to detect apoptotic cell death in thymic epithelial cells. This antibody also detected programmed cell death in the interdigital necrotic zone of the mouse limb plate on day 14 of gestation, and in the ganglion of the trigeminal nerve on day 13 of gestation. These results show that the antibody specific for single-stranded DNA detected both drug-induced apoptosis and programmed cell death during embryogenesis.     

Apoptosis in the Initial Leaf of Etiolated Wheat Seedlings: Influence of the Antioxidant Ionol (BHT) and Peroxides

Apoptosis was observed in the initial leaf of 5-8-day-old etiolated wheat seedlings. A condensation of cytoplasm in apoptotic cells, formation of myelin-like structures, specific fragmentation of cytoplasm, appearance in vacuoles of specific vesicles containing subcellular organelles, condensation and margination of chromatin in the nucleus, and internucleosomal fragmentation of nuclear DNA are ultrastructural features of apoptosis in the initial wheat leaf. Single-membrane vesicles detected in vacuoles of the leaf cells resemble in appearance the vacuolar vesicles in the coleoptile apoptotic cells described earlier (Bakeeva, L. E., et al. (1999) FEBS Lett., 457, 122-125); they contain preferentially plastids but not mitochondria as was observed in coleoptile. The vacuolar vesicles are specific for the apoptotic plant cells. Thus, apoptosis in various tissues is an obligatory element of plant (wheat) growth and development even in the early stages of ontogenesis. Contrary to strong geroprotecting action in coleoptile, the known antioxidant BHT (ionol, 2.27·10^–4 M) does not prevent in the leaf cells the apoptotic internucleosomal DNA fragmentation and appearance of specific vacuolar vesicles containing subcellular organelles. Therefore, the antioxidant action on apoptosis in plants is tissue specific. Peroxides (H₂O₂, cumene hydroperoxide) stimulated apoptosis (internucleosomal DNA fragmentation) in coleoptile and induced it in an initial leaf when apoptosis in a control seedling leaf was not yet detected. Thus, apoptosis that is programmed in plant ontogenesis and controlled by reactive oxygen species (ROS) can be modulated by anti- and prooxidants.     

Induction of Apoptosis in Cardiac Myocytes by an A3Adenosine Receptor Agonist

The effects of the selective adenosine (ADO) A₃receptor agonist IB-MECA (N⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide) on cultured newborn rat cardiomyocytes were examined in comparison with ADO, the ADO A₁receptor-selective agonistR-PIA (N⁶-R-phenylisopropyladenosine), or the ADO A₃selective antagonist MRS 1191 (3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5 dicarboxylate), using digital image analysis of Feulgen-stained nuclei. At high concentration, IB-MECA (10 μM ) and ADO (200 μM) induced apoptosis; however,R-PIA or MRS 1191 did not have any detectable effects on cardiac cells. In addition, DNA breaks in cardiomyocytes undergoing apoptosis following treatment by IB-MECA were identifiedin situusing the nick end labeling of DNA (“TUNEL”-like) assay. In the presence of 10 μM IB-MECA, disorder in the contraction waves appeared, and a decrease in the frequency of beats was observed. Analysis with light microscopy revealed that the number of contracting cells decreased in a concentration-dependent manner. The A₃receptor agonist IB-MECA caused an increase in intracellular free calcium concentration ([Ca²⁺]_i). The drug produced a rapid rise followed by a sustained increase in [Ca²⁺]_i, which lasted for 40–60 s. Finally, cessation of beating and Ca²⁺transients were observed. Full recovery of contractile activity and rhythmical Ca²⁺transients were observed 15–20 min after IB-MECA treatment. The induction of apoptosis in the cardiocytes by IB-MECA led to the appearance of features of apoptotic nuclei: the onset of condensation, compacting, and margination of nuclear chromatin. These effects were accompanied by the disintegration of the structural framework of the nucleus and nuclear breakdown. The results suggest that activation of the A₃adenosine receptor may participate in the process of apoptosis in cardiomyocytes.     

Autoradiographic and ultrastructural study of Cucurbita pepo root cells during their growth and differentiation

Summary Cortex cells of the root meristem of Cucurbita pepo (0.0–0.5 mm from the cap junction), in the 3–4, 5–6 and 7–8 mm segments above the root tip, and the cells of the first three layers of lateral part of root cap were the object of the present study. The volume of cortex cells increases more than 20 times in the 7–8 mm segment as compared with meristematic cells, and the volume of cytoplasm about sevenfold. The largest increment of the cytoplasmic volume occurs between 0.5–6.0 mm. In consecutive root segments the sustained increase of the volume of nuclei takes place. By applying autoradiography the following processess have been investigated: DNA synthesis (³H thymidine uptake), template activity of DNA (³H actinomycin D(³H AMD)-binding), RNA synthesis (³H uridine incorporation), and protein synthesis (³H leucine). In the root cap cells and in segments where meristematic activity is over, DNA is replicated by endomitosis. On the basis of nuclear labelling it appears that nuclei in the 3–4 mm segment reach 4C ploidy state, but in the 7–8 mm segment half of the nuclei reach the 8C ploidy state. Most of the root cap cells are 4C, the remaining cells are 8C. Considering the uptake of ³H thymidine into nucleoli one may suppose that in the root cap cells nucleolar DNA is underreplicated, and to a lesser degree in 5–6 and 7–8 mm segments, while in 3–4 mm segment DNA is overreplicated as compared to meristem cells. Measurements of nucleolar volume, ³H uridine uptake, ³H AMD binding and quantity of granular component, indicate that the most noticeable nucleolar activity takes place in meristematic zone and in root parts showing the highest increase of cytoplasmic volume (3–4 and 5–6 mm segments). ³H leucine is still incorporated intensely into 7–8 mm segment, in which the concentration of ribosomes is low, however they are present in the form of polysomes. Comparison of ³H thymidine uptake into nuclear DNA with ³H AMD binding and ³H uridine incorporation into nuclei indicates that endomitotic DNA replication results in an increase of DNA template activity in root cap cells as well as in 3–4 and 5–6 mm segments; in the 7–8 mm segment binding of ³H AMD slightly decreases, while ³H uridine incorporation is considerably reduced. Divergence between the ploidy state, ³H AMD binding and ³H uridine incorporation can be due to the increment of the condensed chromatin area in differentiated cells. Plastids and mitochondria reach full maturity in 3–4 mm segment. The increasing volume density of ER and diminishing volume density of Golgi structures is accompanied by differentiation of cortex cells.This work was partly supported by Polish Academy of Sciences, Botanical Committee, Grant 217/II     

Sulfalene concentrations in plasma and blood cells of Plasmodium falciparum malaria cases after treatment with metakelfin using high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method using acetonitrile–methanol–1 M perchloric acid–water (25:9:0.8:95, v/v/v) at a flow-rate of 1.0 ml min⁻¹ on LiChrospher 100 RP 18 column (250×4 mm; 5 μm) with UV (254 nm) detection has been developed for the determination of sulfalene in plasma and blood cells after oral administration of the antimalarial drug metakelfin. Calibration curves were linear in the range 0.5–100 μg ml⁻¹. The limit of quantification was 50 ng ml⁻¹. Within-day and day-to-day coefficients of variation averaged 3.84 and 5.31%, respectively. Mean extraction recoveries of sulfalene from plasma and blood cells were 87.21 and 84.65%, respectively. Mean concentrations of sulfalene in plasma of P. falciparum cases on days 2, 7 and 15 were 44.58, 14.90 and 1.70 μg ml⁻¹, respectively; in blood cells concentrations of sulfalene were 7.77, 3.25 and 0.75 μg ml⁻¹, respectively, after oral treatment with two tablets (1000 mg) of metakelfin. Significant difference was recorded on day 2 for sulfalene concentration in blood cells of healthy and P. falciparum cases (t=9.49; P<0.001).     

Apoptosis detected in hybrids between Nicotiana glutinosa and N. repanda expressing lethality

Hybrid lethality is one of the mechanisms for reproductive isolation. Apoptotic features were detected in the cells of hybrid seedlings of Nicotiana glutinosa L. ×N. repanda Willd. Condensation of chromatin and fragmentation of nuclei were observed in the leaf protoplasts isolated from hybrid seedlings expressing this lethality. Fragmentation of nuclei was correlated with the progression of lethal symptoms, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from hybrid leaves showing lethality revealed a specific ladder pattern suggesting nucleosomal fragmentation associated with nuclear fragmentation. In-situ detection of DNA fragmentation using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) showed that this process occurred in all leaf cells. This is the first evidence that apoptosis can induce suicide of hybrid plants, thus leading to reproductive isolation. Received: 18 June 1999 / Accepted: 20 July 1999     

Mechanism of UV-Induced Apoptosis in Human Leukemia Cells: Roles of Ca/Mg-Dependent Endonuclease, Caspase-3, and Stress-Activated Protein Kinases

Ultraviolet light (UV) induced rapid apoptosis of U937 leukemia cells, concurrent with DNA fragmentation and cleavage of poly(ADP-ribose)polymerase (PARP) by activated caspase-3. Thein vitroreconstitution of intact HeLa S3 nuclei and apoptotic U937 cytosolic extract (CE) revealed that (i) Ca²⁺/Mg²⁺-dependent, Zn²⁺-sensitive endonuclease activated in the apoptotic CE induced DNA ladder in HeLa nuclei at pH 6.8–7.4, (ii) activated caspase-3 cleaved PARP in HeLa nuclei, and (iii) when the apoptotic CE was treated with the caspase-3 inhibitor (1 μM Ac-DEVD-CHO) or the caspase-1 inhibitor (10 μM Ac-YVAD-CHO), the former, but not the latter, caused a 50% inhibition of DNA fragmentation and the complete inhibition of PARP cleavage in HeLa nuclei. Similarly, Ac-DEVD-CHO (100 μM) inhibited apoptosis and DNA ladder by 50% and PARP cleavage completely in UV-irradiated U937 cells, but Ac-YVAD-CHO (100 μM) did not. Thus, UV-induced apoptosis of U937 cells involves the Ca²⁺/Mg²⁺-dependent endonuclease pathway and the caspase-3–PARP cleavage–Ca²⁺/Mg²⁺-dependent endonuclease pathway. The former pathway produced directly 50% of apoptotic DNA ladder, and the latter involved activated caspase-3 and PARP cleavage, followed by formation of the remaining 50% DNA ladder by the activated endonuclease. In UV-irradiated B-cell lines, further, p53-dependent increase of Bax resulted in a greater caspase-3 activation compared to its absence. However, UV-induced activation of JNK1 and p38 was not affected by the caspase-1 and -3 inhibitors in U937 cells, so that caspases-1 and -3 do not function upstream of JNK1 and p38.     

Multimeric α-Lactalbumin from Human Milk Induces Apoptosis through a Direct Effect on Cell Nuclei

A fraction from human milk containing spf-multimer α-lactalbumin (MAL) induces apoptosis in tumor cells and immature cells but spares mature cells. The mechanism of apoptosis induction and the molecular basis for the difference in susceptibility between tumor cells and healthy cells have not been defined. In this study we examined the interaction of MAL with different cellular compartments, using confocal microscopy and subcellular fractionation. MAL was shown to accumulate in the nuclei of sensitive cells rather than in the cytosol, the vesicular fraction, or the ER–Golgi complex. Nuclear uptake occurred rapidly in cells that were susceptible to the apoptosis-inducing effect, but not in nuclei of resistant cells. Nuclear uptake was through the nuclear pore complex and was critical for the induction of DNA fragmentation, since inhibition of nuclear uptake with WGA rescued digitonin-permeabilized cells from induction of DNA fragmentation. Ca²⁺was required for MAL-induced DNA fragmentation but nuclear uptake of MAL was independent of Ca²⁺. This way MAL differs from most previously described agents in that it crosses the plasma membrane and cytosol, and enters cell nuclei where it induces DNA fragmentation through a direct effect at the nuclear level.     

Necessity of Superoxide Production for Development of Etiolated Wheat Seedlings

It was found that production of superoxide (O₂ ^{– ·}) is crucial for normal morphogenesis of etiolated wheat seedlings in the early stages of plant development. The development of etiolated wheat seedlings was shown to be accompanied with cyclic changes in the rate of O₂ ^{– ·} production both in the entire intact seedling and in its separated organs (leaf, coleoptile). First increase in the rate of O₂ ^{– ·} production was clearly observed in the period from two to four days of seedling development, then the rate of O₂ ^{– ·} production decreased to the initial level, and then it increased again for two days to a new maximum. An increase in O₂ ^{– ·} production in the period of the first four days of seedling development correlates with an increase in DNA and protein contents in the coleoptile. The second peak of increased rate of O₂ ^{– ·} production observed on the sixth or seventh day of seedling development coincides with a decrease in DNA and protein contents and apoptotic internucleosomal nuclear DNA fragmentation in the coleoptile. Incubation of seedlings in the presence of the antioxidant BHT (ionol) strongly affects their development but it does not influence the increase in DNA and protein contents for the initial four days of seedling life, and it slows down the subsequent age-dependent decrease in protein content and fully prevents the age-dependent decrease in DNA content in the coleoptile. A decrease in the O₂ ^{– ·} amount induced by BHT distorts the seedling development. BHT retards seedling growth, presumably by suppression of cell elongation, and it increases the life span of the coleoptile. It seems that O₂ ^{– ·} controls plant growth by cell elongation at the early stages of seedling development but later O₂ ^{– ·} controls (induces) apoptotic DNA fragmentation and protein disintegration.     

Chronic heat-shock treatment driven differentiation induces apoptosis in Leishmania donovani

The present study investigates the role of apoptosis in the regulation of cell numbers of Leishmania donovani during the in vitro differentiation of promastigote stage to amastigote stage in axenic conditions. We report that apoptosis is induced in Leishmania donovani due to chronic heat-shock treatment of 37 ^°C that also mediates the differentiation of promastigotes to amastigotes. This is characterized by the fragmentation of DNA, blebbing in the parasite cell membrane, nuclear condensation, formation of preapoptotic bodies and involvement of Ca⁺⁺ in the apoptotic process. The flowcytometric analysis shows an early and steep rise in percentage apoptotic nuclei till 48-hour stage of differentiation and then a gradual decline, suggesting synergistic action of Ca⁺⁺ ATPase and probably Hsp70. Hsp70 might be rescuing cells from apoptosis in the death signaling pathway. Incubation of the culture with Ca⁺⁺ chelator EGTA (1 mM) brings down the percentage of apoptotic nuclei considerably showing thereby that calcium is needed for the process of cell death here that occurs by apoptosis. The survival of the infective individuals appears to be decided by the parasite in the early stages of its differentiation. Our studies show the potential of the physiological temperature of 37 ^°C in inducing apoptosis in Leishmania donovani and the therapeutic use it can be put to.     

Successful separation of apoptosis and necrosis pathways in HaCaT keratinocyte cells induced by UVB irradiation

UVB irradiation can induce apoptotic, necrotic, and differentiation pathways in normal human keratinocytes. The present study was undertaken to determine at what dose of UVB each of these pathways is induced and whether these pathways are distinct or overlapping. We have observed that UVB induces fragmentation of DNA in human HaCaT keratinocytes, in a bimodal manner. Low doses of UVB, 5–20 mJ/cm², increase the levels of apoptosis as shown by increased levels of fragmented DNA, Fas, PARP, and FasL protein, and the number of apoptotic cells as assessed by FACS analysis. At higher doses of UVB (20 and 30 mJ/cm²) the number of apoptotic cells becomes reduced, as does the amount of Fas, PARP, and FasL protein. At these higher doses, cell viability is decreased as measured by DNA synthesis (BrdU labeling) neutral red uptake, which represents an increasing necrotic phenotype. Expression of markers of keratinocyte differentiation, involucrin, keratin K1, and keratin K10, are also observed to decrease with increasing UVB dose. These changes are accompanied by a further increase in DNA fragmentation. We conclude that low doses of UVB (5–20 mJ/cm²) induced an apoptotic pathway, whereas increasing doses (greater than 20 mJ/cm²) of UVB produce a direct necrotic effect and inhibit terminal differentiation. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Effects of Phytohormones and 5-Azacytidine on Apoptosis in Etiolated Wheat Seedlings

The development of etiolated wheat (Triticum aestivum L.) seedlings is necessarily accompanied by apoptosis in their coleoptiles and first leaves. Internucleosome DNA fragmentation, which is characteristic of apoptosis, was detected in the coleoptile as soon as six days after germination. After eight days of germination, DNA fragmentation was clearly expressed in the coleoptile and was noticeable in the apical part of the first-leaf blade. Growing of intact seedlings or incubation of their shoots in the presence of such phytohormones as benzyladenine, gibberellin A₃, fusicoccin C, and 2,4-D at the concentration of 10^–5 M did not essentially affect DNA fragmentation in the coleoptile. As distinct from antioxidants, none of the phytohormones used prevented apoptosis in wheat seedlings. In contrast, ABA (10^–5 M) and an ethylene producer, ethrel (2-chloroethylphosphonic acid, 10^–2–10^–3 M), stimulated sharply DNA fragmentation in the coleoptile. An inhibitor of DNA methylation, 5-azacytidine, was very efficient in the stimulation of DNA fragmentation in the coleoptiles of eight-day-old seedlings at its concentration of 100 g/ml. Thus, some phytohormones can regulate apoptosis, and DNA methylation is involved in this process. Our results indicate that apoptosis activation by some phytohormones may be mediated by their regulation of DNA methylation/demethylation, which is responsible for the induction of genes encoding apoptogenic proteins and/or the repression of antiapoptotic genes.     

The chlorophenoxy herbicide dicamba and its commercial formulation banvel induce genotoxicity and cytotoxicity in Chinese hamster ovary (CHO) cells

The sister chromatid exchange (SCE) frequency, the cell-cycle progression analysis, and the single cell gel electrophoresis technique (SCGE, comet assay) were employed as genetic end-points to investigate the geno- and citotoxicity exerted by dicamba and one of its commercial formulation banvel^® (dicamba 57.71%) on Chinese hamster ovary (CHO) cells. Log-phase cells were treated with 1.0–500.0 μg/ml of the herbicides and harvested 24 h later for SCE and cell-cycle progression analyses. All concentrations assessed of both test compounds induced higher SCE frequencies over control values. SCEs increased in a non-dose-dependent manner neither for the pure compound (r = 0.48; P > 0.05) nor for the commercial formulation (r = 0.58, P > 0.05). For the 200.0 μg/ml and 500.0 μg/ml dicamba doses and the 500.0 μg/ml banvel^® dose, a significant delay in the cell-cycle progression was found. A regression test showed that the proliferation rate index decreased as a function of either the concentration of dicamba (r = −0.98, P < 0.05) or banvel^® (r = −0.88, P < 0.01) titrated into cultures in the 1.0–500.0 μg/ml dose-range. SCGE performed on CHO cells after a 90 min pulse-treatment of dicamba and banvel^® within a 50.0–500.0 μg/ml dose-range revealed a clear increase in dicamba-induced DNA damage as an enhancement of the proportion of slightly damaged and damaged cells for all concentrations used (P < 0.01); concomitantly, a decrease of undamaged cells was found over control values (P < 0.01). In banvel^®-treated cells, a similar overall result was registered. Dicamba induced a significant increase both in comet length and width over control values (P < 0.01) regardless of its concentration whereas banvel^® induced the same effect only within 100.0–500.0 μg/ml dose range (P < 0.01). As detected by three highly sensitive bioassays, the present results clearly showed the capability of dicamba and banvel^® to induce DNA and cellular damage on CHO cells.     

Evidence for dosage compensation in parthenogenetic Hymenoptera

Amount of DNA-Feulgen staining in individual somatic nuclei and mature sperm of the parthenogenetic wasps, Habrobracon juglandis, H. serinopae, and Mormoniella vitripennis, were determined with a scanning microdensitometer. The haploid genome for both species of Habrobracon was estimated to be 0.15–0.16×10^–12 g DNA, corresponding to a molecular weight of roughly 10×10¹⁰ daltons. The haploid genome of M. vitripennis is approximately twice this value, 0.33–0.34×10^–12 g, or about 20×10¹⁰ daltons. Measurements made on dividing nuclei from syncytial preblastoderm embryos of H. juglandis and M. vitripennis showed that the chromosomes of impaternate males were present in the haploid number and contained the C amount of DNA; whereas nuclei from female preblastoderm embryos contained the diploid number of chromosomes and the 2C amount of DNA. However, hemocyte and brain cell nuclei from either male or female adult wasps contained 2C and 4C amounts of DNA. Both sexes also showed equivalent levels of polyploidy (8C, 16C, or 32C) in Malpighian tubule nuclei. Therefore, in these parthenogenetic species, a mechanism must exist that compensates during later development for the initial two-fold difference in the chromatin content of somatic nuclei in haploid male and diploid female embryos. Hemocytes from impaternate Mormoniella diploid males and triploid females contain the 2C and 3C amounts of DNA, respectively. Therefore dosage compensation involves an additional cycle of DNA replication only in haploid cells, and it insures that a certain minimum quantity of DNA is received by each somatic cell.     

Detection of apoptotic bodies in nickel-treated root apical cells of <Emphasis Type="Italic">Allium cepa</Emphasis>

Nickel, a phytotoxic metal, is easily taken up by plant roots. Excessive Ni in soil can induce programmed cell death in root tips. DNA fragmentation was assessed by deoxyuracyl nick end labeling with fluorescein mediated by terminal deoxynucleotide transferase (TUNEL). In control roots, programmed cell death was observed by using TUNEL assay in the root cap and xylem vessels. At moderate Ni²⁺ concentrations (10–25 µM), cortical cells also showed TUNEL-positive reaction, indicating the Ni-induced cell death. The Ni-induced cell death differs from natural cell death in the appearance of spherical bodies of condensed chromatin and formation of apoptotic bodies. These apoptotic bodies resemble those appearing during animal apoptosis.From Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 151–153.Original English Text Copyright © 2005 by Samadi, Behboodi.This article was presented by the authors in English.     

Autophagy in embryonic erythroid cells: its role in maturation

Yolk sac-derived embryonic erythroid cells differentiate synchronously in the peripheral blood of Syrian hamster. The stage of differentiation on day 10 of gestation is equivalent to polychromatophilic erythroblast stage and that on day 13 is equivalent to the reticulocyte stage in adult animals. The cytoplasm of embryonic erythroid cells became scant and devoid of most organelles on day 12 of gestation. In addition, there were very few non-erythroid cells in circulation before day 13. Thus the embryonic erythroid cells serve a pure and synchronous system to study the mechanisms of terminal differentiation. The number of mitochondria in the embryonic erythroid cells decreased to about 10% of the initial number during the period between day 10 and day 12 of gestation. In contrast, the frequency of autophagy of mitochondria increased 4.6-fold in the same period. The cytochrome c content of the cell decreased as the mitochondria became extinct. However, release of cytochrome c into the cytoplasm was not detectable through day 10-13 of gestation, suggesting that the mitochondria were digested within a closed compartment. Decomposed mitochondria and ferritin particles were detected in lysosomes by electron microscopy on and after day 12 of gestation, which also suggested digestion in a closed compartment. Mitochondrial ATP synthase subunit c, which is known to be a protease-refractory protein, was retained in the cells even after the disappearance of mitochondria, indicating that most of the mitochondria were not extruded from the cells. The digestion of mitochondria in autolysosomes may allow the cells to escape from rapid apoptotic cell death through concomitant removal of mitochondrial death-promoting factors such as cytochrome c.