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Well-established mechanisms for regulation of protein activity include thiol-mediated oxidoreduction in addition to protein-protein interactions and phosphorylation. Nucleoredoxin (NRX), glutaredoxin (GRX), and thioredoxin (TRX) have been shown to act as a potent thiol reductase and reactive oxygen species regulator. They constitute a oxidoreductase superfamily and have been suggested as a candidate operating in the redox regulation of gene expression. We demonstrated here that intracellular localization of these redox molecules differ from each other and that the redox molecules differentially regulate NF-kappaB, AP-1, and CREB activation induced by TNFalpha, PMA, and forskolin and by expression of signaling intermediate kinases, NIK, MEKK, and PKA in HEK293 cells. This is a first report that describes involvement of NRX and GRX and differences from TRX in transcriptional regulation of NF-kappaB, AP-1, and CREB in living cells.

The CD4 molecule acts as a receptor for class II MHC molecules to stabilize Ag recognition by the TCR and as a high affinity receptor for HIV-1. In this study, we investigated the effect of oxidative stress on the level of CD4 expression on cultured peripheral blood T lymphocytes (PBL blasts). As previously reported, we observed that the surface CD4 was down-regulated by PMA. Unexpectedly, treatment of PBL blasts with hydrogen peroxide (H2O2) or a sulfhydryl oxidative reagent, diamide, almost completely inhibited PMA-induced CD4 down-regulation, although these oxidants per se did not affect the level of CD4 expression. We next assessed the serine phosphorylation of CD4, which is reported to be an indispensable process for PMA-induced CD4 endocytosis. PMA could induce the serine phosphorylation even in the presence of oxidants. We also found that these oxidants had an additive effect on PMA-induced dissociation between CD4 and p56(lck), which is known to be another necessary step for CD4 endocytosis. These results indicate that in T cells, oxidants inhibit protein kinase C-mediated CD4 down-regulation due to perturbing a signaling process other than the above two steps, implying that oxidative stress may tune the functions of CD4+ T cells and their susceptibility to HIV-1 through the control of CD4 expression.

Thiol redox status modulates various aspects of cellular function. We demonstrate that oxidation of cellular sulfhydryl (SH) groups induces apoptosis. In Jurkat T cells and human PBL blasts, the fraction of apoptotic nuclei increased after treatment with an SH-specific oxidant, diamide. Analysis of DNA fragmentation and nuclear morphology also indicated that SH oxidation could induce apoptosis. In the apoptosis induced by SH oxidation, the decrease of cellular glutathione was transient and the increase of glutathione disulfide was observed only after apoptotic changes had occurred. Depletion of cellular glutathione with buthionine sulfoximine failed to induce apoptosis, despite a marked decrease of cellular glutathione, which was greater than that observed in apoptosis induced by diamide. Thus, the changes of cellular glutathione or glutathione disulfide may not be the major cause of apoptosis induced by diamide. Intracellular adult T cell leukemia-derived factor/human thioredoxin, another thiol-related antioxidant protein, was oxidized by incubation with diamide. These results suggest that thiol redox status is one of the key factors of the apoptotic pathway in which thiols other than glutathione may play even more critical roles.

SUMMARYThe serum levels of soluble IL-2 receptor (sIL-2R), IL-4 and IgE-binding factors were examined in children with allergic diseases, and compared with those in non-allergic controls of the same age and sex. The results showed age-related decreases in the serum levels of sIL-2R and IgE-binding factors, but not in that of IL-4 in both allergic and non-allergic individuals. Significant elevation of sIL-2R was observed in sera from children with atopic eczema or history of an anaphylactic reaction to food, as compared with that in non-allergic controls. The serum concentration of IL-4 was elevated in all allergic groups, including cases of atopic eczema, bronchial asthma and anaphylaxis to food, compared with non-allergic controls, and was correlated significantly with the serum level of IgE (r= 0.59). The IgE-binding factor levels in sera from patients aged 6-10 years with bronchial asthma, or patients aged 1-5 years with a history of food anaphylaxis were elevated as compared with those in non-allergic controls of same age. There was no significant correlation between the serum levels of IgE-binding factors and IgE. Since sIL-2R is released by activated T cells, the present study is in favour of T cell activation causing allergic skin disorders. The serum levels of IL-4 as well as IgE did not differ among allergic patients of different clinical categories. The role of IgE in atopic eczema and other allergic diseases is not clearly established; however, it seems likely that IL-4 is deeply involved in the increased production of IgE seen in allergic individuals. The possible involvement of IgE-binding factors in the age-related changes of clinical manifestations in childhood allergic diseases was also discussed.

An increase in intracellular cAMP level induced the expression of IL-2R alpha-chain, the 55-kDa component of IL-2R complex, in a human NK-like cell line, YT. We show here that forskolin also induces the expression of IL-2R alpha-chain on mouse large granular lymphocytes (LGL) but not on T cells. In contrast, treatment with a combination of phorbol ester and calcium ionophore, which is a strong inducer of IL-2R alpha-chain on T cells, does not induce the expression of the alpha-chain on LGL cells. Forskolin was shown to activate the transcription of IL-2R alpha-chain gene in YT cells as revealed by the chloramphenicol acetyltransferase assay. Chemical cross-linking experiments using radio-iodinated IL-2 also supported the enhanced expression of IL-2R alpha-chain by treatment with forskolin. In contrast to the alpha-chain, IL-2R beta-chain was not induced by forskolin as revealed by flow cytofluorometry with a mAb against the beta-chain molecule. These results indicate that the activation of adenylate cyclase induces or/and enhance the expression of IL-2R alpha-chain at the transcriptional level in LGL/NK cells including mouse LGL and human YT cell, which leads to the enhanced expression of high affinity IL-2 receptors.

Expression of low-affinity receptors for IgE (FcɛRII) on T and B cells was examined with monoclonal antibodies to cell surface antigens of FcɛRII (CD23), T cells (CD3) or B cells (CD 19) by two-dimensional analysis under laser flow cytometry system. The majority of cells bearing FcɛRII were B cells; however, a small proportion of T cells (1. 6%) bore FcɛRII in children with atopic asthma and elevated serum level of IgE. The occurrence of FcɛRII positive B cells was also increased in children with atopic asthma compared with non-atopic controls (p < 0.001). The numbers of T and B cells hearing Fc receptors for IgG (FcɛR) did not differ between atopic and non-atopic children. T cells were isolated from venous blood by rosetting with sheep erythrocytes, and then cultured in vitro with phytohemagglutinin plus phorbol-myristate-acetate. The amount of IgE-binding factors (IgE-BF), a soluble form of FcɛRII, was determined by means of enzyme-linked immunosorbent assay. The level of IgE-BF was increased in cultures from children with a atopic asthma compared with non-atopic controls (p < 0.01). The possible involvements of FcɛRII on T cells and their soluble products, IgE-BF, in the pathogenesis of atopic asthma is discussed.

Thioredoxin (TRX) is a stress-inducible protein with diverse intracellular functions, which is expressed under conditions of oxidative stress. Exercise is known to cause oxidative stress by the generation of oxygen radicals from various biological pathways. The purpose of this study was to determine the level of TRX induction of cellular extracts prepared from peripheral blood mononuclear cells after a 30-min swimming exercise in mice. Plasma corticosterone concentration, considered to be a marker for exercise-induced various stress, rose significantly (p0.05) 0.5 h after exercise and rapidly dropped down following recovery. The carbonyl proteins as a marker of oxidative stress were significantly (p0.05) higher after 6 and 12 h of recovery in cytosolic extracts. The cytoplasm and nucleus TRX expressions were slightly higher to resting values after 12 and 24 h of recovery. The nucleus TRX expression was significantly (p0.05) higher after 24 h of recovery. These findings demonstrate that exercise-induced oxidative stress may be associated with increased intracellular TRX expression after 12 and/or 24 h after exercise in peripheral blood mononuclear cells. It is implied that this delayed and prolonged over-expression of TRX may play some roles in response to exercise-induced oxidative stress.

Thioredoxin (TRX) is an ubiquitous protein disulfide reductase, which is known to be involved in the implantation development of mouse embryos. In the present study, recombinant human TRX was used to evaluate its effect on the promotion of preimplantation development of bovine embryos derived from in vitro maturation and fertilization. Supplementation of the medium 24h post insemination with TRX significantly (P

To evaluate the clinical significance of thioredoxin in diabetic patients, serum thioredoxin levels measured with a recently established sandwich enzyme-linked immunosorbent assay kit were compared with clinical laboratory data and complications in 174 patients with Type 2 diabetes. Thioredoxin levels were significantly higher in diabetic patients (mean value, 38 ng/ml) than in healthy controls (21 ng/ml) (p < 0.05). Fasting blood sugar and hemoglobin A1c did not correlate with thioredoxin. Plasma non-esterified fatty acids levels were significantly higher in patients with higher thioredoxin levels (≥ 40 ng/ml) than in those with lower thioredoxin levels (< 40 ng/ml) (p < 0.001). There was a significant correlation both between thioredoxin and non-esterified fatty acids in patients with diet/exercise therapy (p < 0.01) and between thioredoxin and fasting immunoreactive insulin in those treated with diet/exercise or oral hypoglycemic agents (p

The ontogenesis of two anti-oxidative enzymes, thioredoxin (TRX) and glutaredoxin (GRX), was examined immunohistochemically in mouse embryos and fetuses at various developmental stages. They were found to be localized in various tissues, with some tissue specificity and temporal sequence. Both TRX and GRX began to be expressed in many tissues at embryonic (E) day E10 or E11 and tended to increase as the developmental stage advanced. In the heart and neuroepithelium, however, their immunoreactivity was already positive at E8.5. In some fetal organs like the liver, pancreas and kidney, TRX and GRX showed heterogeneous localization, suggesting that their expression may reflect the variable functional states of the cell. These results suggest that TRX and GRX may be associated with tissue differentiation in embryos and fetuses are involved in the acquisition of the capacity of resistance against oxygen radicals.

Compelling evidence has suggested that oxidative stress mediates various cellular responses, and control of reduction/oxidation (redox) is important in maintaining the homeostasis of an organism. The thioredoxin (TRX) system, as well as the glutathione system, is one of the key systems in controlling cellular redox status. TRX is a small ubiquitous protein with the redox-active site sequence -Cys-Gly-Pro-Cys-. It has been demonstrated to be a multifunctional protein, which has regulatory roles in cellular signaling and gene transcription in addition to cytoprotective activities through the quenching of reactive oxygen species. Various oxidative stimuli, such as UV irradiation, cytokines and some chemicals, promptly induce the expression of TRX. Overexpression of TRX correlates with a wide variety of oxidative stress conditions and, in some cases, TRX has shown promising effects for clinical use, for instance in the attenuation of tissue injury in ischemia reperfusion models. The modulation of TRX functions in association with other redox-regulatory molecules should give us a new therapeutic strategy in the treatment of oxidative stress-mediated disorders and diseases.

Estrogen receptor (ER) and p53 are important transcription factors in the growth regulation of tumor cells in breast cancer. We reported previously that thioredoxin (TRX) regulates the DNA binding activities of ER and p53 in vitro. The expression of pS-2, a trefoil factor, is also correlated with that of ER. To clarify the regulation mechanism of tumor growth in breast cancer, here we investigated the expression of TRX, ER, pS-2, and p53 and the mitotic index (MI) in 147 breast cancer tissues using immunohistochemical analysis. Of 123 TRX+ cases, ER+ cases (n = 62) showed a higher pS-2 score and lower MI than did ER- cases (n = 61). Furthermore, p53- cases (no mutation in p53; n = 76) also showed a lower MI than did p53+ cases (n = 47). There was no significant correlation between pS-2 and ER, MI and ER, or p53 and MI in the TRX- group. Among the ER+ and p53- cases (ER+/p53- group; n = 61), MI was lower in the TRX+ group (n = 46) than in the TRX- group (n = 15). However, in all other groups (n = 86) with abnormalities in the immunohistochemical expression of either p53 or ER, there was no significant correlation between MI and TRX expression. In the TRX+ and ER +/p53- group (n = 46), histological grading was lower than that in all other groups (n = 101). These findings suggest that TRX expression is linked to the ER- and p53-dependent regulation of tumor growth in breast cancer. In addition, TRX expression in ER+ and p53 intact (wild-type p53+) groups may mean better prognosis than in other conditions.

Serum thioredoxin (TRX) levels have recently been established as an indicator of oxidative stress in various diseases. The aim of the present study was to clarify the clinical significance of serum ferritin in chronic liver diseases.Levels of ferritin, transferrin saturation (TS), aspartate aminotransferase (AST), and TRX were measured in the sera of patients with chronic hepatitis C (CH-C, n=92), chronic hepatitis B (CH-B, n=28), nonalcoholic fatty liver (FL, n=31), or alcoholic liver diseases (ALD, n=17). Serum TRX levels were evaluated with a recently established sandwich enzyme-linked immunosorbent assay kit.Serum TRX levels were significantly higher in CH-C, FL, and ALD than in healthy volunteers. A larger proportion of patients with CH-C, FL, and ALD had elevated levels of serum ferritin than CH-B. Serum ferritin levels were positively correlated with levels of TS, AST, and TRX in CH-C, but were merely correlated with TS values in CH-B. Ferritin levels were also well correlated with AST and TRX, but not with TS in FL and ALD.Oxidative stress, which was evaluated by measuring serum TRX, in addition to storage iron and hepatocyte damage is a cause of increasing serum ferritin levels in chronic liver diseases. An elevated serum ferritin level, which was correlated with TS, indicates that iron-induced oxidative stress contributes to CH-C. Elevated ferritin levels in FL and ALD may be mostly due to iron-unrelated stresses.

Bleomycin (BLM) is an anticancer drug, administration of which leads to severe lung injury, in which the generation of intracellular reactive oxygen species (ROS) is thought to participate in that. Thioredoxin (TRX) has been found to function as a powerful antioxidant by reducing ROS, and thus protecting against ROS-mediated cytotoxicity. However, a protective role of TRX in BLM-induced lung injury has not been determined. In the present study, we therefore attempted to clarify this issue. Human TRX-transfected L929 murine fibrosarcoma cells were more resistant to BLM-induced cytotoxicity than the parental and the control transfected cells, indicating that TRX plays the protective role in BLM-induced cytotoxicity. Next, we examined TRX expression in the lung of in vivo model of BLM-induced lung injury and BLM-stimulated bronchial epithelial cells in vitro to clarify the role of TRX in BLM-induced lung injury. In the lungs of BLM-treated mice, the expression of TRX was strongly induced in bronchial epithelial cells. TRX expression was also up-regulated at both the mRNA and protein levels in cultured BEC with the treatment with BLM. However, the expression of other major antioxidants, such as Cu/Zn-SOD, Mn-SOD, catalase and glutathione peroxidase, was not affected by BLM. These observations suggest that the cellular reduction and oxidation (redox) state modified by TRX is involved in the BLM resistancy and the induction of TRX in bronchial epithelial cells might play a protective role in BLM-induced lung injury.

Thioredoxin plays an important role in various cellular processes through redox regulation. Here, we have demonstrated that thioredoxin expression is transcriptionally induced in K562 cells by hemin (ferriprotoporphyrin IX) through activation of a regulatory region positioned from -452 to -420 bp of the thioredoxin gene. Insertion of a mutation in the antioxidant responsive element (ARE)/AP-1 consensus binding sequence in this region abolished the response to hemin. With electrophoretic mobility shift and DNA affinity assays, we have shown that the NF-E2p45/small Maf complex constitutively binds to the ARE. The binding of the Nrf2/small Maf complex to ARE was induced by hemin, whereas the binding of Jun/Fos proteins to ARE was induced by phorbol 12-myristate 13-acetate, but not hemin. Hemin induced nuclear translocation of Nrf2 but did not affect nuclear expression of Jun/Fos proteins. Overexpression of Nrf2 augmented the response to hemin in a dose-dependent manner. In contrast, overexpression of the dominant negative mutant of Nrf2 suppressed hemin-induced activation through the ARE. We show here hemin-induced activation of the thioredoxin gene by Nrf2 through the ARE and propose a novel mechanism of the regulation of the ARE through a switch of its binding factors.

To investigate whether lipid peroxides play a role in retinal cell death due to ischemia-reperfusion injury, whether recombinant human thioredoxin (rhTRX) treatment reduces production of lipid peroxides of the retina, and whether such treatment reduces the number of cells expressing c-Jun and cyclin D1.Retinal ischemia was induced in rats by increasing the intraocular pressure to 110 mm Hg for 60 minutes. After reperfusion, immunohistochemical staining for lipid peroxide, peroxynitrite, c-Jun, and cyclin D1 and propidium iodide (PI) staining were performed on retinal sections from animals treated intravenously with and without rhTRX, a free radical scavenger. Quantitative analyses of PI-, c-Jun-, and cyclin D1-positive cells were performed after the ischemic insult. Concentration of lipid peroxides in the retina was determined by the thiobarbituric acid assay.Specific immunostaining for lipid peroxides was seen in the ganglion cell layer at 6 hours after reperfusion, in the inner nuclear layer at 12 hours, and in the outer nuclear layer at 48 hours. Time course studies for PI-positive cells in the three nuclear layers coincided with those of specific immunostaining for lipid peroxides. The specific immunostaining was weakened by pre- and posttreatment with 0.5 mg of rhTRX. The number of PI-, c-Jun-, and cyclin D1-positive cells and the concentration of lipid peroxides were significantly decreased by treatment with rhTRX compared with those of vehicle-treated control rats (P:0. 01).Lipid peroxides formed by free radicals may play a role in neuronal cell death in retinal ischemia-reperfusion injury.

Pancreatic cancer (pancreatic ductal carcinoma) is one of the most malignant solid tumors with poor prognosis. There is accumulating evidence that cellular reduction/oxidation (redox) status is deeply involved in the growth promotion and drug resistance of cancer cells. We therefore investigated the expression of redox-regulating proteins, such as thioredoxin (TRX) and glutaredoxin (GRX) in surgically resected pancreatic tissues and cis-diamminedichloroplatinum (CDDP)-resistant cells. Plasma levels of TRX were also measured in subjects with pancreatic diseases. Pancreatic ductal carcinoma tissues were immunohistochemically more positive for TRX (24/32 cases) and GRX (29/32 cases) than pancreatic cystadenocarcinoma or normal pancreas tissues. Plasma levels of TRX (mean +/- SD) measured by ELISA were significantly higher in patients with pancreatic ductal carcinoma (54.8 +/- 37.6 ng/ml, n = 60) than in healthy controls (24.4 +/- 12.9 ng/ml, n = 81). CDDP-resistant subclones of HeLa cells, HeLa-CP5 cells, had higher expression of TRX (1.5 fold) and GRX (1.6 fold) compared with parental HeLa cells by immunoblotting. These results indicate the possible association of TRX and GRX with malignant potential of pancreatic ductal carcinoma.

To examine the expression of the thioredoxin (TRX)-thioredoxin reductase (TR) system in patients with rheumatoid arthritis (RA) and patients with other rheumatic diseases.Levels of TRX in plasma and synovial fluid (SF) were measured using enzyme-linked immunosorbent assay. Cellular distribution of TRX was determined by flow cytometry and histochemistry. Cellular expression of TR was studied by in situ messenger RNA (mRNA) hybridization. The effect of oxidative stress and tumor necrosis factor alpha (TNF alpha) on TRX expression by cultured rheumatoid fibroblast-like synoviocytes was studied.Significantly increased TRX levels were found in the SF from 22 patients with RA, when compared with plasma levels in the same patients (P0.001) and compared with SF TRX levels in 15 patients with osteoarthritis (P0.001), 13 patients with gout (P0.05), and 9 patients with reactive arthritis (P0.0001). The presence of TRX could be demonstrated within the SF-derived mononuclear cells and synovial tissue (ST) of RA patients. Concordantly, expression of TR mRNA was observed in the ST of these patients. Stimulation of synovial fibroblast-like synoviocytes with either H2O2 or TNF alpha induced an increase in the production of TRX.The data demonstrate significantly increased concentrations of TRX in the SF and ST of RA patients when compared with the levels in patients with other joint diseases. Evidence is presented that the local environment in the rheumatic joint contributes to increased TRX production. Based on its growth-promoting and cytokine-like properties, it is proposed that increased expression of TRX contributes to the disease activity in RA.

Oxidative stresses such as UV irradiation to mammalian cells triggers a variety of oxistress responses including activation of transcription factors. Recently, activation of nuclear factor-kappaB (NF-kappaB) has been shown to be under oxidoreduction (redox) regulation controlled by thioredoxin (TRX), which is one of major endogenous redox-regulating molecules with thiol reducing activity. In order to elucidate where in the cellular compartment TRX participates in NF-kappaB regulation, we investigated the intracellular localization of TRX. UVB irradiation induced translocation of TRX from the cytoplasm into the nucleus. In our in vitro diamide-induced cross-linking study, we showed that TRX can associate directly with NF-kappaB p50. Overexpression of wild-type TRX suppressed induction of luciferase activity under NF-kappaB-binding sites in response to UV irradiation compared with the mock transfectant. In contrast, overexpression of nuclear-targeted TRX enhanced the luciferase activity. Thus, TRX seems to play dual and opposing roles in the regulation of NF-kappaB. In the cytoplasm, it interferes with the signals to IkappaB kinases and blocks the degradation of IkappaB. In the nucleus, however, TRX enhances NF-kappaB transcriptional activities by enhancing its ability to bind DNA. This two-step TRX-dependent regulation of the NF-kappaB complex may be a novel activation mechanism of redox-sensitive transcription factors.

Human thioredoxin (hTrx) is a cellular redox-active protein that catalyzes dithiol/disulfide exchange reactions, thus controlling multiple biological functions, including cell growth-promoting activity. Here we show that the expression of hTrx protein and messenger RNA was up-regulated by incubation with 17beta-estradiol (E2) in primary culture of stromal cells isolated from human endometrium. Maximal enhancement of hTrx protein and messenger RNA was observed after 6-12 h of incubation with 10-100 nM E2, and the enhancing effect was suppressed by tamoxifen, an estrogen antagonist. Release of hTrx into the culture medium was markedly augmented after 5-day exposure of E2 plus progesterone (P) accompanied by in vitro differentiation of endometrial stromal cells (decidualization). Immunocytochemical studies showed that hTrx was localized in the nucleus, nucleolus, and cytosol in the stromal cells. Strongly enhanced immunoreactivity for hTrx was observed in the E2-treated cells, whereas there was no apparent difference in the pattern of subcellular localization among the untreated and E2- and/or P-treated cells. Although 1-50 microg/ml recombinant hTrx alone did not promote endometrial stromal cell growth, epidermal growth factor-dependent mitogenesis was additively enhanced by hTrx. Our results indicate that hTrx modulates endometrial cell growth, acting as a comitogenic factor for epidermal growth factor, which is known to be a mediator of estrogen action. It is also suggested that hTrx is deeply involved in the hormonal control of the endometrium by E2 and P, playing a regulatory role in endometrial cell growth and differentiation.

Oxidative stress induces a variety of cellular responses, including apoptosis, and caspase family proteases are known to be involved in apoptosis. Caspase-3(-like) protease activity was examined in Jurkat T cells to investigate the mechanism of apoptosis induced by a thioloxidant, diamide. Caspase-3 was activated when cells were cultured with 200 microM diamide that induced apoptosis, whereas no caspase-3 activation was detected with 500 microM diamide that induced necrosis. When apoptosis was induced in cells with exposure to 200 microM diamide, the intracellular thioredoxin (TRX) levels were maintained and the intracellular generation of reactive oxygen intermediates was marginal. The cytosolic fractions of cytochrome c were increased earlier than the activation of caspase-3. In contrast, when cells were exposed to 500 microM diamide, intracellular reactive oxygen intermediate generation was increased and processing of caspase-3 was not detected despite cytochrome c release, resulting in necrosis. Caspase-3 activity in cell lysate precultured with anti-Fas Ab was suppressed dose dependently by diamide and restored by thiol-reducing agents, DTT or TRX. When cells were precultured with 5 mM of buthionine sulfoximine, an inhibitor of glutathione synthesis, intracellular TRX levels were maintained, and as low as 20 microM diamide could induce apoptosis associated with the increase of cytosolic cytochrome c and the activation of caspase-3. These results indicate that the activation of caspase-3 in diamide-induced apoptosis is mediated, at least partly, by cytochrome c release from mitochondria, and the cellular reducing environment maintained by TRX, as well as glutathione, is required for caspase-3 activity to induce apoptosis.

Thioredoxin (TRX) is an intracellular enzyme that has a variety of activities as a hydrogen donor for various intracellular molecules. In the present study, we investigated the role of TRX in atherosclerotic lesions. In human atherosclerotic specimens, TRX and TRX mRNA were enhanced in endothelial cells and macrophages in the atherosclerotic plaques. In balloon-injured rat arteries, TRX expression increased from 2 to 6 weeks after injury; TRX was induced in the neointimal regenerating endothelial cells. In hybridization experiments, TRX mRNA was also induced from 2 to 6 weeks in the endothelium. In this model, inducible nitric oxide synthase immunoreactivity in the neointimal smooth muscle cells and endothelial cells increased from 2 to 6 weeks after surgical procedures were performed. During this period, the immunoreactivity of nitrotyrosine, which is a marker of nitric oxide (NO) production, also increased. We focused on the association between TRX and NO. In vitro studies using a murine endothelial cell line showed TRX and TRX mRNA induction by NO and peroxynitrite donors. Enhanced expression of TRX was detected mainly within the cytoplasm in immunocytochemical studies. In addition, TRX-transfected cells showed resistance to peroxynitrite-induced cytotoxicity. These findings indicate that TRX and the cellular redox state modified by TRX play a crucial role in arterial neointima formation in atherosclerosis.

To evaluate the protective effects of recombinant adult T-cell leukemia- derived factor (ADF)-human thioredoxin against ischemia-reperfusion injury in the rat retina.Retinal ischemia was induced in rats by increasing the intraocular pressure to 110 mm Hg for 60 minutes. Various doses of recombinant human ADF (rhADF) or vehicle were administered intravenously before ischemia induction and immediately after reperfusion. The degree of retinal damage was assessed by electroretinogram (ERG) recording, by measuring the inner retinal thickness, and by counting the number of TdT-dUTP terminal nick-end labeling (TUNEL)-positive cells in the inner nuclear layer.The amplitudes of the ERG b-wave and oscillatory potentials were increased significantly by treatment before ischemia and after reperfusion with 0.5 mg or 5 mg rhADF and by treatment after reperfusion with 1 mg rhADF, compared with those of vehicle-treated control rats (P0.01). On day 28 after reperfusion, the thickness of the inner retina of control rats and of rats treated before ischemia and after reperfusion with 0.5 mg rhADF were 46.1+/-6.4 microm and 78.5+/-8.9 microm, respectively (P0.01). The number of TUNEL-positive cells on days 1 and 2 after reperfusion was decreased significantly by treatments with 0.5 mg rhADF compared with the number of TUNEL-positive cells in control rats (P0.01).Electrophysiologic and histologic studies showed that ischemia for 60 minutes produces severe damage in vehicle-treated control rat retina, particularly in the inner retinal layer. Intravenous injection of rhADF protects the rat retina from ischemia-reperfusion injury.

We have shown that cell cycle progression of human T-cell leukemia virus type I (HTLV-I)-transformed T-cell lines was inhibited by 13-cis-retinoic acid (13cRA). In the present study, we report that 13cRA inhibited proliferation and induced cell death of peripheral blood mononuclear cells obtained from four patients with acute adult T-cell leukemia but not of mitogen- or interleukin 2-activated peripheral blood mononuclear cells from HTLV-I-negative healthy donors. Because HTLV-I-infected lymphocytes are susceptible to oxidative stress, we examined the role of the intracellular redox state in 13cRA-induced cell death using a HTLV-I-positive T-cell line, ATL2, as a model. 13cRA induced apoptosis in ATL2 cells within 48 h in a dose-dependent manner. The ability of 13cRA to induce apoptosis was more potent than that of all-trans-retinoic acid. Apoptosis induction by 13cRA was significantly enhanced by buthionine sulfoximine (BSO), which decreased the levels of intracellular reduced glutathione, although 13cRA by itself did not alter them, suggesting that intracellular reduced glutathione may modulate 13cRA-induced apoptosis. In addition, flow cytometric analysis revealed that 13cRA increased intracellular peroxides in 24 h and that the addition of BSO further enhanced them. Although N-acetylcysteine had only a marginal effect, pretreatment with catalase markedly inhibited 13cRA-induced apoptosis. These results suggest that peroxide generation, ie., oxidative stress, may play a crucial role in the induction of apoptosis by 13cRA and further demonstrate that combined treatment with 13cRA and BSO induces apoptosis of HTLV-I-positive lymphocytes even more potently.

We have previously shown that tumor necrosis factor-alpha (TNF-alpha) reduces interleukin-4 (IL-4)-induced Fc epsilon RII expression in human monocytes. It has been shown that TNF-alpha activates nuclear transcriptional factors through the generation of reactive oxygen intermediates (ROIs), and antioxidant N-acetylcysteine (NAC) inhibits TNF-alpha-induced activation of nuclear transcriptional factors. Therefore, we hypothesized that TNF-alpha-dependent reduction of IL-4-induced Fc epsilon RII expression in monocytes might be mediated through the ROIs-activated mechanism. In the present study, to test our hypothesis, we examined the effect of NAC on TNF-alpha-dependent reduction of IL-4-induced Fc epsilon RII expression in human monocytes. NAC attenuated TNF-alpha-dependent reduction of IL-4-induced Fc epsilon RII expression by attenuating TNF-alpha-dependent reduction of Fc epsilon RII mRNA expression. Similarly, the structurally unrelated antioxidant, pyrrolidine dithiocarbamate (PDTC), also effectively attenuated this-reduction. These results indicate that an ROIs-activated and antioxidant-sensitive mechanism might be involved in TNF-alpha-dependent reduction of IL-4-induced Fc epsilon RII expression in monocytes.

Adult T-cell leukemia-derived factor (ADF), originally defined as an interleukin-2 receptor inducer, is a human thioredoxin (TRX). ADF/TRX is a conserved multifunctional reductant presumably associated with redox regulation of the cellular environment; it works in vitro as a cytokine, free radical scavenger, activator of transcription factors, and substrate for several enzymes. In the present series of experiments, we studied the expression and intracellular localization of ADF/TRX in mouse kidney from two different renal tubular injury models: a free radical-associated model with ferric nitrilotriacetate (Fe-NTA) and a free radical-independent model with HgCl2. Markers of oxidative damage, such as 8-hydroxy-2'-deoxyguanosine, thiobarbituric acid-reactive substances, and 4-hydroxy-2-nonenal-modified proteins, were significantly increased in kidney from male C57B/6 mice 1 hour after a single intraperitoneal injection of Fe-NTA (5 mg iron/kg). However, in kidney from mice given a subcutaneous injection of HgCl2 (5 mg Hg/kg), none of these markers were increased, despite tubular necrosis with sulfhydryl depletion. In the Fe-NTA model only, Northern and Western blot analyses of the kidney revealed induction of ADF/TRX (2.5-fold) after 16 hours and translocation of ADF/TRX from the cytoplasmic to nuclear fraction (3.5-fold) after 24 hours. Immunohistochemistry showed a patchy distribution of ADF/TRX in the normal renal proximal tubules. In ex vivo experiments using serial normal kidney frozen sections, it was found that renal proximal tubules with low expression of ADF/TRX were more vulnerable to oxidative stress mediated by Fe-NTA. Collectively, these data suggest that: (a) ADF/TRX is induced and translocated to nuclei by oxidative damage mediated by Fe-NTA in the renal proximal tubules; (b) induction of ADF/TRX is independent of tubular necrosis or sulfhydryl depletion; and (c) ADF/TRX is involved in the cellular defense mechanisms in vivo against oxidative damage.

Transcription factor PEBP2/CBF consists of a DNA binding subunit, alpha, and a regulatory subunit, beta. The alpha subunit has an evolutionarily conserved 128-amino acid region termed "Runt domain" that is responsible for both DNA binding and heterodimerization with the beta subunit. The Runt domain in all mammalian submembers of the alpha subunit contains two conserved cysteine residues, and its DNA binding activity undergoes redox regulation. To investigate the mechanism of this redox regulation, we performed site-directed mutagenesis of the two conserved cysteines in the Runt domain of the mouse PEBP2alphaA homolog. Substitution of Cys-115 to serine resulted in a partially impaired DNA binding, which remained highly sensitive to a thiol-oxidizing reagent, diamide. Conversely, the corresponding substitution of Cys-124 caused an increased DNA binding concomitant with an increased resistance to diamide. In contrast, substitution of either cysteine to aspartate was destructive to DNA binding to marked extents. These results have revealed that both Cys-115 and Cys-124 are responsible for the redox regulation in their own ways with low and high oxidizabilities, respectively. We have also found that two cellular thiol-reactive proteins, thioredoxin and Ref-1, work effectively and synergistically for activation of the Runt domain. Interestingly, the beta subunit further enhanced the activation by these proteins and reciprocally prevented the oxidative inactivation by diamide. These findings collectively suggest the possibility that the Runt domain's function in vivo could be dynamically regulated by the redox mechanism with Trx, Ref-1, and the beta subunit as key modulators.

The authors examined the regulatory effects of tumour necrosis factor-alpha (TNF-alpha) on granulocyte macrophage colony stimulating factor (GM-CSF)-, interleukin-3 (IL-3)- or macrophage colony stimulating factor (M-CSF)-induced gene expression of the low affinity receptor for IgE (Fc epsilon RII) on human monocytes and GM-CSF-, IL-3- or M-CSF-induced soluble Fc epsilon RII (sFc epsilon RII) release from monocytes. The expression of GM-CSF-, IL-3- or M-CSF-induced Fc epsilon RII on the surface of monocytes was reduced by TNF-alpha. The present analysis was designed to examine whether or not TNF-alpha could suppress GM-CSF-, IL-3- or M-CSF-induced Fc epsilon RII messenger RNA (mRNA) expression and enhance the release of sFc epsilon RII induced by these cytokines. The addition of TNF-alpha to monocyte cultures with GM-CSF, IL-3 or M-CSF significantly reduced Fc epsilon RII expression on the surface of monocytes and significantly increased sFc epsilon RII release from monocytes. These results suggest that TNF-alpha-dependent reduction of GM-CSF-, IL-3- or M-CSF-induced Fc epsilon RII expression on the surface of monocytes resulted, at least in part, from the suppression of Fc epsilon RII mRNA and the enhancement of sFc epsilon RII release.

Thymic ID cells are involved in the differentiation of mature T cells which are resistant against apoptosis. TRX/ADF is a potent thiol-related reducing agent, acts as a redox regulator, and it can attenuate the induction of apoptosis of T lineage lymphocytes. In the present study, 42 thymoma-free thymus and 40 thymoma samples were examined to identify the expression of TRX/ADF in human thymic tissue. TRX/ADF high-producer (TRXh) cells with cytoplasmic protrusions were found distributed in the thymic medulla. These TRXh cells were negative for CD3, a lymphocyte marker, keratin, an epithelial cell marker, and CD68 or lysozyme, macrophage/monocyte markers, but were positive for S100 protein and HLA-DR complex. Our results revealed that the TRXh cells in the thymic medulla were ID cells. As TRX/ADF has an important and fundamental role in cellular responses acting against oxidative stress, TRX/ADF may provide an explanation of cellular interaction between the medullary ID cells and the mature T cells.

Fc epsilon R II/CD23 is a low affinity Fc receptor for IgE. It plays various biological roles. In this paper, we reviewed recent knowledge of this molecule.

Thioredoxin, a cellular thiol, functions as a self-defense mechanism in response to environmental stimuli, including oxidative stress. We first determined cellular levels of thioredoxin in several human bladder and prostatic cancer cell lines resistant to cis-diamminedichloroplatinum(II) (cisplatin). All cisplatin-resistant cell lines had much higher levels of thioredoxin than those in their drug-sensitive parental counterpart. We then, by introducing thioredoxin antisense expression plasmids into human bladder cancer T24 cells, established two bladder cancer cell lines that had decreased levels of thioredoxin. These thioredoxin antisense transfectants showed increased sensitivity to cisplatin and also to other superoxide-generating agents, i.e., doxorubicin, mitomycin C, etoposide, and hydrogen peroxide, as well as to UV irradiation, but not to the tubulin-targeting agents, vincristine, and colchicine. Cellular levels of thioredoxin thus appear to limit sensitivity to various superoxide-generating anticancer drugs in cancer cells.

We examined the regulatory effects of TNF-alpha on IL-4-induced gene expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) in human monocytes and IL-4-induced soluble Fc epsilon RII (sFc epsilon RII) release from monocytes. IL-4-induced Fc epsilon RII expression on the surface of monocytes was reduced by TNF-alpha as early as 1 day after culture and the effect of TNF-alpha increased with prolonged culture. The present analysis was designed to examine whether or not TNF-alpha could suppress IL-4-induced Fc epsilon RII mRNA expression and enhanced IL-4-induced sFc epsilon RII release. The addition of TNF-alpha to monocyte cultures with IL-4 significantly reduced Fc epsilon RII expression on the surface of monocytes and significantly increased sFc epsilon RII release from monocytes. Over time, there was an inverse relationship between the disappearance of cell surface Fc epsilon RII and the appearance of sFc epsilon RII in culture supernatants. Fc epsilon RII mRNA expression in monocytes cultured with IL-4 was not affected by TNF-alpha when examined at 6 h after cultivation. When the cells were cultured with TNF-alpha for more than 24 h, however, TNF-alpha down-regulated IL-4-induced Fc epsilon RII mRNA levels. This correlated with the kinetics of down-regulation of IL-4-induced Fc epsilon RII expression on the surface of monocytes by TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

This study was undertaken to define intracellular signaling pathways upstream to glycogen synthase activation. First, we examined the role of the two pathways of insulin signaling, Ras-dependent and wortmannin/LY294002-sensitive, in glycogen synthase activation. Although negative dominant Ras (Ras17N) induction in PC12 cells markedly decreased activities of mitogen-activated protein kinase (MAP) and pp90 S6 kinase in response to insulin or insulin-like growth factor I (IGF-I), activation of glycogen synthase by these agents was unaffected by negative dominant Ras induction. In contrast, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), inhibitors of phosphatidylinositol 3-kinase, antagonized glycogen synthase activation in response to insulin or IGF-I. Next, we examined the contribution of pp70 S6 kinase, one of the wortmannin/LY294002-sensitive signaling molecules on glycogen synthase activation. Immunosuppressant rapamycin completely blocked activation of pp70 S6 kinase by insulin or IGF-I, but rapamycin alone or in combination with induction of negative dominant Ras failed to antagonize glycogen synthase activation by these hormones. These data suggest that 1) activation of Ras-MAP kinase is not necessary for stimulation of glycogen synthase and 2) activation of wortmannin/LY294002-sensitive pathway, independent of pp70 S6 kinase, plays a key role in glycogen synthase regulation in PC12 cells.

Adult T cell leukemia derived factor (ADF), which was first reported as a cytokine-like factor produced by human T lymphotropic virus I (HTLV-I)-transformed T cells, is a human homologue of thioredoxin (TRX). ADF/TRX has multiple functions including growth promoting, antiapoptotic and radical scavenging activities, and is also involved in a wide variety of intracellular processes as a dithiol reducing agent in cooperation with the NADPH-TRX reductase system. In HTLV-1(+) T cell lines, HuT 102 and MT-2, which are ADF/TRX high producing cells, we found that the expression of ADF/TRX was dependent on the cell cycle and peaked at S phase. The reducing activity of ADF/TRX in these cells was also dependent on the cell cycle and elevated in S phase as determined by NADPH-dependent insulin degradation assay. Furthermore, inhibitors of TRX reductase, 13-cis-retinoic acid (13-cis-RA) and azelaic acid, inhibited the DNA synthesis of these cells. In contrast, the residual expression and reducing activity of ADF/TRX in HTLV-I(-) T cell lines did not show any significant correlation with the cell cycle. There was no distinct inhibitory effect of 13-cis-RA or azelaic acid on the growth of these ADF/TRX low producing cells. These results indicate that a high level of reducing activity of the ADF/TRX system may be required for the cell division of these virally transformed cells. This suggests that the TRX reductase inhibitors including retinoid derivatives have a potential therapeutic utility for treatment of HTLV-1(+) T cell leukemia without any effect on HTLV-I(-) cells.

The adult T cell leukemia (ATL)-derived factor (ADF) was first described as an interleukin 2 receptor alpha chain (IL-2R alpha) inducing factor which is produced by an HTLV-I infected cell line. Subsequent purification and gene cloning proved that it is a human homologue of a bacterial reducing coenzyme, thioredoxin (TRX). ADF/human TRX (hTRX) has multiple functions both in the extracellular and intracellular compartments, such as cytokine activity, dithiol-reducing activity and radical scavenging activity. ADF/hTRX can facilitate the interactions between the transcriptional factors and its target DNA sequences, which may result in the overexpression of IL -2R alpha in HTLV-I infected cells. Recently, we have detected the presence of ADF/hTRX in human serum (sADF) obtained from healthy volunteers using the insulin reducing assay and Western blotting analysis. Another endogenous redox regulator, glutathione (GSH) system, has long been studied for its relation to cell proliferation and activation. Our recent data showed that thiol compounds such as L-cysteine and GSH may be involved in the activation and cell cycle progression of stimulated lymphocytes. We have also found that ADF/hTRX promotes L-cysteine transport into the cells and increases intracellular GSH content, indicating the close association between ADF/hTRX and GSH systems. Redox regulation by ADF/hTRX and GSH systems seems to play an important role in regulating cell proliferation and activation. To assess the possible alteration of the sADF level in pathological conditions, such as viral infections, an ELISA system for ADF/hTRX was recently established using two different monoclonal antibodies against rADF.(ABSTRACT TRUNCATED AT 250 WORDS)

The proliferative response of PBMC to PHA, Con A, OKT3 mAb and IL-2-dependent proliferation of PHA-blasts was examined in a thiol-free environment (cultured in a L-cystine- and GSH-free medium). [3H]TdR incorporation assay and cell cycle analysis revealed that stimulated PBMC could not enter the S phase when deprived of these thiol compounds. In thiol-free cultures, an increase in intracellular free Ca2+ concentration and IL-2R alpha-chain/p 55 (Tac) induction was still observed, whereas transferrin receptor induction was markedly reduced, suggesting that the proliferative response of mitogenically stimulated PBMC was arrested in the late G1 phase in which transferrin receptor is induced. In GSH-depleted cultures, a similar reduction of the proliferative response of PBMC and PHA-blasts was observed when the concentration of L-cystine was lowered, in a dose-dependent manner. Each reduction or loss of proliferative response was partially restored by supplementation of 2-ME or adult T cell leukemia-derived factor (ADF)/human thioredoxin which is considered to be an endogenous dithiol-reducing factor. L-Cystine transport analysis showed that mitogenically stimulated PBMC and PHA blasts incorporated L-cystine, whereas resting PBMC did not. Furthermore, ADF as well as 2-ME exhibited an enhancing activity on the L-cystine transport in PHA blasts. Together with the fact that L-cystine transport is a limiting step in glutathione synthesis, these findings suggest that GSH and ADF might cooperate in the thiol-mediated redox regulation process and might also play key roles in cell cycle (late G1 to S) progression of activated lymphocytes.

Adult T cell leukemia derived factor (ADF) is a human homologue of thioredoxin (hTx), which exhibits scavenging activity with reactive oxygen intermediates. In their previous study, the authors found that after transient retinal ischemia, the expression of thioredoxin in rat retinal pigment epithelium (RPE) layer increased markedly. The present investigation is to determine intracellular ADF localization in RPE after transient ischemia and in cultured human RPE cells after oxidative insult by H2O2.The authors employed immunoelectron microscopy to examine ADF localization in RPE. Labeling density analysis was performed to supplement the main observation in the experiment of transient retinal ischemia. 3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazolium bromide (MTT) assay was performed to verify the protective role of recombinant ADF (rADF) against H2O2.In immunogold electron microscopy, sparse ADF-positive labeling was seen in the cytosol and mitochondria in normal rat RPE and in untreated cultured RPE cells. After oxidative stress, it was concentrated in mitochondria in both groups. MTT assay proved that rADF protected cultured RPE from the toxicity of H2O2.This study shows the induction of ADF/hTx in mitochondria of RPE after oxidative stresses and its protective effect on cultured RPE exposed to H2O2. The data indicate the possibly important role of ADF/hTx in the protection of retinal cells from the oxidative stresses associated with retinal ischemic disease and probably with regular visual activity.

Adult T cell leukemia derived factor (ADF), originally defined as an interleukin-2 receptor inducer, is a human thioredoxin homolog. ADF/thioredoxin has several biologic functions, such as defense against cellular damage, activity to scavenge oxidative stress by hydrogen peroxide radicals and cytokine-like properties. We hypothesized that under certain conditions, ischemia followed by reperfusion and/or light exposure, ADF may be induced in the retina.To test this hypothesis, we prepared experimental animal models in which oxidative stress could be applied to the retina. In the present study, we examined ADF expression in the rat retina using temporary ischemia and reperfusion, and photodynamic retinal vascular thrombosis by immunohistochemical and Western blotting methods.ADF expression was strongly induced in the retinal pigment epithelial cells after 2 hours of ischemia followed by 2, 24, or 48 hours of reperfusion. ADF was also expressed in retinal pigment epithelial cells as early as 3 hours after light exposure followed by rose bengal injection, and this expression increased with time. Western blotting data showed that ADF expression increased in the retinal pigment epithelial cells in the experimental group, as compared with the control group. These results indicate that ADF is actively induced in retinal pigment epithelial cells upon oxidative stress.ADF induction in retinal pigment epithelial cells may be involved in the defense mechanism against cellular damage caused by active oxygen species generated during oxygen stress to the retina.

The low-affinity Fc receptor for IgE (Fc epsilon RII/CD23) and its soluble form (sCD23, IgE-binding factor) have multiple functions, and enhanced levels of these are associated with various immunological diseases. We established two sensitive ELISA systems using enzyme-conjugated mAb and biotinylated mAb. The detection limits of the ELISA systems were 0.03 and 1.0 ng/ml, which showed good correlation in the range 1.0-10 ng/ml. In the ELISA system using enzyme-conjugated mAb, the average sCD23 concentration in 303 normal healthy volunteers was 1.4 +/- 0.3 ng/ml. In the ELISA system using biotinylated mAb, sCD23 levels in normal healthy volunteers showed almost the same values. In patients with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Sjögren syndrome, progressive systemic sclerosis, and mixed connective tissue disease, the sCD23 levels were significantly higher than those in normal individuals. Furthermore, in Epstein-Barr virus-related disorders after liver transplantation with immunosuppression, plasma levels of sCD23 rapidly increased to more than 12 ng/ml when clinical symptoms were evident. In addition, the sCD23 values remained high, although elevated GOT levels gradually decreased to standard values and EBV hepatitis improved. These data suggest that sCD23 levels are a sensitive marker of autoimmune diseases and EBV-related disorders in addition to allergic disorders. The ELISA system for sCD23 may be an additional diagnostic tool in estimating the clinical courses of these diseases.

Adult T cell leukemia-derived factor (ADF), originally defined as an IL-2 receptor alpha-chain (IL-2R alpha)/p55 (Tac) inducer, is a human thioredoxin homologue and has many cytokine-like activities. In this study, we examined the regulatory effect of ADF on eosinophil migration using human eosinophils and an eosinophilic subline of HL-60 human promyelocytic leukemia cells, YY-1. rADF induced migration of eosinophils from patients with hypereosinophilia, although rADF exhibited little activity on eosinophils from healthy donors. When human eosinophils were incubated with rADF (0.1-10 micrograms/ml) at 37 degrees C for 24 h, both chemotactic and chemokinetic activity of the complement anaphylatoxin peptide C5a on eosinophil migration was markedly enhanced in a dose-dependent manner. Similarly, this enhancing effect of rADF was observed in the migration assay using YY-1 cells. In contrast, rADF showed no modulation of migratory behavior of human eosinophils and YY-1 cells by IL-3, IL-5, nor granulocyte-macrophage colony-stimulating factor. Scatchard analysis of C5a receptors on YY-1 cells using 125I-C5a showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. Furthermore, mutant ADF (mADF), which had no reducing activity, had no enhancing effect on C5a-induced eosinophil migration. These results indicate a possible involvement of ADF in the recruitment of eosinophils through redox regulation by a dithiol reductase activity.

Protein tyrosine phosphorylation was examined after T cells were exposed to oxidative stress in vitro to investigate the possible involvement of redox regulation in T-cell signaling. Oxidative reagents such as hydrogen peroxide (H2O2) and diamide, which oxidize the free sulfhydryl groups in the cells, markedly induced tyrosine phosphorylation of multiple cellular proteins, especially a 55-kDa protein, of cultured peripheral blood T lymphocytes (PBL blasts). The 55-kDa molecule phosphorylated by diamide turned out to be a src family protein tyrosine kinase, p56lck. The immune complex kinase assay showed that the kinase activity of p56lck of diamide-treated PBL blasts was enhanced. The tryptic peptide mapping of p56lck demonstrated that diamide induced the phosphorylation both at Tyr-394 (autophosphorylation site) and at Tyr-505 (negative regulatory site). Taken together, the tyrosine phosphorylation and presumably kinase activity of p56lck were swiftly enhanced by oxidative stress, indicating that T cells have a redox-sensitive signaling mechanism, which is partly mediated by the lymphocyte-specific protein tyrosine kinase p56lck.