DDT induces apoptosis in rat thymocytes

We have isolated an Arabidopsis BBM II isomerase cDNA from an Arabidopsis cDNA library, by means of functional complementation of the E. coli hisA mutant strain HfrG6. The isolated cDNA encodes a polypeptide of 304 amino acids with a calculated molecular weight of 33,363. Sequence comparison with the HIS6 proteins of yeasts revealed that Arabidopsis BBM II isomerase contains an N-terminal extension of approximately 40 amino acids that shows the general properties of chloroplast transit peptides. This finding is consistent with the localization of other histidine biosynthetic enzymes, such as imidazoleglycerolphosphate dehydratase and histidinol dehydrogenase, in the chloroplasts in higher plants. The primary structure of the mature protein was 50% and 42% identical, respectively, to the HIS6 proteins of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively, while no prominent sequence similarity to the bacterial BBM II isomerase was found. That the isolated Arabidopsis cDNA actually encodes a functionally active BBM II isomerase activity was confirmed in an in vitro enzyme assay using a crude extract prepared from strain HfrG6 transformed with the Arabidopsis BBM II isomerase cDNA.     

Adriamycin induces apoptosis in rat thymocytes

Apoptosis is a controlled form of cell death accompanied by distinct morphological and biochemical changes. In this study the nature of cytotoxicity induced by adriamycin (ADM) in rat thymocytes was evaluated. Morphological and biochemical changes characteristic of apoptosis were found to precede adriamycin-induced cell death. Our findings demonstrate the involvement of c-Myc, c-Jun, antioxidant enzymes CuZn superoxide dismutase and catalase, and perhaps poly ADP ribosylation in ADM-induced cell death.     

Identification of factors from rat neutrophils responsible for cytotoxicity to isolated hepatocytes

Activated polymorphonuclear neutrophils (PMNs) have been shown to be cytotoxic to rat hepatic parenchymal cells in vitro. This cytotoxicity could be observed without direct cell-cell contact, since the conditioned medium from PMNs activated with formyl-Met-Leu-Phe (fMLP) was effective in hepatocyte killing. To identify the toxic factor(s) released by PMNs, degranulation was induced by fMLP in PMNs pretreated with cytochalasin B. The contents released from the phagocytes were subjected to gel filtration on a Sephadex G-100 column. Resulting fractions were tested for cytotoxicity to isolated hepatocytes by using release of alanine aminotransferase as a marker for hepatocyte injury. Cytotoxicity was associated with fractions containing cathepsin G and elastase and not with other fractions, including those containing myeloperoxidase. The former two enzymes were purified to homogeneity with a carboxymethyl cellulose column. Each of these enzymes demonstrated concentration-dependent cytotoxicity to hepatocytes at concentrations > 2 microgram/mL. Moreover, they exhibited an additive cytotoxic effect. Effective concentrations for the combined cathepsin G and elastase in the incubation mixture were similar to the concentrations of these enzymes in PMN-conditioned medium that produced cytotoxicity to hepatocytes. Cytotoxicity of either purified enzyme or of conditioned medium could be prevented by plasma alpha-1-antitrypsin or soybean trypsin-chymotrypsin inhibitor, which were also potent inhibitors of enzymic activity of both cathepsin G and elastase. By contrast, the serine protease inhibitors, aprotinin and 4-(2-aminoethyl)-benzene-sulfonyl fluoride, were less effective in inhibiting cathepsin G and elastase activities as well as cytotoxicity caused by the purified proteases or PMN-conditioned medium. These results support the hypothesis that cathepsin G and elastase are important mediators of hepatic parenchymal cell killing produced by activated PMNs in vitro.     

Vav regulates peptide-specific apoptosis in thymocytes

Homologies between vertebrate forebrain subdivisions are still uncertain. In particular the identification of homologs of the mammalian neocortex or the dorsal ventricular ridge (DVR) of birds and reptiles is still a matter of dispute. To get insight about the organization of the primordia of the main telencephalic subdivisions along the anteroposterior axis of the neural tube, a fate map of the dorsal prosencephalon was obtained in avian chimeras at the 8- to 9-somite stage. At this stage, the primordia of the pallium, DVR and striatum were located on the dorsal aspect of the prosencephalon and ordered caudorostrally along the longitudinal axis of the brain. Expression of homeobox-containing genes of the Emx, Dlx and Pax families were used as markers of anteroposterior developmental subdivisions of the forebrain in mouse, chick, turtle and frog. Their expression domains delineated three main telencephalic subdivisions in all species at the onset of neurogenesis: the pallial, intermediate and striatal neuroepithelial domains. The fate of the intermediate subdivisions diverged, however, between species at later stages of development. Homologies between forebrain subdivisions are proposed based on the conservation and divergence of these gene expression patterns.     

Linomide enhances apoptosis in CD4+CD8+ thymocytes

Linomide, a quinoline-3-carboxamide, has a pleiotropic immune modulating capacity and inhibits development as well as progression of disease in animal models of autoimmunity. Linomide treatment of mice resulted in a dramatic, dose-dependent decrease of the thymic cell number shortly after the start of administration. Flow cytometric analysis revealed that the major thymocyte subset, the early immature type CD4+CD8+ thymocytes, were reduced in number by 75%, mature CD4+CD8- or CD4-CD8+ thymocytes were less sensitive to treatment. The polyclonal T cell activator Con A (Concanavalin A) was used together with IL-2 to evaluate the potential proliferative responsiveness of ex vivo thymocytes. Thymocytes from mice treated with Linomide exhibited a more vigorous proliferation than control cultures. An effect shown to not only be due to the enrichment of mature thymocytes in the cultures from Linomide treated animals, but also when purified, mature thymocytes (CD4+CD8- and CD4-CD8+) were cultured with Con A and IL-2, these cells responded with a significantly enhanced proliferation. In vivo Linomide treatment did not result in increased plasma concentrations of corticosterone and treatment of adrenalectomized mice resulted in a reduction of thymocytes which was comparable to the effect in intact mice, indicating that glucocorticoids (GC) are not major mediators of Linomide-induced thymocyte deletion. In addition to this, and supporting a glucocorticoid independent mode of action, Linomide treatment of thymocytes in vitro resulted in a significant increase in the number of apoptotic cells, specifically in the CD4+CD8+ subset, implicating apopotosis as one component in the course of thymocyte reduction. In addition to this, in vivo treatment with Linomide resulted in an identical pattern to that seen in vitro in that there was significantly increased apoptosis only in the CD4+CD8+. These data indicate that Linomide modifies thymocyte development using a glucocorticoid independent pathway and results in the increased apoptosis of the CD4+CD8+ subset.     

Identification of mutations at DNA topoisomerase I responsible for camptothecin resistance

A camptothecin-resistant cell line that exhibits more than 600-fold resistance to camptothecin, designated CPT(R)-2000, was established from mutagen-treated A2780 ovarian cancer cells. CPT(R)-2000 cells also exhibit 3-fold resistance to a DNA minor groove-binding ligand Ho33342, a different class of mammalian DNA topoisomerase I inhibitors. However, CPT(R)-2000 cells exhibit no cross-resistance toward drugs such as Adriamycin, amsacrine, vinblastine, and 4'-dimethyl-epipodophyllotoxin. The mRNA, protein levels, and enzyme-specific activity of DNA topoisomerase I are relatively the same in parental and CPT(R)-2000 cells. However, unlike the DNA topoisomerase I activity of parental cells, which can be inhibited by camptothecin, that of CPT(R)-2000 cells cannot. In addition, parental cells after camptothecin treatment results in a decrease in the level of DNA topoisomerase I, whereas CPT(R)-2000 cells are insensitive to camptothecin treatment. These results suggested that the mechanism of camptothecin resistance is most likely due to a DNA topoisomerase I structural mutation. This notion is supported by DNA sequencing results confirming that DNA topoisomerase I of CPT(R)-2000 is mutated at amino acid residues Gly717 to Val and Thr729 to Ile. We also used the yeast system to examine the mutation(s) responsible for camptothecin resistance. Our results show that each single amino acid change results in partial resistance, and the double mutation gives a synergetic effect on camptothecin resistance. Because both mutation sites are near the catalytic active center, this observation raises the possibility that camptothecin may act at the vicinity of the catalytic active site of the enzyme-camptothecin-DNA complex.     

Identification and expression of multidrug transporters responsible for fluconazole resistance in Candida dubliniensis

Candida dubliniensis is a recently described Candida species associated with oral candidosis in human immunodeficiency virus (HIV)-infected and AIDS patients, from whom fluconazole-resistant clinical isolates have been previously recovered. Furthermore, derivatives exhibiting a stable fluconazole-resistant phenotype have been readily generated in vitro from fluconazole-susceptible isolates following exposure to the drug. In this study, fluconazole-resistant isolates accumulated up to 80% less [3H] fluconazole than susceptible isolates and also exhibited reduced susceptibility to the metabolic inhibitors 4-nitroquinoline-N-oxide and methotrexate. These findings suggested that C. dubliniensis may encode multidrug transporters similar to those encoded by the C. albicans MDR1, CDR1, and CDR2 genes (CaMDR1, CaCDR1, and CaCDR2, respectively). A C. dubliniensis homolog of CaMDR1, termed CdMDR1, was cloned; its nucleotide sequence was found to be 92% identical to the corresponding CaMDR1 sequence, while the predicted CdMDR1 protein was found to be 96% identical to the corresponding CaMDR1 protein. By PCR, C. dubliniensis was also found to encode homologs of CDR1 and CDR2, termed CdCDR1 and CdCDR2, respectively. Expression of CdMDR1 in a fluconazole-susceptible delta pdr5 null mutant of Saccharomyces cerevisiae conferred a fluconazole-resistant phenotype and resulted in a 75% decrease in accumulation of [3H]fluconazole. Northern analysis of fluconazole-susceptible and -resistant isolates of C. dubliniensis revealed that fluconazole resistance was associated with increased expression of CdMDR1 mRNA. In contrast, most studies showed that overexpression of CaCDR1 was associated with fluconazole resistance in C. albicans. Increased levels of the CdMdr1p protein were also detected in fluconazole-resistant isolates. Similar results were obtained with fluconazole-resistant derivatives of C. dubliniensis generated in vitro, some of which also exhibited increased levels of CdCDR1 mRNA and CdCdr1p protein. These results demonstrate that C. dubliniensis encodes multidrug transporters which mediate fluconazole resistance in clinical isolates and which can be rapidly mobilized, at least in vitro, on exposure to fluconazole.     

A Mg(2+)-dependent endonuclease is responsible for internucleosomal DNA fragmentation in human B lymphoblastic IM9 cells

We have identified a Mg(2+)-dependent endonuclease activity from human B lymphoblastic IM9 cell lysates and nuclei using autodigestion method and DNA-native-polyacrylamide gel electrophoresis (DNA-native-PAGE) nuclease assay system. The level of the endonuclease activity in cell lysates was significantly decreased at certain stage by treatment of the cells with cycloheximide. However, the enzyme activity consistently remained for over 12 hours in the isolated nuclei of the apoptotic IM9 cells. The Mg(2+)-dependent endonuclease isolated from the nuclei by native-PAGE elution was able to catalyze the conversion of supercoiled plasmid DNA into linear form. This particular endonuclease activity was not detected in cycloheximide treated-U937 cells. Several lines of experimental evidence suggest that the Mg(2+)-dependent endonuclease localized in the nucleus may be responsible for the DNA fragmentation of apoptotic IM9 cells.     

Preincubation of thymocytes with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) induces apoptosis in non-stimulated thymocytes

Previous studies have reported that cocaine exposure in utero results in structural and functional alterations in the development of the anterior cingulate cortex (ACC). In the present study, the effects of maternal cocaine dosage and of cocaine-elicited maternal seizures on the progeny were studied. The incidence of maternal generalized tonic clonic seizures (GTCSs) elicited by cocaine was recorded. No GTCSs were elicited in pregnant rabbits by doses of 2 or 3 mg/kg of cocaine, but GTCSs were sometimes elicited by the highest dose (4 mg/kg per injection). We analyzed the offspring of cocaine-exposed and control animals using three assays of ACC development: (i) the structure of apical dendrites of pyramidal neurons, (ii) the distribution of a calcium binding protein (parvalbumin) in the dendrites of GABAergic neurons, and (iii) coupling of D1-like receptors and their G proteins. In all progeny of rabbits exposed to 3 or 4 mg/kg of cocaine during pregnancy, there was a significant change in the structure of apical dendrites, a significant increase in the number of dendrites of GABAergic neurons which were parvalbumin immunoreactive, and a significant reduction in D1/G protein coupling. In assays of apical dendrites, the effects on offspring of rabbits given 2 mg/kg cocaine were as pronounced as in offspring of rabbits given 3 or 4 mg/kg, but the effects on parvalbumin immunoreactivity and D1/G protein coupling were reduced at this low dose. Thus, previous findings of ACC developmental abnormalities in offspring of rabbits given a dose of 4 mg/kg were replicated, the effects were shown to be dose-related and to be independent of maternal seizures. A mechanism by which dysfunction of the D1 receptor system could mediate cocaine-associated changes in all three parameters of ACC structure and function is discussed.     

CD40 expressed on thymic epithelial cells provides costimulation for proliferation but not for apoptosis of human thymocytes

Human thymic epithelial cells express CD40, so we examined the possible role of CD40 in activation of thymocytes. We observed that both CD4+CD8- and CD4-CD8+ thymocytes proliferate after stimulation by anti-CD3 mAb in the presence of cultured thymic epithelial cells. Costimulation of CD4+ thymocytes by thymic epithelial cells is partly inhibited by an anti-CD40 mAb, but this mAb has no effect on costimulation of CD8+ thymocytes. The selective costimulatory ability of CD40 for CD4+ thymocytes was confirmed in experiments in which thymocytes were stimulated with anti-CD3 in the presence of murine P815 cells transfected with CD40 cDNA. The level of costimulation induced by P815-CD40 was comparable with that induced by P815 cells expressing CD80 (B7.1). Treatment of thymocytes with the Ca2+ ionophore ionomycin and the phorbol ester PMA or with anti-CD3 mAb resulted in up-regulation of the CD40 ligand, suggesting that this molecule is involved in CD40-mediated costimulation of human thymocytes. Costimulation of thymocytes by CD80 strongly increased anti-CD3-induced death of fetal thymocytes. In contrast, costimulation by CD40 did not increase anti-CD3-mediated apoptosis of these thymocytes. To confirm that CD40 does not affect anti-CD3-induced cell death, we established a variant of the Jurkat T leukemic cell line that constitutively expresses CD40L and analyzed the sensitivity of this cell line for activation-induced apoptosis. In contrast to CD80, CD40 failed to increase anti-CD3-mediated apoptosis in CD40L+ Jurkat cells, whereas both CD40 and CD80 strongly increased IL-2 production induced by anti-CD3. These findings suggest that costimulation by CD40 is involved in clonal expansion of CD4+ thymocytes but not in activation-induced cell death.     

Calcium channel subtypes responsible for voltage-gated intracellular calcium elevations in embryonic rat motoneurons

The central role of electrical activity and Ca2+ influx in motoneuron development raises important questions about the regulation of Ca2+ signalling induced by voltage-dependent Ca2+ influx. In the purified embryonic rat motoneuron preparation, we recorded barium currents through voltage-activated Ca2+ channels using the whole-cell configuration of the patch-clamp technique. We found that motoneurons express at least four types of high-voltage-activated Ca2+ channels, based on their kinetics, voltage-dependences and pharmacological properties. Of the sustained Ca2+ current activated at 0 mV from a holding potential of -100 mV, approximately 45% was omega-conotoxin-GVIA (1 microM) sensitive, 25% was omega-agatoxin-IVA (30 nM) sensitive and 20% was nitrendipine (250 nM) sensitive. The residual current, after applying these three antagonists, was an inactivating current that differs from classical T-type Ca2+ currents. Based on this pharmacology, changes in intracellular free Ca2+ concentrations were then monitored by Fura 2 digital imaging microspectrofluorimetry. Upon K+ depolarization, the intracellular Ca2+ transient induced by the activation of each type of Ca2+ channel appeared to be quantitatively proportional to their Ca2+ influx. The existence of a calcium-induced calcium release mechanism through activation of caffeine-, ryanodine-sensitive intracellular stores was then investigated. High doses of caffeine and low doses of ryanodine failed to increase intracellular free calcium concentrations and low concentrations of caffeine and high concentrations of ryanodine did not affect K+-induced intracellular free calcium concentration transients indicating both the absence of Ca2+-gated Ca2+-release channels and of a Ca2+-induced Ca2+ release mechanism. Together, these data provide evidence that embryonic motoneurons express multiple Ca2+ channels that function as important regulators of intracellular Ca2+ signalling and may be involved in their development.     

Cholesterol induced variations of membrane dynamics related to the induction of apoptosis in mouse thymocytes

PURPOSE: To evaluate the involvement of cholesterol induced variations of membrane dynamics in mouse thymocyte apoptosis. MATERIALS AND METHODS: Membranes of thymocytes of RK mice were enriched with cholesterol using methyl-beta-cyclodextrins as carriers. Spontaneous apoptosis was compared with apoptosis induced either by X-irradiation, by treatment with dexamethasone (DEX), and by phorbol-12-myristate-13-acetate (PMA). Apoptotic cells were quantified by means of flow cytofluorometry. RESULTS: Small amounts of incorporated cholesterol enhance the cellular sensitivity for spontaneous apoptosis whereas larger amounts of incorporated cholesterol protect against spontaneous apoptosis and apoptosis induced by irradiation, DEX, or PMA. CONCLUSIONS: Cholesterol exerts specific rigidity effects on lipid membranes which have been shown to be involved in thymocyte apoptosis. The general effect of higher concentrations of cholesterol protecting against apoptosis hints towards a central protective mechanism. This study believes that either cholesterol paralyses great parts of the cell metabolism or that the apoptotic chain reaction is interrupted at a central point due to protection of membrane lipid regions from oxidative stress.     

Role of proteolysis in apoptosis: involvement of serine proteases in internucleosomal DNA fragmentation in immature thymocytes

Three chemically distinct serine, but not cysteine, protease inhibitors (phenylmethylsulphonyl fluoride, N-tosyl-L-phenylalanylchloromethyl ketone and 3,4-dichloroisocoumarin) prevented, in a dose-dependent manner, the characteristic apoptotic internucleosomal DNA cleavage (DNA ladder) typically observed in thymocytes in response to dexamethasone and teniposide VM-26. This effect was not the result of a direct inhibition of the Ca2+,Mg(2+)-dependent endonuclease, since oligonucleosomal DNA cleavage occurred in the presence of these inhibitors in isolated nuclei. The proteolytic step occurred at a very early stage of apoptosis, and preincubation of thymocytes with the inhibitors before dexamethasone or teniposide VM-26 were added irreversibly suppressed ladder formation. This implied that the cellular effector(s) of these compounds preexisted and were not resynthesized in response to the inducers of apoptosis. Serine protease inhibitors also suppressed apoptotic cell shrinkage and complete nuclear collapse, suggesting that these morphological changes were directly related to internucleosomal fragmentation of DNA. However, the serine protease inhibitors did not prevent high molecular weight DNA cleavage (> 50 kilobases) that preceded the ladder formation and thymocytes still died by apoptosis. This supported the view that internucleosomal DNA cleavage, considered to be the biochemical marker of apoptosis, might in fact be a late and dispensable step and that the newly described high molecular weight DNA cleavage might be a better indicator of apoptosis.     

The region responsible for stroke on chromosome 4 in the stroke-prone spontaneously hypertensive rat

To detect genetic loci responsible for stroke susceptibility, we produced 107 male F2 progenies crossed between stroke-prone spontaneously hypertensive rats (SHRSP/Izm) and normotensive Wistar Kyoto rats (WKY/Izm) and followed them up until they developed cerebral stroke. One hundred and twenty-five simple sequence repeat (SSR) markers were analyzed in these F2 rats. Nine of 107 F2 rats suffered from macroscopically overt stroke. In these 9 rats, the segregation ratio of 3 genotypes at 6 SSR marker loci on chromosomes 2, 4, 9, and 10 was highly distorted from the expected value (the observed sp/sp:sp/wky:wky/ wky ratio was either 6:3:0 or 6:2:1, while the expected was 1:2:1, p < 0.01 by chi 2 test). Further, the brain weight was significantly heavier (p < 0.001) in the F2 rats suffering from stroke, suggesting that the brain weight was a parameter for stroke. The brain weight of F2 rats cosegregated with D4Mit19, D4Mgh7, and D4Mgh8 (p = 0.0015, 0.0014, and 0.0040 by ANOVA, respectively) on chromosome 4 supporting genetic effects of this genetic loci on the pathogenesis of cerebral stroke. Blood pressure did not cosegregate with these markers on chromosome 4. These results suggest that a region on chromosome 4, independently of hypertension, determines genetic susceptibility to cerebral stroke.     

Priming of immature thymocytes to CD3-mediated apoptosis by infection with murine cytomegalovirus

Cytomegalovirus (CMV) causes severe clinical manifestations in immunocompromised hosts; however, it remains unclear whether the virus itself is a cause of immunosuppression or whether it is involved as an opportunistic bystander pathogen. This study was performed to elucidate the effect of CMV infection on the host's immune system. The double-positive thymocytes of BALB/c mice inoculated with a sublethal dose of murine CMV (MCMV) were extensively depleted by a 10-micrograms amount of anti-CD3 monoclonal antibody, while such an amount was unable to induce any apparent elimination of thymocytes in noninfected mice. In immature thymocytes of infected hosts, a markedly high level of susceptibility to apoptosis induction was found on treatment with anti-CD3 monoclonal antibody. Analysis of the signal transduction pathway of such double-positive thymocytes demonstrated a profound elevation of the intracellular Ca2+ level after anti-CD3 stimulation, implying that this aberrant mobilization of Ca2+ plays a crucial role in the signaling pathway leading these cells to an extensive apoptosis. Examination of the thymus by PCR was able to detect a low copy number of MCMV DNAs in thymic stromal cells but none at all in thymocytes. Therefore, it is suggested that a mechanism which is not associated with virus replication within the cells exerts a critical effect on rendering the thymocytes highly apoptosis sensitive in hosts infected with MCMV.     

Diterpenoids from germander, an herbal medicine, induce apoptosis in isolated rat hepatocytes

BACKGROUND & AIMS: Germander was withdrawn from the market after its use for weight control caused an epidemic of hepatitis. Its toxicity was shown to be caused by diterpenoids and their cytochrome P4503A-mediated metabolic activation into electrophilic metabolites that deplete cellular thiols. The aim of the present study was to determine the mechanisms of cell death. METHODS: Isolated rat hepatocytes were incubated for 2 hours with germander diterpenoids (100 micrograms/mL). RESULTS: Diterpenoids decreased cell glutathione, increased cytosolic [Ca2+], activated Ca(2+)-dependent tissue transglutaminase forming a cross-linked protein scaffold, and caused internucleosomal DNA fragmentation and the ultrastructural features of apoptosis. Cell death was prevented by decreasing metabolic activation (with troleandomycin), preventing depletion of glutathione (with cystine), blocking activation of Ca(2+)-modulated enzymes (with calmidazolium), or inhibiting internucleosomal DNA fragmentation (with aurintricarboxylic acid). Apoptosis was increased and diterpenoids caused overexpression of p53 and interleukin 1 beta-converting enzyme in rats treated with dexamethasone (cytochrome P4503A inducer). Apoptosis was also increased by a diet deficient in sulfur amino acids. CONCLUSIONS: The germander furano diterpenoids cause apoptosis within 2 hours in isolated rat hepatocytes. Electrophilic metabolites may stimulate apoptosis by decreasing thiols, increasing [Ca2+], and activating Ca(2+)-dependent transglutaminase and endonucleases.