Exposure of N-acetylglucosamine decreases early in dexamethasone-induced apoptosis in thymocytes, demonstrated by flow cytometry using wheat germ agglutinin and pokeweed mitogen. (1/100)

In the present paper we describe changes in the exposure of oligosaccharides containing N-acetylglucosamine (Glc-NAc) during apoptosis of mouse thymocytes. The structures containing this sugar were probed with fluorescein isothiocyanate-labelled lectins, wheat germ agglutinin and pokeweed mitogen in flow cytometric assays. Both lectins bind to structures containing Glc-NAc. The present report describes experiments in which two different dual-staining techniques were used to simultaneously identify apoptotic cells and measure their lectin exposure. In these experiments, we observed an early and substantial decrease in the exposure of Glc-NAc-containing structures associated with the onset of apoptosis, before or simultaneously with phosphatidylserine exposure. This was followed by an increase in the exposure of Glc-NAc-containing structures after longer incubation times, when a large proportion of cells was demonstrated to have fragmented DNA. These results provide evidence for major changes in the structure of plasma membrane oligosaccharides during apoptosis. The initial decrease may be a by-product of the hydrolysis of glycosphingolipids to yield ceramide for apoptotic signalling or a deliberate process related to the removal of cell adhesion signalling structures, associated with the separation of the apoptotic cell from its neighbours. The later increase in Glc-NAc-containing structures may be the result of the incorporation of internal membranes into the plasma membrane or a deliberate production of prophagocytic signals by a still-functioning Golgi apparatus.  (+info)

Increased cell surface exposure of phosphatidylserine on propidium iodide negative thymocytes undergoing death by necrosis. (2/100)

Phosphatidylserine (PS) exposure on propidium iodide negative cells using FITC labelled annexin-V has been used to quantify apoptosis in vitro and in vivo. Detection of PS within cells undergoing necrosis is also possible if labelled annexin-V specific for PS enters the cell following early membrane damage. Necrotic or late apoptotic cells can be excluded from flow cytometric analysis using propidium iodide which enters and stains cells with compromised membrane integrity. Here we show that thymocytes undergoing death exclusively by necrosis show early exposure of PS prior to loss of membrane integrity. This early exposure of PS occurs in cells treated with agents which both raise intracellular calcium levels and are also capable of interacting with protein thiol groups. We also demonstrate that PS exposure in thymocytes induced to undergo apoptosis by three different agents does not correlate with calcium rises but correlates with and precedes DNA fragmentation.  (+info)

The secondary fungal metabolite gliotoxin targets proteolytic activities of the proteasome. (3/100)

BACKGROUND: The fungal epipolythiodioxopiperazine metabolite gliotoxin has a variety of toxic effects such as suppression of antigen processing, induction of macrophagocytic apoptosis and inhibition of transcription factor NF-kappaB activation. How gliotoxin acts remains poorly understood except that the molecule's characteristic disulfide bridge is important for immunomodulation. As this fungal metabolite stabilizes the NF-kappaB inhibitor IkappaBalpha in the cytoplasm, we decided to investigate its molecular mechanism of action. RESULTS: We show that gliotoxin is an efficient, noncompetitive inhibitor of the chymotrypsin-like activity of the 20S proteasome in vitro. Proteasome inhibition can be reversed by dithiothreitol, which reduces gliotoxin to the dithiol compound. In intact cells, gliotoxin inhibits NF-kappaB induction through inhibition of proteasome-mediated degradation of IkappaBalpha. CONCLUSIONS: Gliotoxin targets catalytic activities of the proteasome efficiently. Inhibition by gliotoxin may be countered by reducing agents, which are able to inactivate the disulfide bridge responsible for the inhibitory capacity of gliotoxin.  (+info)

NF-kappaB determines between apoptosis and proliferation in hepatocytes during liver regeneration. (4/100)

Tumor necrosis factor (TNF)-alpha is a potent inducer of apoptotic cell death in various tissues, whereas the transcription factor nuclear factor (NF)-kappaB is essential to protect against TNF-alpha-induced apoptosis. Human hepatoma cell lines were used to investigate the effectiveness and specificity of the fungal metabolite gliotoxin in inhibiting TNF-alpha-induced NF-kappaB activation in transformed cells. Gliotoxin-TNF-alpha cotreatment induced massive apoptosis in these otherwise TNF-alpha-resistant cell lines. With the use of the mouse partial hepatectomy model, we were also able to demonstrate in vivo the capacity of gliotoxin to act as inhibitor of NF-kappaB activation. Bromodeoxyuridine staining of liver sections showed that the lack of NF-kappaB activation correlated with 80% reduction of DNA synthesis 48 h after hepatectomy compared with untreated controls. Additionally, animals treated with gliotoxin showed nuclear condensation and DNA laddering of hepatocytes indicative of apoptosis 24 h after hepatectomy. In summary, our results demonstrate that NF-kappaB is essential in defining the fate of liver cells in response to TNF-alpha in vivo and furthermore implicate gliotoxin as a potential new response modifier for TNF-alpha-based therapy.  (+info)

Gliotoxin-induced cytotoxicity proceeds via apoptosis and is mediated by caspases and reactive oxygen species in LLC-PK1 cells. (5/100)

Renal failure associated with aspergillosis is caused by pathogenic fungi. Gliotoxin is a toxic epipolythiodioxopiperazine metabolite produced by the pathogens. The present study investigated the cytotoxicity and underlying mechanisms induced by gliotoxin in LLC-PK1 cells, a porcine renal proximal tubular cell line. Gliotoxin at 100 ng/ml did not show a cytotoxic effect, but unmasked a dose-dependent cell death induced by TNF-alpha. TNF-alpha-induced cell death in the presence of gliotoxin was associated with hypodiploid nuclei and activation of caspase-3-like proteases. Blockade of caspases by boc-aspartyl (OMe)-fluoromethylketone and z-DEVD.fmk inhibited TNF-alpha-induced cell death. As the concentrations of gliotoxin were increased, gliotoxin killed the cells directly in a dose-dependent manner. Further analyses of DNA fragmentation, hypodiploid nuclei, mitochondrial membrane potential, and plasma membrane integrity revealed that cell death proceeded via apoptosis. Gliotoxin-induced apoptosis was associated with dose-dependent and time-dependent activation of caspase-3-like proteases. Boc-aspartyl (OMe)-fluoromethylketone attenuated the killing effect. Gliotoxin also increased the intracellular levels of reactive oxygen species as measured by flow cytometry. N-acetylcysteine, a well-known antioxidant, completely abolished the gliotoxin-induced caspase-3-like activity, cytotoxicity, and reactive oxygen species. In conclusion, (1) gliotoxin at 100 ng/ml unmasks the ability of TNF-alpha-induced apoptosis, and the effect of TNF-alpha is mediated by caspase-3-like proteases; and (2) at higher concentrations gliotoxin itself induces cell death, which is via apoptosis and dependent on caspase-3-like activity and reactive oxygen species.  (+info)

Nuclear factor-kappa B activity and intestinal inflammation in dextran sulphate sodium (DSS)-induced colitis in mice is suppressed by gliotoxin. (6/100)

In acute DSS-induced colitis nuclear factor (NF)-kappaB-dependent inflammatory cytokines including IL-1 and tumour necrosis factor-alpha (TNF-alpha) are up-regulated. Here we examined the effects of gliotoxin, a fungal metabolite known to inhibit NF-kappaB activity, on cytokine production by a mouse cell system in vitro and on intestinal inflammation and NF-kappaB activation in vivo. In vitro gliotoxin decreased TNF-alpha gene expression and protein production by RAW-264.7 mouse macrophage-like cells stimulated with lipopolysaccharide. In vivo, gliotoxin treatment of mice was begun on day 3 of 5% DSS application dissolved in the drinking water and continued until day 8. Gliotoxin treatment dose-dependently down-regulated colonic inflammation as assessed histologically and in parallel there was a suppression of colonic TNF-alpha and IL-1alpha mRNA expression on day 8 as analysed by semiquantitative reverse transcriptase-polymerase chain reaction (P < 0.01). Furthermore, colonic NF-kappaB DNA-binding activity was increased in DSS-induced colitis and was suppressed by gliotoxin. These results demonstrate the essential role of NF-kappaB in DSS-induced colitis and indicate a molecular approach to the treatment of intestinal inflammatory disorders.  (+info)

Inactivation of rabbit muscle creatine kinase by reversible formation of an internal disulfide bond induced by the fungal toxin gliotoxin. (7/100)

The biological activity of gliotoxin is dependent on the presence of a strained disulfide bond that can react with accessible cysteine residues on proteins. Rabbit muscle creatine kinase contains 4 cysteines per 42-kDa subunit and is active in solution as a dimer. Only Cys-282 has been identified as essential for activity. Modification of this residue results in loss of activity of the enzyme. Treatment of creatine kinase with gliotoxin resulted in a time-dependent loss of activity abrogated in the presence of reducing agents. Activity was restored when the inactivated enzyme was treated with reducing agents. Inactivation of creatine kinase by gliotoxin was accompanied by the formation of a 37-kDa form of the enzyme. This oxidized form of creatine kinase was rapidly reconverted to the 42-kDa species by the addition of reducing agents concomitant with restoration of activity. A 1:1 mixture of the oxidized and reduced monomer forms of creatine kinase as shown on polyacrylamide gel electrophoresis was equivalent to the activity of the fully reduced form of the enzyme consistent with only one reduced monomer of the dimer necessary for complete activity. Conversion of the second monomeric species of the dimer to the oxidized form by gliotoxin correlated with loss of activity. Our data are consistent with gliotoxin inducing the formation of an internal disulfide bond in creatine kinase by initially binding and possibly activating a cysteine residue on the protein, followed by reaction with a second neighboring thiol. The recently published crystal structure of creatine kinase suggests the disulfide is formed between Cys-282 and Cys-73.  (+info)

Ligand activation of nerve growth factor receptor TrkA protects monocytes from apoptosis. (8/100)

Nerve growth factor (NGF) receptors are expressed in different cell types outside the nervous system, and increasing evidence indicates that NGF can act as a regulatory molecule during inflammatory and immune responses. In this study, we show that triggering of the high-affinity NGF receptor TrkA with agonists protects monocytes from apoptosis induced by gliotoxin or UVB radiation. TrkA stimulation up-regulates the expression of the anti-apoptotic Bcl-2 family members, Bcl-2, Bcl-XL, and Bfl-1. On the other hand, TrkA stimulation does not change the expression of MHC, CD80, CD86, CD40, and CD54 molecules, nor the antigen-presenting function of monocytes. In addition, during in vitro monocyte to dendritic cell differentiation TrkA expression is progressively lost, suggesting that NGF selectively affects monocyte but not dendritic cell survival.  (+info)

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