(1/2675) JunB forms the majority of the AP-1 complex and is a target for redox regulation by receptor tyrosine kinase and G protein-coupled receptor agonists in smooth muscle cells.

To understand the role of redox-sensitive mechanisms in vascular smooth muscle cell (VSMC) growth, we have studied the effect of N-acetylcysteine (NAC), a thiol antioxidant, and diphenyleneiodonium (DPI), a potent NADH/NADPH oxidase inhibitor, on serum-, platelet-derived growth factor BB-, and thrombin-induced ERK2, JNK1, and p38 mitogen-activated protein (MAP) kinase activation; c-Fos, c-Jun, and JunB expression; and DNA synthesis. Both NAC and DPI completely inhibited agonist-induced AP-1 activity and DNA synthesis in VSMC. On the contrary, these compounds had differential effects on agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression. NAC inhibited agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression except for platelet-derived growth factor BB-induced ERK2 activation. In contrast, DPI only inhibited agonist-induced p38 MAP kinase activation and c-Fos and JunB expression. Antibody supershift assays indicated the presence of c-Fos and JunB in the AP-1 complex formed in response to all three agonists. In addition, cotransfection of VSMC with expression plasmids for c-Fos and members of the Jun family along with the AP-1-dependent reporter gene revealed that AP-1 with c-Fos and JunB composition exhibited a higher transactivating activity than AP-1 with other compositions tested. All three agonists significantly stimulated reactive oxygen species production, and this effect was inhibited by both NAC and DPI. Together, these results strongly suggest a role for redox-sensitive mechanisms in agonist-induced ERK2, JNK1, and p38 MAP kinase activation; c-Fos, c-Jun, and JunB expression; AP-1 activity; and DNA synthesis in VSMC. These results also suggest a role for NADH/NADPH oxidase activity in some subset of early signaling events such as p38 MAP kinase activation and c-Fos and JunB induction, which appear to be important in agonist-induced AP-1 activity and DNA synthesis in VSMC.  (+info)

(2/2675) N,N'-Diacetyl-L-cystine-the disulfide dimer of N-acetylcysteine-is a potent modulator of contact sensitivity/delayed type hypersensitivity reactions in rodents.

Oral N-acetyl-L-cysteine (NAC) is used clinically for treatment of chronic obstructive pulmonary disease. NAC is easily oxidized to its disulfide. We show here that N,N'-diacetyl-L-cystine (DiNAC) is a potent modulator of contact sensitivity (CS)/delayed type hypersensitivity (DTH) reactions in rodents. Oral treatment of BALB/c mice with 0.003 to 30 micromol/kg DiNAC leads to enhancement of a CS reaction to oxazolone; DiNAC is 100 to 1000 times more potent than NAC in this respect, indicating that it does not act as a prodrug of NAC. Structure-activity studies suggest that a stereochemically-defined disulfide element is needed for activity. The DiNAC-induced enhancement of the CS reaction is counteracted by simultaneous NAC-treatment; in contrast, the CS reaction is even more enhanced in animals treated with DiNAC together with the glutathione-depleting agent buthionine sulfoximine. These data suggest that DiNAC acts via redox processes. Immunohistochemically, ear specimens from oxazolone-sensitized and -challenged BALB/c mice treated with DiNAC display increased numbers of CD8(+) cells. DiNAC treatment augments the CS reaction also when fluorescein isothiocyanate is used as a sensitizer in BALB/c mice; this is a purported TH2 type of response. However, when dinitrofluorobenzene is used as a sensitizer, inducing a purported TH1 type of response, DiNAC treatment reduces the reaction. Treatment with DiNAC also reduces a DTH footpad-swelling reaction to methylated BSA. Collectively, these data indicate that DiNAC in vivo acts as a potent and effective immunomodulator that can either enhance or reduce the CS or DTH response depending on the experimental conditions.  (+info)

(3/2675) Protective alterations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People's Republic of China.

BACKGROUND: Residents of Qidong, People's Republic of China, are at high risk for development of hepatocellular carcinoma, in part due to consumption of foods contaminated with aflatoxins, which require metabolic activation to become carcinogenic. In a randomized, placebo-controlled, double-blind phase IIa chemoprevention trial, we tested oltipraz, an antischistosomal drug that has been shown to be a potent and effective inhibitor of aflatoxin-induced hepatocarcinogenesis in animal models. METHODS: In 1995, 234 adults from Qidong were enrolled. Healthy eligible individuals were randomly assigned to receive by mouth 125 mg oltipraz daily, 500 mg oltipraz weekly, or a placebo. Sequential immunoaffinity chromatography and liquid chromatography coupled to mass spectrometry or to fluorescence detection were used to identify and quantify phase 1 and phase 2 metabolites of aflatoxin B1 in the urine of study participants. Reported P values are two-sided. RESULTS: One month of weekly administration of 500 mg oltipraz led to a 51% decrease in median levels of the phase 1 metabolite aflatoxin M1 excreted in urine compared with administration of a placebo (P = .030), but it had no effect on levels of a phase 2 metabolite, aflatoxin-mercapturic acid (P = .871). By contrast, daily intervention with 125 mg oltipraz led to a 2.6-fold increase in median aflatoxin-mercapturic acid excretion (P = .017) but had no effect on excreted aflatoxin M1 levels (P = .682). CONCLUSIONS: Intermittent, high-dose oltipraz inhibited phase 1 activation of aflatoxins, and sustained low-dose oltipraz increased phase 2 conjugation of aflatoxin, yielding higher levels of aflatoxin-mercapturic acid. While both mechanisms can contribute to protection, this study highlights the feasibility of inducing phase 2 enzymes as a chemopreventive strategy in humans.  (+info)

(4/2675) Proteasome-dependent degradation of the human estrogen receptor.

In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for the targeted degradation of proteins with short half-lives. The covalent attachment of ubiquitin to lysine residues of targeted proteins is a signal for the recognition and rapid degradation by the proteasome, a large multi-subunit protease. In this report, we demonstrate that the human estrogen receptor (ER) protein is rapidly degraded in mammalian cells in an estradiol-dependent manner. The treatment of mammalian cells with the proteasome inhibitor MG132 inhibits activity of the proteasome and blocks ER degradation, suggesting that ER protein is turned over through the ubiquitin-proteasome pathway. In addition, we show that in vitro ER degradation depends on ubiquitin-activating E1 enzyme (UBA) and ubiquitin-conjugating E2 enzymes (UBCs), and the proteasome inhibitors MG132 and lactacystin block ER protein degradation in vitro. Furthermore, the UBA/UBCs and proteasome inhibitors promote the accumulation of higher molecular weight forms of ER. The UBA and UBCs, which promote ER degradation in vitro, have no significant effect on human progesterone receptor and human thyroid hormone receptor beta proteins.  (+info)

(5/2675) Critical role of glass fiber length in TNF-alpha production and transcription factor activation in macrophages.

Recent studies have demonstrated that dielectrophoresis is an efficient method for the separation of fibers according to fiber length. This method allows the investigation of fiber-cell interactions with fiber samples of the same composition but of different lengths. In the present study, we analyzed the effects of length on the interaction between glass fibers and macrophages by focusing on production of the inflammatory cytokine tumor necrosis factor (TNF)-alpha in a mouse macrophage cell line (RAW 264.7). The underlying molecular mechanisms controlling TNF-alpha production were investigated at the gene transcription level. The results show that glass fibers induced TNF-alpha production in macrophages and that this induction was associated with activation of the gene promoter. Activation of the transcription factor nuclear factor (NF)-kappaB was responsible for this induced promoter activity. The inhibition of both TNF-alpha production and NF-kappaB activation by N-acetyl-L-cysteine, an antioxidant, indicates that generation of oxidants may contribute to the induction of this cytokine and activation of this transcription factor by glass fibers. Long fibers (17 micrometer) were significantly more potent than short fibers (7 micrometer) in inducing NF-kappaB activation, the gene promoter activity, and the production of TNF-alpha. This fiber length-dependent difference in the stimulatory potency correlated with the fact that macrophages were able to completely engulf short glass fibers, whereas phagocytosis of long glass fibers was incomplete. These results suggest that fiber length plays a critical role in the potential pathogenicity of glass fibers.  (+info)

(6/2675) Evidence for proteasome involvement in polyglutamine disease: localization to nuclear inclusions in SCA3/MJD and suppression of polyglutamine aggregation in vitro.

Spinocerebellar ataxia type 3, also known as Machado-Joseph disease (SCA3/MJD), is one of at least eight inherited neurodegenerative diseases caused by expansion of a polyglutamine tract in the disease protein. Here we present two lines of evidence implicating the ubiquitin-proteasome pathway in SCA3/MJD pathogenesis. First, studies of both human disease tissue and in vitro models showed redistribution of the 26S proteasome complex into polyglutamine aggregates. In neurons from SCA3/MJD brain, the proteasome localized to intranuclear inclusions containing the mutant protein, ataxin-3. In transfected cells, the proteasome redistributed into inclusions formed by three expanded polyglutamine proteins: a pathologic ataxin-3 fragment, full-length mutant ataxin-3 and an unrelated GFP-polyglutamine fusion protein. Inclusion formation by the full-length mutant ataxin-3 required nuclear localization of the protein and occurred within specific subnuclear structures recently implicated in the regulation of cell death, promyelocytic leukemia antigen oncogenic domains. In a second set of experiments, inhibitors of the proteasome caused a repeat length-dependent increase in aggregate formation, implying that the proteasome plays a direct role in suppressing polyglutamine aggregation in disease. These results support a central role for protein misfolding in the pathogenesis of SCA3/MJD and suggest that modulating proteasome activity is a potential approach to altering the progression of this and other polyglutamine diseases.  (+info)

(7/2675) N-acetyl-L-cysteine inhibits primary human T cell responses at the dendritic cell level: association with NF-kappaB inhibition.

N-acetyl-L-cysteine (NAC) is an antioxidant molecule endowed with immunomodulatory properties. To investigate the effect of NAC on the induction phase of T cell responses, we analyzed its action on human dendritic cells (DC) derived from adherent PBMC cultured with IL-4 and granulocyte-macrophage CSF. We first found that NAC inhibited the constitutive as well as the LPS-induced activity of the transcription factor NF-kappaB. In parallel, NAC was shown to down-regulate the production of cytokines by DC as well as their surface expression of HLA-DR, CD86 (B7-2), and CD40 molecules both at the basal state and upon LPS activation. NAC also inhibited DC responses induced by CD40 engagement. The inhibitory effects of NAC were not due to nonspecific toxicity as neither the viability of DC nor their mannose receptor-mediated endocytosis were modified by NAC. Finally, we found that the addition of NAC to MLR between naive T cells and allogeneic DC resulted in a profound inhibition of alloreactive responses, which could be attributed to a defect of DC as APC-independent T cell responses were not inhibited by NAC. Altogether, our results suggest that NAC might impair the generation of primary immune responses in humans through its inhibitory action on DC.  (+info)

(8/2675) Modulation of proteasomal activity required for the generation of a cytotoxic T lymphocyte-defined peptide derived from the tumor antigen MAGE-3.

We have analyzed the presentation of human histocompatability leukocyte antigen-A*0201-associated tumor peptide antigen MAGE-3271-279 by melanoma cells. We show that specific cytotoxic T lymphocyte (CTL)-recognizing cells transfected with a minigene encoding the preprocessed fragment MAGE-3271-279 failed to recognize cells expressing the full length MAGE-3 protein. Digestion of synthetic peptides extended at the NH2 or COOH terminus of MAGE-3271-279 with purified human proteasome revealed that the generation of the COOH terminus of the antigenic peptide was impaired. Surprisingly, addition of lactacystin to purified proteasome, though partially inhibitory, resulted in the generation of the antigenic peptide. Furthermore, treatment of melanoma cells expressing the MAGE-3 protein with lactacystin resulted in efficient lysis by MAGE-3271-279-specific CTL. We therefore postulate that the generation of antigenic peptides by the proteasome in cells can be modulated by the selective inhibition of certain of its enzymaticactivities.  (+info)

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