Retinal stimulates ATP hydrolysis by purified and reconstituted ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt disease.
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Many substrates for P-glycoprotein, an ABC transporter that mediates multidrug resistance in mammalian cells, have been shown to stimulate its ATPase activity in vitro. In the present study, we used this property as a criterion to search for natural and artificial substrates and/or allosteric regulators of ABCR, the rod photoreceptor-specific ABC transporter responsible for Stargardt disease, an early onset macular degeneration. ABCR was immunoaffinity purified to apparent homogeneity from bovine rod outer segments and reconstituted into liposomes. All-trans-retinal, a candidate ligand, stimulates the ATPase activity of ABCR 3-4-fold, with a half-maximal effect at 10-15 microM. 11-cis- and 13-cis-retinal show similar activity. All-trans-retinal stimulates the ATPase activity of ABCR with Michaelis-Menten behavior indicative of simple noncooperative binding that is associated with a rate-limiting enzyme-substrate intermediate in the pathway of ATP hydrolysis. Among 37 structurally diverse non-retinoid compounds, including nine previously characterized substrates or sensitizers of P-glycoprotein, only four show significant ATPase stimulation when tested at 20 microM. The dose-response curves of these four compounds are indicative of multiple binding sites and/or modes of interaction with ABCR. Two of these compounds, amiodarone and digitonin, can act synergistically with all-trans-retinal, implying that they interact with a site or sites on ABCR different from the one with which all-trans-retinal interacts. Unlike retinal, amiodarone appears to interact with both free and ATP-bound ABCR. Together with clinical observations on Stargardt disease and the localization of ABCR to rod outer segment disc membranes, these data suggest that retinoids, and most likely retinal, are the natural substrates for transport by ABCR in rod outer segments. These observations have significant implications for understanding the visual cycle and the pathogenesis of Stargardt disease and for the identification of compounds that could modify the natural history of Stargardt disease or other retinopathies associated with impaired ABCR function. (+info)
Prevention and therapy of cancer by dietary monoterpenes.
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Monoterpenes are nonnutritive dietary components found in the essential oils of citrus fruits and other plants. A number of these dietary monoterpenes have antitumor activity. For example, d-limonene, which comprises >90% of orange peel oil, has chemopreventive activity against rodent mammary, skin, liver, lung and forestomach cancers. Similarly, other dietary monoterpenes have chemopreventive activity against rat mammary, lung and forestomach cancers when fed during the initiation phase. In addition, perillyl alcohol has promotion phase chemopreventive activity against rat liver cancer, and geraniol has in vivo antitumor activity against murine leukemia cells. Perillyl alcohol and d-limonene also have chemotherapeutic activity against rodent mammary and pancreatic tumors. As a result, their cancer chemotherapeutic activities are under evaluation in Phase I clinical trials. Several mechanisms of action may account for the antitumor activities of monoterpenes. The blocking chemopreventive effects of limonene and other monoterpenes during the initiation phase of mammary carcinogenesis are likely due to the induction of Phase II carcinogen-metabolizing enzymes, resulting in carcinogen detoxification. The post-initiation phase, tumor suppressive chemopreventive activity of monoterpenes may be due to the induction of apoptosis and/or to inhibition of the post-translational isoprenylation of cell growth-regulating proteins. Chemotherapy of chemically induced mammary tumors with monoterpenes results in tumor redifferentiation concomitant with increased expression of the mannose-6-phosphate/insulin-like growth factor II receptor and transforming growth factor beta1. Thus, monoterpenes would appear to act through multiple mechanisms in the chemoprevention and chemotherapy of cancer. (+info)
Role of metabolic activation in the pathogenesis of chemically induced pulmonary disease: mechanism of action of the lung-toxic furan, 4-ipomeanol.
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Many xenobiotics produce hepatic injury due to their metabolism in the liver to highly reactive electrophile intermediates which form covalent conjugates with nucleophilic cellular constituents. This presentation describes studies indicating that the production of chemically reactive metabolites by pulmonary metabolism of xenobiotics can also play a fundamental role in the pathogenesis of chemically induced lung disease. (+info)
Disparate T cell requirements of two subsets of lupus-specific autoantibodies in pristane-treated mice.
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Intraperitoneal injection of pristane induces a lupus-like disease in BALB/c and other non-autoimmune mice characterized by autoantibody production and the development of immune complex disease closely resembling lupus nephritis. Two subsets of autoantibodies are induced by pristane: IgG anti-DNA DNA and -chromatin autoantibodies are strongly IL-6-dependent, whereas IgG anti-nRNP/Sm and -Su antibodies are not. The present studies were carried out to examine the role of T cells in establishing this dichotomy between the production of anti-nRNP/Sm/Su versus anti-DNA/chromatin autoantibodies. Autoantibody production and renal disease were evaluated in athymic (nude) mice treated with pristane. BALB/c nu/nu mice spontaneously developed IgM and IgG anti-single-stranded (ss)DNA and -chromatin, but not anti-nRNP/Sm or -Su, autoantibodies. Pristane treatment increased the levels of IgG anti-chromatin antibodies in nu/nu mice, but did not induce production of anti-nRNP/Sm or -Su antibodies. In contrast, BALB/c nu/+ and +/+ control mice did not spontaneously produce autoantibodies, whereas anti-nRNP/Sm and -Su autoantibodies were induced by pristane in approx. 50% of nu/+ and +/+ mice and anti-DNA/chromatin antibodies at lower frequencies. Nude mice spontaneously developed mild renal lesions that were marginally affected by pristane, but were generally milder than the lesions developing in pristane-treated nu/+ and +/+ mice. The data provide further evidence that two distinct pathways with different cytokine and T cell requirements are involved in autoantibody formation in pristane-induced lupus. This dichotomy may be relevant to understanding differences in the regulation of anti-DNA versus anti-nRNP/Sm autoantibodies in systemic lupus erythematosus, as well as the association of anti-DNA, but not anti-nRNP/Sm, with lupus nephritis. (+info)
Apoptosis and cell-cycle arrest in human and murine tumor cells are initiated by isoprenoids.
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Diverse classes of phytochemicals initiate biological responses that effectively lower cancer risk. One class of phytochemicals, broadly defined as pure and mixed isoprenoids, encompasses an estimated 22,000 individual components. A representative mixed isoprenoid, gamma-tocotrienol, suppresses the growth of murine B16(F10) melanoma cells, and with greater potency, the growth of human breast adenocarcinoma (MCF-7) and human leukemic (HL-60) cells. beta-Ionone, a pure isoprenoid, suppresses the growth of B16 cells and with greater potency, the growth of MCF-7, HL-60 and human colon adenocarcinoma (Caco-2) cells. Results obtained with diverse cell lines differing in ras and p53 status showed that the isoprenoid-mediated suppression of growth is independent of mutated ras and p53 functions. beta-Ionone suppressed the growth of human colon fibroblasts (CCD-18Co) but only when present at three-fold the concentration required to suppress the growth of Caco-2 cells. The isoprenoids initiated apoptosis and, concomitantly arrested cells in the G1 phase of the cell cycle. Both suppress 3-hydroxy-3-methylglutaryl CoA reductase activity. beta-Ionone and lovastatin interfered with the posttranslational processing of lamin B, an activity essential to assembly of daughter nuclei. This interference, we postulate, renders neosynthesized DNA available to the endonuclease activities leading to apoptotic cell death. Lovastatin-imposed mevalonate starvation suppressed the glycosylation and translocation of growth factor receptors to the cell surface. As a consequence, cells were arrested in the G1 phase of the cell cycle. This rationale may apply to the isoprenoid-mediated G1-phase arrest of tumor cells. The additive and potentially synergistic actions of these isoprenoids in the suppression of tumor cell proliferation and initiation of apoptosis coupled with the mass action of the diverse isoprenoid constituents of plant products may explain, in part, the impact of fruit, vegetable and grain consumption on cancer risk. (+info)
Activation of the transforming growth factor beta signaling pathway and induction of cytostasis and apoptosis in mammary carcinomas treated with the anticancer agent perillyl alcohol.
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The mechanisms of action of the anticancer agent perillyl alcohol (POH), presently in Phase II clinical trials, were investigated in advanced rat mammary carcinomas. Gross and ultrastructural morphology of POH-mediated tumor regression indicated that apoptosis accounted for the marked reduction in the epithelial compartment. Characterization of cell growth and death indices revealed that apoptosis was induced within 48 h of chemotherapy, before the induction of cytostasis. RNA expression studies, based on a multiplexed-nuclease protection assay, demonstrated that cell cycle- and apoptosis-related genes were differentially expressed within 48 h of POH treatment; p21(Cip1/WAF1), bax, bad, and annexin I were induced; cyclin E and cyclin-dependent kinase 2 were repressed; and bcl-2 and p53 were unchanged. Next, a potential role for transforming growth factor beta (TGF-beta) signaling in POH-mediated carcinoma regression was explored. RNA expression studies, again based on a multiplexed-nuclease protection assay, showed that TGF-beta-related genes were induced and temporally regulated during POH treatment: (a) c-jun and c-fos were transiently induced within 12 h of chemotherapy; (b) TGF-beta1 was induced within 24 h of chemotherapy; (c) the mannose 6-phosphate/insulin-like growth factor II receptor and the TGF-beta type I and II receptors were induced within 48 h of chemotherapy; and (d) smad3 was induced during active carcinoma regression. In situ protein expression studies, based on fluorescence-immunohistochemistry in concert with confocal microscopy, confirmed up-regulation and demonstrated colocalization of TGF-beta1, the mannose 6-phosphate/insulin-like growth factor II receptor, the TGF-beta type I and II receptors, and Smad2/Smad3 in epithelial cells. Nuclear localization of Smad2/Smad3 indicated that the TGF-beta signaling pathway was activated in regressing carcinomas. Subpopulations of Smad2/Smad3-positive and apoptotic nuclei colocalized, indicating a role for Smads in apoptosis. Thus, Smads may serve as a potential biomarker for anticancer activity. Importantly, none of the POH-mediated anticancer activities were observed in normal mammary gland. (+info)
Stimulation of pregnant rat uterine contraction by the polychlorinated biphenyl (PCB) mixture aroclor 1242 may be mediated by arachidonic acid release through activation of phospholipase A2 enzymes.
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The polychlorinated biphenyl (PCB) mixture Aroclor 1242 (A1242) increases frequency of contractions of pregnant rat uteri, suggesting a possible mechanism for decreased gestational age and increased spontaneous abortion in women and animals exposed to PCBs. In the present study, we hypothesized that A1242-induced stimulation of uterine contraction is mediated by arachidonic acid released by phospholipase A2 (PLA2) enzymes. Isometric uterine contraction was measured in longitudinal uterine strips isolated from gestation day 10 rat. Pretreatment of uterine strips with the PLA2 inhibitor (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (HELSS) or manoalide, or an inhibitor of the G protein of PLA2, isotetrandrine, completely prevented the increase of contractile frequency induced by 50 microM A1242. However, the phospholipase C inhibitors 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC) and neomycin were unable to block stimulation of uterine contraction by A1242. In accordance, A1242 (100 microM) did not release inositol phosphates from myo-[3H]inositol-labeled myometrial cells, whereas myometrial cells prelabeled with [3H]arachidonic acid released arachidonic acid in a concentration- and time-dependent manner after exposure to A1242 (10-100 microM). A1242 significantly stimulated arachidonic acid release in the absence of extracellular calcium, although the release was attenuated. Analysis of the eicosanoids released by A1242 indicated that only 0.83% of released [3H]arachidonic acid was metabolized to eicosanoids and 99.07% remained as free arachidonate. Uterine contraction increased in strips exposed to exogenous arachidonic acid (1-100 microM). This study suggests that A1242 stimulates contraction in pregnant rat uterus by a mechanism involving PLA2-mediated arachidonic acid release, and that arachidonic acid, rather than eicosanoids, may mediate A1242 uterotonic action in the uterus. (+info)
Metabolism of (R)-(+)-pulegone and (R)-(+)-menthofuran by human liver cytochrome P-450s: evidence for formation of a furan epoxide.
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(R)-(+)-Pulegone, a monoterpene constituent of pennyroyal oil, is a hepatotoxin that has been used in folklore medicine as an abortifacient despite its potential lethal effects. Pulegone is metabolized by human liver cytochrome P-450s to menthofuran, a proximate hepatotoxic metabolite of pulegone. Expressed human liver cytochrome (CYP) P-450s (1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4) were tested for their ability to catalyze the oxidations of pulegone and menthofuran. Expressed CYP2E1, CYP1A2, and CYP2C19 oxidized pulegone to menthofuran, with respective Km and Vmax values of 29 microM and 8.4 nmol/min/nmol P-450 for CYP2E1, 94 microM and 2.4 nmol/min/nmol P-450 for CYP1A2, and 31 microM and 1.5 nmol/min/nmol P-450 for CYP2C19. The human liver P-450s involved in the metabolism of menthofuran are the same as pulegone except for the addition of CYP2A6. These P-450s were found to oxidize menthofuran to a newly identified metabolite, 2-hydroxymenthofuran, which is an intermediate in the formation of the known metabolites mintlactone and isomintlactone. Based on studies with 18O2 and H218O, 2-hydroxymenthofuran arises predominantly from a dihydrodiol formed from a furan epoxide. CYP2E1, CYP1A2, and CYP2C19 oxidized menthofuran with respective Km and Vmax values of 33 microM and 0.43 nmol/min/nmol P-450 for CYP2E1, 57 microM and 0.29 nmol/min/nmol P-450 for CYP1A2, and 62 microM and 0.26 nmol/min/nmol P-450 for CYP2C19. (+info)