Cell death patterns of the rat spermatogonial cell progeny induced by sertoli cell geometric changes and Fas (CD95) agonist.
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Spermatogonial-Sertoli cell co-cultures, prepared from sexually immature rats (7-10 days old) and maintained for experimental purposes for a maximum period of time of eight days, were used to determine whether Sertoli cell geometry can influence spermatogonial cell growth, viability and differentiation. We have found that when Sertoli cells are allowed to stretch, spermatogonial cell cohorts attached to Sertoli cell surfaces remain viable and exhibit typical cell oscillatory movements with a maximal oscillation radial length of 0.8 microm throughout the duration of the experiments. However, spermatogonial cell viability decreased when Sertoli cells were compelled to contract by preventing cell spreading onto a non-adhesive substrate. A video-microscopy analysis of spermatogonial cells progenies cocultured with contracted Sertoli cells revealed that conjoined members of the cohorts displayed a typical apoptotic sequence preceded by vigorous oscillatory cell movements (maximal oscillation radial length: 1.5 microm) followed by the release of apoptotic bodies and cessation of cell movements. This sequence of events occurred in a single cell. Upon completion of this sequence, another member of the cohort initiated the same cell death course until all members completed the cell death sequence. A similar apoptotic sequence was observed following addition of Fas (CD95/APO-1) antibody (ligand agonist) to the cocultures. Fragmentation of the actin-containing cytoskeleton was observed by indirect immunofluorescence in apoptotic spermatogonial cell cohorts, independent from the activating mechanism. We conclude that by forcing Sertoli cells to contract or by adding an apoptosis inducer to the cocultures, individual members of a spermatogonial cell cohort switch on a death (apoptosis) program in a coordinated fashion. (+info)
Nonproteolytic neuroprotection by human recombinant tissue plasminogen activator.
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Human recombinant tissue plasminogen activator (tPA) may benefit ischemic stroke patients by dissolving clots. However, independent of thrombolysis, tPA may also have deleterious effects on neurons by promoting excitotoxicity. Zinc neurotoxicity has been shown to be an additional key mechanism in brain injuries. Hence, if tPA affects zinc neurotoxicity, this may provide additional insights into its effect on neuronal death. Independent of its proteolytic action, tPA markedly attenuated zinc-induced cell death in cortical culture, and, when injected into cerebrospinal fluid, also reduced kainate seizure-induced hippocampal neuronal death in adult rats. (+info)
Li+ protects nerve cells against destabilization of Ca2+ homeostasis and delayed death caused by removal of external Na+.
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In experiments with fura-2 loaded cultured rat cerebellar granule cells we have compared the changes in [Ca2+]i homeostasis produced by replacement of external Na+ with the organic cation N-methyl-D-glucamine (NMDG) or Li+. The Na+/NMDG replacement caused an increase in baseline [Ca2+]i and a considerable delay in [Ca2+]i recovery following a glutamate (Glu) pulse in almost all the cells. In contrast Na+/Li+ replacement usually did not change baseline [Ca2+]i and produced only a small (if any) delay in the post-glutamate [Ca2+]i recovery. Previously [Storozhevykh et al. (1998) FEBS Lett. 431, 215-218] we revealed that perturbation of [Ca2+]i homeostasis caused by Na+/NMDG replacement cannot be explained by a reversal of the Na+/Ca2+ exchange but is mainly due to Ca2+ influx through NMDA channels activated by Na+ dependent release of endogenous excitatory amino acids ('reversed Glu uptake'). In the present work we confirmed this conclusion and obtained evidence suggesting that in contrast to NMDG Li+ interferes with the 'reversed Glu uptake' triggered by removal of external Na+. Thus it has been shown that the addition of Li+ (20 mM) to a Na+-free NMDG-containing solution suppressed both the perturbation of [Ca2+]i homeostasis and delayed neuronal death caused by Na+/NMDG replacement. Li+ is also able to abolish the [Ca2+]i response induced by PDC which at high concentrations (> 200 microM) is shown to stimulate the release of endogenous Glu. In contrast to Na+/Li+, Na+/NMDG replacement greatly enhances [Ca2+]i increase caused by PDC. Control experiments showed that Na+/Li+ replacement does not decrease the [Ca2+]i response to the Glu pulse. Therefore we concluded that a considerable quantitative difference between the effects of Na+/NMDG and Na+/Li+ replacements on both [Ca2+]i homeostasis and cell viability resulted mainly from the ability of Li+ to attenuate the release of endogenous Glu in response to the removal of external Na+. (+info)
P2Z purinoreceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alterations of cell death.
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Myeloic cells express a peculiar surface receptor for extracellular ATP, called the P2Z/P2X7 purinoreceptor, which is involved in cell death signalling. Here, we investigated the role of caspases, a family of proteases implicated in apoptosis and the cytokine secretion. We observed that extracellular ATP induced the activation of multiple caspases including caspase-1, -3 and -8, and subsequent cleavage of the caspase substrates PARP and lamin B. Using caspase inhibitors, it was found that caspases were specifically involved in ATP-induced apoptotic damage such as chromatin condensation and DNA fragmentation. In contrast, inhibition of caspases only marginally affected necrotic alterations and cell death proceeded normally whether or not nuclear damage was blocked. Our results therefore suggest that the activation of caspases by the P2Z receptor is required for apoptotic but not necrotic alterations of ATP-induced cell death. (+info)
Regulation of reactive oxygen species by nerve growth factor but not Bcl-2 as a novel mechanism of protection of PC12 cells from superoxide anion-induced death.
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Although neurotrophins protect PC12 cells and neurons from oxidative stress-induced death, the molecular mechanism of this effect is largely unknown. Xanthine (XA)+xanthine oxidase (XO) increased the production of the superoxide anion (O2-) and hydrogen peroxide (H2O2), and the death of PC12 cells. Catalase but not superoxide dismutase (SOD) nor a NO scavenger protected PC12 cells from death, indicating that H2O2 is the main effector responsible for this cell death. Both nerve growth factor (NGF) and Bcl-2 protected PC12 cells from O2--induced toxicity. NGF enhanced the production of O2- and suppressed that of H2O2, suggesting that it inhibits the conversion of O2- to H2O2, while Bcl-2 had no such effect. These results suggested that NGF protected the cells from oxidative stress by altering the composition of the reactive oxygen species (ROS) without affecting their total level. (+info)
Genetic vulnerability of cortical neurons isolated from stroke-prone spontaneously hypertensive rats in hypoxia and oxygen reperfusion.
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Severe hypertension and cerebrovascular diseases develop in stroke-prone spontaneously hypertensive rats (SHRSP). Cortical neurons from SHRSP are more vulnerable than those from Wistar Kyoto rats (WKY) to the effects of nitric oxide (NO)- and N-methyl-D-aspartate (NMDA)-mediated neurotoxic agents. Growth factors, idebenone, and nilvadipine (a Ca2+ channel blocker) can reduce neuronal damage caused by hypoxia or neurotoxic agents. This study was designed to determine 1) whether cortical neurons from SHRSP are more vulnerable than those from WKY and 2) whether neuronal damage is minimized by the so-called neuroprotective agents in cells exposed to hypoxia and oxygen reperfusion. We demonstrated that 6 to 24 h of hypoxia did not increase cell death in either WKY or SHRSP, whereas 36 h of hypoxia significantly increased cell death in SHRSP (p < 0.01). Furthermore, 6 to 36 h of hypoxia and 1.5 to 5 h of reperfusion heavily damaged cells from both strains of rats, and most cells became apoptotic or necrotic. We also verified that the ability to protect neurons in hypoxia and oxygen reperfusion was as follows: idebenone > insulin-like growth factor-1 (IGF-1) > nilvadipine. These data indicate that oxygen radical generation occurs and the free radicals heavily damage neurons in hypoxia and oxygen reperfusion. SHRSP neurons are weaker than WKY neurons in these conditions. Furthermore, we surmise that idebenone, an antioxidant, decreases free radicals, and IGF-I attenuates p53-mediated apoptosis and thereby prevents cell death. We conclude that antioxidants are more potent than IGF-1 in protecting cortical neurons from damage caused by hypoxia and oxygen reperfusion, although both are very useful in minimizing damage to cortical neurons. (+info)
Mechanism of the intracellular killing and modulation of antibiotic susceptibility of Listeria monocytogenes in THP-1 macrophages activated by gamma interferon.
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Listeria monocytogenes, a facultative intracellular pathogen, readily enters cells and multiplies in the cytosol after escaping from phagosomal vacuoles. Macrophages exposed to gamma interferon, one of the main cellular host defenses against Listeria, become nonpermissive for bacterial growth while containing Listeria in the phagosomes. Using the human myelomonocytic cell line THP-1, we show that the combination of L-monomethyl arginine and catalase restores bacterial growth without affecting the phagosomal containment of Listeria. A previous report (B. Scorneaux, Y. Ouadrhiri, G. Anzalone, and P. M. Tulkens, Antimicrob. Agents Chemother. 40:1225-1230, 1996) showed that intracellular Listeria was almost equally sensitive to ampicillin, azithromycin, and sparfloxacin in control cells but became insensitive to ampicillin and more sensitive to azithromycin and sparfloxacin in gamma interferon-treated cells. We show here that these modulations of antibiotic activity are largely counteracted by L-monomethyl arginine and catalase. In parallel, we show that gamma interferon enhances the cellular accumulation of azithromycin and sparfloxacin, an effect which is not reversed by addition of L-monomethyl arginine and catalase and which therefore cannot account for the increased activity of these antibiotics in gamma interferon-treated cells. We conclude that (i) the control exerted by gamma interferon on intracellular multiplication of Listeria in THP-1 macrophages is dependent on the production of nitric oxide and hydrogen peroxide; (ii) intracellular Listeria may become insensitive to ampicillin in macrophages exposed to gamma interferon because the increase in reactive oxygen and nitrogen intermediates already controls bacterial growth; and (iii) azithromycin and still more sparfloxacin cooperate efficiently with gamma interferon, one of the main cellular host defenses in Listeria infection. (+info)
Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal.
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After recognition of Ag/MHC and ligation of a costimulatory molecule, resting T cells will clonally expand and then delete to very low levels. Previously, it was shown that deletion can be prevented by coinjection of cytokines or proinflammatory agents such as adjuvants. Here, we demonstrate that ligation of 4-1BB blocks deletion of superantigen-activated T cells in the absence of adjuvant or additional cytokine treatment. Nearly 10 times as many staphylococcal enterotoxin A-specific T cells were detected in the spleens of mice injected 21 days previously with staphylococcal enterotoxin A and an agonist anti-4-1BB Ab compared with mice given staphylococcal enterotoxin A and a control IgG. Even though both CD4- and CD8-activated T cells expressed 4-1BB, a higher proportion of CD8 T cells were rescued compared CD4 T cells. These data suggest that although 4-1BB provides costimulation, it may also promote long-term T cell survival. (+info)