Increased serum anti-CYP2E1 IgG autoantibody levels may be involved in the pathogenesis of occupational trichloroethylene hypersensitivity syndrome: a case-control study.

Occupational exposure to trichloroethylene (TCE) causes a systemic skin disorder with hepatitis known as TCE hypersensitivity syndrome (TCE-HS). Human Leukocyte Antigen (HLA)-B*13:01 is its susceptibility factor; however, the immunological pathogenesis of TCE-HS remains unknown. We herein examined the hypothesis that autoantibodies to CYP2E1 are primarily involved in TCE-HS. A case-control study of 80 TCE-HS patients, 186 TCE-tolerant controls (TCE-TC), and 71 TCE-nonexposed controls (TCE-nonEC) was conducted to measure their serum anti-CYP2E1 antibody (IgG) levels. The effects of TCE exposure indices, such as 8-h time-weighted-average (TWA) airborne concentrations, urinary metabolite concentrations, and TCE usage duration; sex; smoking and drinking habits; and alanine aminotransferase (ALT) levels on the antibody levels were also analyzed in the two control groups. There were significant differences in anti-CYP2E1 antibody levels among the three groups: TCE-TC > TCE-HS patients > TCE-nonEC. Antibody levels were not different between HLA-B*13:01 carriers and noncarriers in TCE-HS patients and TCE-TC. The serum CYP2E1 measurement suggested increased immunocomplex levels only in patients with TCE-HS. Multiple regression analysis for the two control groups showed that the antibody levels were significantly higher by the TCE exposure. Women had higher antibody levels than men; however, smoking, drinking, and ALT levels did not affect the anti-CYP2E1 antibody levels. Anti-CYP2E1 antibodies were elevated at concentrations lower than the TWA concentration of 2.5 ppm for TCE exposure. Since HLA-B*13:01 polymorphism was not involved in the autoantibody levels, the possible mechanism underlying the pathogenesis of TCE-HS is that TCE exposure induces anti-CYP2E1 autoantibody production, and HLA-B*13:01 is involved in the development of TCE-HS.

Hydrogen gas improves left ventricular hypertrophy in Dahl rat of salt-sensitive hypertension.

PURPOSE: Hypertension is an important risk factor for death resulting from stroke, myocardial infarction, and end-stage renal failure. Hydrogen (H2) gas protects against many diseases, including ischemia-reperfusion injury and stroke. The effects of H2 on hypertension and its related left ventricular (LV) function have not been fully elucidated. The purpose of this study was to investigate the effects of H2 gas on hypertension and LV hypertrophy using echocardiography. METHODS: Dahl salt-sensitive (DS) rats were randomly divided into three groups: those fed an 8% NaCl diet until 12 weeks of age (8% NaCl group), those additionally treated with 2% H2 gas (8% NaCl + 2% H2 group), and control rats maintained on a diet containing 0.3% NaCl until 12 weeks of age (0.3% NaCl group). H2 gas was supplied through a gas flowmeter and delivered by room air (2% hydrogenated room air, flow rate of 10 L/min) into a cage surrounded by an acrylic chamber. We evaluated interventricular septal wall thickness (IVST), LV posterior wall thickness (LVPWT), and LV mass using echocardiography. RESULTS: IVST, LVPWT, and LV mass were significantly higher in the 8% NaCl group than the 0.3% NaCl group at 12 weeks of age, whereas they were significantly lower in the 8% NaCl + 2% H2 group than the 8% NaCl group. There was no significant difference in systolic blood pressure between the two groups. CONCLUSION: Our findings suggest that chronic H2 gas inhalation may help prevent LV hypertrophy in hypertensive DS rats.

Non-thermal atmospheric pressure plasmas as a novel candidate for preventive therapy of melanoma.

Due to the increased ultraviolet radiation, the incidence of melanoma is increasing worldwide more than that of any other cancer. In this study, the effects of irradiation of non-thermal atmospheric pressure plasmas (NEAPPs) on benign melanocytic tumors from our original hairless model mice (HL-RET-mice), in which benign melanocytic tumors and melanomas spontaneously develop in the skin stepwise, were examined. Expression levels of melanoma cell adhesion molecule (MCAM) and matrix metalloproteinase-2 (MMP-2) mRNA in melanomas were higher than those in benign melanocytic tumors in the mice. Repeated irradiation of non-thermal atmospheric pressure plasmas (NEAPPs) for the benign tumors decreased the expression levels of MCAM and MMP-2 mRNA in the tumors from the mice. Previous studies showed that MCAM sites are upstream of MMP-2, that MCAM regulates transcription of MMP-2 in melanoma cells and that MMP-2 is associated with the conversion of a benign tumor to a malignant tumor. Therefore, our results suggest that the NEAPP irradiation-mediated decrease in the expression level of MMP-2 in benign melanocytic tumors is associated with decreased expression levels of MCAM. Moreover, NEAPP irradiation might be a potential candidate for therapy to prevent melanoma development through suppression of malignant conversion in benign melanocytic tumors.

Hippocampal theta wave activity during configural and non-configural tasks in rats.

This study examined hippocampal theta power during configural and non-configural tasks in rats. Experiment 1 compared hippocampal theta power during a negative patterning task (A+, B+, AB-) to a configural task and a simple discrimination task (A+, B-) as a non-configural task. The results showed that hippocampal theta power during the non-reinforcement trial (non-RFT) of the negative patterning task was higher than that during the simple discrimination task. However, this hippocampal power may reflect sensory processing for compound stimuli that have cross-modality features (the non-RFT of the negative patterning task was presented together with visual and auditory stimuli, but the non-RFT of the simple discrimination task was presented with visual or auditory stimulus alone). Thus, in experiment 2, we examined whether the experiment 1 results were attributable to sensory processing of a compound stimulus by comparing hippocampal theta power during negative patterning (A+, B+, AB-), simultaneous feature-negative (A+, AB-), and simple discrimination tasks (A+, B-). Experiment 2 showed that hippocampal theta activity during the non-RFT in the negative patterning task was higher than that in the simultaneous feature-negative and simple discrimination tasks. Thus, we showed that hippocampal theta activity increased during configural tasks but not during non-configural tasks.

Effects of long sleep time and irregular sleep-wake rhythm on cognitive function in older people.

Sleep disturbances and cognitive decline are common in older adults. We aimed to investigate the effects of the total sleep time (TST) and sleep-wake rhythm on executive function and working memory in older adults. In 63 older participants, we measured the TST, wake after sleep onset (WASO), and sleep timing (midpoint between bedtime and wake-up time) using actigraphy. Executive function was evaluated with the trail making test B (TMT-B) and Wisconsin card sorting test (WCST). The number of back task (N-back task) was used to measure working memory. Participants with a TST ≥ 8 h had a significantly lower percentage of correct answers (% correct) on the 1-back task than those with a TST < 8 h. The % correct on the 1-back task was significantly correlated with the TST, WASO, and sleep timing. Multiple regression analyses revealed that the TST and sleep timing were significant factors of the % correct on the 1-back task. The TMT-B score was significantly correlated with the sleep timing. Category achievement on the WCST was significantly correlated with the standard deviation of the sleep timing. Therefore, a long sleep time and an irregular sleep-wake rhythm could have adverse effects on executive function and working memory in older people.

A redox-linked novel pathway for arsenic-mediated RET tyrosine kinase activation.

We examined the biochemical effects of arsenic on the activities of RET proto-oncogene (c-RET protein tyrosine kinases) and RET oncogene (RET-MEN2A and RET-PTC1 protein tyrosine kinases) products. Arsenic activated c-RET kinase with promotion of disulfide bond-mediated dimerization of c-RET protein. Arsenic further activated RET-MEN2A kinase, which was already 3- to 10-fold augmented by genetic mutation compared with c-RET kinase activity, with promotion of disulfide bond-mediated dimerization of RET-MEN2A protein (superactivation). Arsenic also increased extracellular domain-deleted RET-PTC1 kinase activity with promotion of disulfide bond-mediated dimerization of RET-PTC1 protein. Arsenic increased RET-PTC1 kinase activity with cysteine 365 (C365) replaced by alanine with promotion of dimer formation but not with cysteine 376 (C376) replaced by alanine. Our results suggest that arsenic-mediated regulation of RET kinase activity is dependent on conformational change of RET protein through modulation of a special cysteine sited at the intracellular domain in RET protein (relevant cysteine of C376 in RET-PTC1 protein). Moreover, arsenic enhanced the activity of immunoprecipitated RET protein with increase in thiol-dependent dimer formation. As arsenic (14.2 microM) was detected in the cells cultured with arsenic (100 microM), direct association between arsenic and RET in the cells might modulate dimer formation. Thus, we demonstrated a novel redox-linked mechanism of activation of arsenic-mediated RET proto-oncogene and oncogene products.

Peptides containing the MXXCW motif inhibit oncogenic RET kinase activity with a novel mechanism of action.

REarranged during Transition (RET) is a tyrosine kinase associated with the development of several malignancies. Identification of RET kinase inhibitors promises valuable therapeutic tools for the intervention of RET-driven tumors. Most currently available tyrosine kinase inhibitors target the ATP binding site, but there are several drawbacks of these ATP-competitive drugs. Therefore, there is a need to develop new kinase inhibitors with alternative mechanisms of action. We have previously reported that a conserved cysteine in the MXXCW motif of RET is crucial to the disulfide-bonded dimerization-linked activation of RET kinases. Reagents which bind to this cysteine may inhibit the activity of RET kinases through disulfide-bond mediated dimerization. Here, we examine the potential of MXXCW motif-containing peptides as candidate kinase inhibitors. We demonstrate that MXXCW motif-containing peptides bind to RET in a redox-sensitive manner and block enzymatic activity, causing inhibition of the RET-dependent activity of extracellular signal-regulated kinases and effectively reducing the malignant potential of RET-papillary thyroid carcinoma-1 (PTC)-expressing cells. These motif-containing peptides were also found to be effective against the drug resistant mutant of RET. The inhibition of RET kinase activity by these peptides resulted in suppression of RET-PTC-1-mediated cancer growth. The great potency of these cysteine targeted peptides could indicate promising approaches for novel molecular-targeted therapies for RET-associated cancers.

Inhibition of mast cell degranulation by melanin.

Melanin is a dark naturally occurring pigment produced in nature and in many organisms. Although several reports have demonstrated applications for melanins in various therapeutic treatments, to date, no research has examined the anti-allergic effect of melanin. In this study, we for the first time found that solubilized or synthesized soluble melanin acts as a potent inhibitor of the degranulation of mast cells. We found that squid-ink-derived melanin significantly inhibited antigen-IgE-FcεRI-mediated degranulation of the mucosal mast cell line RBL-2H3. A homogenized melanin nanoparticle prepared by laser ablation also clearly suppressed antigen-induced mast cell degranulation. We also successfully solubilized synthetic melanin in a neutral biochemical buffer and found that it also significantly inhibited IgE-sensitized mast cells. The anti-degranulation activity of synthesized melanin was abolished in the melanin fraction below 50-kD molecular weight. All melanins used in this study did not exert significant cell death. Signal transduction analysis revealed that melanin suppressed antigen-triggered phosphorylation of signaling molecules as well as calcium influx. Transmission electron microscopy revealed that homogenized melanin nanoparticles partially attached to the cell surface and some nanoparticles were internalized to the cell. Flow cytometry revealed that the number of FcεRI-bound IgE molecules was decreased by melanin. Fluorescence recovery after photobleaching analysis indicated that melanin attenuated both plasma membrane and cytoplasmic fluidity, implying that melanin increased their viscosities. In vivo experiments using passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA) mouse models demonstrated that oral administration of melanin accelerated the recovery of decreased body temperature after antigen infection in PSA, and combination sensitization of IgE with melanin attenuated antigen-induced extravasation in PCA. These findings indicated that melanin exhibits preventative effects against IgE-mast cell-mediated anaphylaxis. This study provides the first evidence that homogenized melanin may be a potential therapeutic agent for diseases involving mast cells.

The vacuolar V1/V0-ATPase is involved in the release of the HOPS subunit Vps41 from vacuoles, vacuole fragmentation and fusion.

At yeast vacuoles, phosphorylation of the HOPS subunit Vps41 depends on the Yck3 kinase. In a screen for mutants that mimic the yck3Delta phenotype, in which Vps41 accumulates in vacuolar dots, we observed that mutants in the V0-part of the V0/V1-ATPase, in particular in vma16Delta, also accumulate Vps41. This accumulation is not due to a phosphorylation defect, but to reduced release of Vps41 from vma16Delta vacuoles. One reason could be a connection to vacuole fission, which is blocked in V-ATPase mutants. Vacuole fusion is not impaired between vacuoles lacking the V0-subunits Vma16 or Vma6 and wild-type vacuoles, whereas fusion between mutant vacuoles is reduced. Our data suggest a connection between vacuole biogenesis and membrane fusion.

Improvement of balance in young adults by a sound component at 100 Hz in music.

About 80% of young people use personal listening devices (PLDs) including MP3 players to listen to music, which consists of sound components with various frequencies. Previous studies showed that exposure to noise of high intensities affected balance in humans. However, there is no information about a frequency-dependent effect of sound components in music from a PLD on balance in young people. In this study, we determined the associations between sound component levels (dB) at 100, 1000 and 4000 Hz in music from a portable listening device (PLD) and balance objectively determined by posturography in young adults (n = 110). We divided the subjects into two groups (low and high exposure groups) based on cut-off values of sound component levels at each frequency using receiver operating characteristic (ROC) curves. Balance in the high exposure group (≥46.6 dB) at 100 Hz was significantly better than that in low exposure group in logistic regression models adjusted for sex, BMI, smoking status and alcohol intake, while there were no significant associations at 1000 and 4000 Hz. Thus, this study demonstrated for the first time that the sound component at 100 Hz with more than 46.6 dB in music improved balance in young adults.

[Ultraviolet irradiation-mediated malignant melanoma induction with RET tyrosine kinase activation].

We previously established a RET-transgenic mouse line (304/B6), in which skin melanosis, benign melanocytic tumors and malignant melanoma spontaneously develop. We found that the activities of RET tyrosine kinase, Erk and c-Jun are definitely upregulated in malignant melanoma in the RET-transgenic mice of line 304/B6. We also established another RET-transgenic mouse line (192), in which skin melanosis and benign melanocytic tumors, but not malignant melanoma, spontaneously develop. Ultraviolet irradiation induced malignant melanoma from benign tumors in the RET-transgenic mice of line 192, and promoted RET tyrosine kinase, Erk and c-Jun activities. These results suggest that the ultraviolet irradiation-mediated enhancement of RET and the activity of its downstream molecules play important roles in malignant melanoma development.

A PKC-mediated backup mechanism of the MXXCW motif-linked switch for initiating tyrosine kinase activities.

The cysteine in the M/IXXCW motif is conserved in all but one (threonine in place of cysteine) of the human protein tyrosine kinases (PTKs). We showed that all RET-PTC-1 mutants in which the C in this motif (C376) was replaced with glycine, lysine, threonine or serine lost their activity in vitro. However, the C376T/S mutants showed normal tyrosine phosphorylation in vivo (in cells). Further analyses reveled that protein kinase C (PKC) initiated the activities of the C376T/S mutants in cells. We conclude that the M/IXXCW motif-mediated mechanisms which initiate PTK activities are partially replaced by a PKC-mediated mechanism.

c-Kit-targeting immunotherapy for hereditary melanoma in a mouse model.

The role of c-Kit in the development of melanoma was studied in line 304/B6 of RET-transgenic mice, in which melanoma spontaneously develops. In Wv/Wv-RET (304/B6)-transgenic mice, in which c-Kit function was severely impaired, development of melanoma was strongly suppressed. Although 31 of the 44 original RET-transgenic mice died of rapidly growing melanoma within 12 months after birth, only 8 of the 44 Wv/Wv-RET-transgenic mice developed slowly growing melanocytic tumors with a greatly prolonged mean tumor-free period, 2 of which died of melanoma at a late stage. Even Wv/+-RET-transgenic mice had a clearly prolonged tumor-free period and definitely reduced frequency (6 of 61) of tumor death within 12 months after birth. Melanin production in the skin of these mice was not strongly impaired, suggesting that c-Kit affects the development of melanomas in these mice with only minor effects in melanin production. c-Kit expression in skin soon after birth was promoted in RET-transgenic mice, and c-Kit was expressed at high levels at the benign but not malignant stage of the tumor. A single injection of anti-c-Kit antibody (ACK2) into RET-transgenic mice soon after birth caused a surprisingly long-lasting suppression of development of melanoma, greatly prolonging the tumor-free period, and none of the 28 ACK2-treated RET-transgenic mice died from tumors at 12 months of age. The c-Kit function needed for melanin production was also suppressed for an unusually long time in ACK2-treated, RET-transgenic mice. These results suggest that c-Kit can be a unique target molecule for melanoma treatment.

Repair by Src kinase of function-impaired RET with multiple endocrine neoplasia type 2A mutation with substitutions of tyrosines in the COOH-terminal kinase domain for phenylalanine.

An oncogenic mutant of c-RET as a receptor-type tyrosine kinase, termed RET-MEN2A, displays both cell-transforming activity in vivo and strong catalytic activity in vitro. In this study, we compared the activities of mutant RET-MEN2A with substitutions of each of nine tyrosines for phenylalanine (Y1062F, Y1015F, Y981F, Y952F, Y928F, Y905F, Y900F, Y864F, and Y826F), which had been transfected into NIH 3T3 cells. In RET-MEN2A with the Y905F mutation, the cell-transforming activity was drastically reduced with a great reduction in the in vitro catalytic activity. Unexpectedly, we found that in vitro kinase activity was severely impaired in RET-MEN2A with Y981F, Y952F, or Y928F mutation, which displayed near-normal cell-transforming activity and only a partially impaired tyrosine phosphorylation level in vivo. Phosphoamino acid analysis actually demonstrated some increase in phosphotyrosine in the Y905F mutant but no or barely detectable increase in the Y981F, Y952F, or Y928F mutant after incubation for in vitro kinase assay. This suggested a crucial role of the Y981/Y952/Y928-linked structural integrity of the COOH end of the catalytic domain of RET in starting Y905 autophosphorylation. Interestingly, the apparent defect in intrinsic kinase activity in vitro in the Y981F, Y952F, or Y928F mutant, but not the reduction in activity in the Y905F mutant, could be partially repaired or restored by c-Src or, more extensively, by v-Src, which promoted Y905 phosphorylation in trans. A complex was shown to be formed between v-Src and RET-MEN2A through association of both with a cholesterol-rich membrane microdomain known as "a raft," possibly for efficient contact of submembranous domains of Src and RET to promote phosphorylation of Y905 of the latter. Finally, endogenous c-Src was shown to promote Y905 phosphorylation of the Y981F mutant in vivo. These results reveal a novel Src kinase-mediated repair mechanism of otherwise function-impaired mutant RET kinases.

Prevention of allergic rhinitis by ginger and the molecular basis of immunosuppression by 6-gingerol through T cell inactivation.

The incidence of allergies has recently been increasing worldwide. Immunoglobulin E (IgE)-mediated hypersensitivity is central to the pathogenesis of asthma, hay fever and other allergic diseases. Ginger (Zingiber officinale Roscoe) and its extracts have been valued for their medical properties including antinausea, antiinflammation, antipyresis and analgesia properties. In this study, we investigated the antiallergic effects of ginger and 6-gingerol, a major compound of ginger, using a mouse allergy model and primary/cell line culture system. In mice with ovalbumin (OVA)-induced allergic rhinitis, oral administration of 2% ginger diet reduced the severity of sneezing and nasal rubbing by nasal sensitization of OVA and suppressed infiltration of mast cells in nasal mucosa and secretion of OVA-specific IgE in serum. 6-Gingerol inhibited the expression of not only Th2 cytokines but also Th1 cytokines in OVA-sensitized spleen cells. Accordingly, 6-gingerol suppressed in vitro differentiation of both Th1 cells and Th2 cells from naïve T cells. In addition, 6-gingerol suppressed both superantigen staphylococcal enterotoxin B (SEB)- and anti-CD3-induced T cell proliferation. 6-Gingerol also abrogated PMA plus ionomycin- and SEB-induced IL-2 production in T cells, suggesting that 6-gingerol affected T cell receptor-mediated signal transduction rather than the antigen-presentation process. Indeed, 6-gingerol inhibited the phosphorylation of MAP kinases, calcium release and nuclear localization of c-fos and NF-κB by PMA and ionomycin stimulation. Thus, our results demonstrate that 6-gingerol suppresses cytokine production for T cell activation and proliferation, thereby not causing B cell and mast cell activation and resulting in prevention or alleviation of allergic rhinitis symptoms.

Correlated expression levels of endothelin receptor B and Plexin C1 in melanoma.

Discussion concerning the effect of endothelin receptor B (Ednrb) on melanoma continues because Ednrb has been reported to have both tumor promoting and suppressive effects for melanoma. In order to examine Ednrb-related signaling in melanomagenesis, DNA microarray analysis for a melanoma from a RFP/RET-transgenic mouse (RET-mouse) and a melanoma from an Ednrb-heterozygously deleted RET-mouse [Ednrb(+/-);RET-mouse], in both of which melanoma spontaneously develops, was performed in this study. We found that the expression level of Plexin C1 (PlxnC1), a suppressor for melanoma, in a melanoma from an Ednrb(+/-);RET-mouse was drastically decreased compared to that in a melanoma from a RET-mouse. Therefore, we further examined the correlation between Ednrb and PlxnC1 expression levels in melanomas. PlxnC1 transcript expression levels in melanomas from Ednrb(+/-);RET-mice were lower than those in melanomas from RET-mice. A strong correlation between Ednrb and PlxnC1 transcript expression levels (R = 0.78, p < 0.01) was also found in melanomas from both RET-mice and Ednrb(+/-);RET-mice. Correspondingly, there was a significant correlation between transcript (R = 0.80; p < 0.01) and protein (R = 0.60; p < 0.01) expression levels of EDNRB and PLXNC1 in human primary melanomas. Together with our results showing that the expression level of PLXNC1 transcript was reduced in EDNRB-depleted human melanoma cells, our results showing positively correlated expression levels of Ednrb/EDNRB and PlxnC1/PLXNC1 in melanoma suggest that PlxnC1/PLXNC1 is involved in the Ednrb/EDNRB-mediated suppressive effect on melanoma.

4-hydroxynonenal triggers multistep signal transduction cascades for suppression of cellular functions.

4-hydroxynonenal (HNE), an aldehyde product of membrane lipid peroxidation, has been suggested to mediate a number of oxidative stress-linked pathological events in humans, including cellular growth inhibition and apoptosis induction. Because HNE is potentially reactive to a number of both cell surface and intracellular proteins bearing sulfhydryl, amino and imidazole groups, it seems that there are multiple signal transduction cascades. Here we briefly review the HNE-triggered signal transduction cascades that lead to suppression of cellular functions and to cell death, based mainly on our own recent study results. We first showed that formation of HNE-cell surface protein adducts, which mimicked ligand-cell surface receptor binding, induced activation of receptor-type protein tyrosine kinases such as epithelial growth factor receptor (EGFR) and that this caused growth inhibition through a cascade of activation of EGFR, Shc and ERK. Next, we showed that HNE-mediated scavenging of cellular glutathione led to activation of caspases and to DNA fragmentation through a Fas-independent and mitochondria-linked pro-apoptotic signal pathway. More recently, we have obtained evidence that the HNE-triggered signal cascade for caspase activation encounters complex positive feedback regulatory mechanisms that are linked to the inhibition of anti-apoptotic signals and are dependent on caspase activity. Underlying multiple regulatory mechanisms, including mechanisms of activation of Akt-dephosphorylating PP2A activity, activities of protein tyrosine kinases have been shown to be biphasically controlled by HNE. In addition, we have obtained results suggesting that HNE inhibits phosphorylation of IkappaB, possibly by targeting some elements upstream of IkappaB, which might downregulate the NF-kappaB-mediated cellular responses, including serum deprivation-induced iNOS expression and generation of anti-apoptotic signals. These results suggest that HNE reacts with multiple cell surface and intracellular sites for triggering a network of signal transduction that is ultimately focused on suppression of cellular functions.

Caspase activation is accelerated by the inhibition of arsenite-induced, membrane rafts-dependent Akt activation.

Renewed interest in arsenic has been shown recently due to its dual nature of being a potent toxin and a drug for treatment of acute promyelocytic leukemia (APL) because of its ability to trigger caspase activation. Here, we found that sodium arsenite (NaAsO(2)) also triggers the signal for activation of Akt and downstream glycogen synthase 3beta (GSK3beta). Such Akt/GSK3beta activation was abrogated completely by wortmannin, an inhibitor of PI-3 kinase, and greatly by pertussis toxin, a G-protein inhibitor. Arsenite-induced Akt phosphorylation also was inhibited by sequestrating membrane cholesterol with beta cyclodextrin. Reducing reagents/reactive oxygen species (ROS) scavengers reduced arsenite-induced Akt phosphorylation and beta cyclodextrin reduced arsenite-mediated ROS production, suggesting that arsenite-induced G-protein/Akt/GSK3beta pathway is membrane raft dependent and redox linked. We also found that a combination of a low concentration (1 microM) of arsenite and wortmannin triggers the signal for caspase activation, whereas neither of these elements alone did so. These results suggested that selective blockade of the arsenite-provoked PI-3 kinase/Akt pathway can promote the arsenite-triggered pathway for caspase activation, and this may open a new study area for wider applications of arsenic as a drug for treating various kinds of leukemia.

Redox-linked signal transduction pathways for protein tyrosine kinase activation.

The signaling for activation of protein tyrosine kinases (PTKs) is usually started by binding of ligands to cell-surface receptors. However, recent evidence suggests the presence of ligand binding-independent signaling pathways that are mediated by oxidative stress. Oxidation and reduction of protein cysteine sulfhydryl (SH) groups may work as a molecular switch to start or to stop the signaling. It is known that oxidation of cysteine SH groups on protein tyrosine phosphatases switches off the action of protein tyrosine phosphatases. This event may not, however, signal for initial autophosphorylation of previously unphosphorylated PTKs, whereas it certainly prevents dephosphorylation of once-phosphorylated PTKs. We have suggested new mechanisms for oxidative stress-mediated PTK activation. First, cell-surface glycosylphosphatidylinositol-anchoring proteins and a phosphoglycolipid/cholesterol-enriched membrane microdomain termed a "raft" can be the direct targets of oxidative stress for inducing their clustering through an S-S-bonded or S-X-S-bonded crosslinking of cell-surface proteins and subsequent activation of raft-associating Src family PTKs. Second, intracellular specific cysteine SH groups on PTK proteins can be another target of oxidative stress for inducing a conformational change necessary for initial activation of PTKs. A possible relationship between cell-surface and intracellular events is that the former frequently induces superoxide production as the second messenger for the latter.