Extracellular ATP decreases trophoblast invasion, spiral artery remodeling and immune cells in the mesometrial triangle in pregnant rats.

PubMed

Spaans, F; Melgert, B N; Chiang, C; Borghuis, T; Klok, P A; de Vos, P; van Goor, H; Bakker, W W; Faas, M M

2014-08-01

Preeclampsia is characterized by deficient trophoblast invasion and spiral artery remodeling, a process governed by inflammatory cells. High levels of the danger signal extracellular adenosine triphosphate (ATP) have been found in women with preeclampsia and infusion of ATP in pregnant rats induced preeclampsia-like symptoms such as albuminuria and placental ischemia. We hypothesized that ATP inhibits trophoblast invasion and spiral artery remodeling and affects macrophages and natural killer (NK) cells present in the rat mesometrial triangle. Pregnant rats were infused with ATP or saline (control) on day 14 of pregnancy. Rats were sacrificed on day 15, 17 or 20 of pregnancy and placentas with mesometrial triangle were collected. Sections were stained for trophoblast cells, α-smooth muscle actin (spiral artery remodeling), NK cells and various macrophage populations. Expression of various cytokines in the mesometrial triangle was analyzed using real-time RT-PCR. ATP infusion decreased interstitial trophoblast invasion on day 17 and spiral artery remodeling on day 17 and 20, increased activated tartrate resistant acid phosphatase (TRAP)-positive macrophages on day 15, decreased NK cells on day 17 and 20, and decreased inducible nitric oxide synthase (iNOS)-positive and CD206-positive macrophages and TNF-α and IL-33 expression at the end of pregnancy (day 20). Interstitial trophoblast invasion and spiral artery remodeling in the rat mesometrial triangle were decreased by infusion of ATP. These ATP-induced modifications were preceded by an increase in activated TRAP-positive macrophages and coincided with NK cell numbers, suggesting that they are involved. Trophoblast invasion and spiral artery remodeling may be inhibited by ATP-induced activated macrophages and decreased NK cells in the mesometrial triangle in rat pregnancy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Embryotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): Theembryonic vasculature is a physiological target for TCDD-induced DNA damage and apoptotic cell death in medaka (Orizias latipes)

USGS Publications Warehouse

Cantrell, Susannah M.; Lutz, Linda H.; Tillitt, Donald E.; Hannink, Mark

1996-01-01

Vertebrate embryos are extremely sensitive to environmental contaminants known as planar halogenated hydrocarbons (PHHs). The physiological targets that mediate PHH-induced embryotoxicity are not known. We have characterized embryotoxicity in medaka (Orizias latipes) caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototypic PHH. DNA degradation in cells of the embryonic vasculature and loss of functional integrity of the medial yolk vein were demonstrated in TCDD-exposed embryos. Pharmacological intervention with piperonyl butoxide inhibited TCDD-induced DNA degradation, restored the functional integrity of the medial yolk vein, and protected against the embryotoxicity of TCDD. Treatment of TCDD-exposed embryos with the antioxidant N-acetylcysteine also provided significant protection against the embryotoxicity of TCDD. These results demonstrate that DNA damage and consequent cell death in the embryonic vasculature are key physiological mediators of TCDD-induced embryotoxicity.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the endogenous metabolism of all-trans-retinoic acid in the rat.

PubMed

Schmidt, Carsten K; Hoegberg, Pi; Fletcher, Nicholas; Nilsson, Charlotte B; Trossvik, Christina; Håkansson, Helen; Nau, Heinz

2003-07-01

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is known to influence vitamin A homeostasis. In order to investigate the mechanism behind this retinoid disruption, male Sprague-Dawley rats were exposed to TCDD at doses ranging from 0.1 to 100 micro g/kg body weight, and were killed 3 days after exposure. Additional groups of rats were killed 1 and 28 days after a single oral dose of 10 micro g TCDD/kg body weight. Serum, kidney, and liver were investigated for retinoid levels, as well as gene expression and enzyme activities relevant for retinoid metabolism. Besides the well known effects of TCDD on apolar retinoids, i.e. decreased hepatic and increased renal retinyl ester (RE) levels, we have found dose-dependent elevation of all- trans-retinoic acid (all- trans-RA) levels in all investigated tissues. In the liver, 9- cis-4-oxo-13,14-dihydro-RA was drastically decreased by TCDD in a dose-dependent manner. In serum, cis-isomers of all- trans-RA, including 9,13-di- cis-RA, were significantly reduced already at the lowest dose level. Protein and mRNA levels of cellular retinol binding protein I (CRBP-I) in liver or kidneys were not significantly altered by TCDD exposure at doses at which retinoid levels were affected, making CRBP-I an unlikely candidate to account for the alterations in retinoid metabolism caused by TCDD. The expression and activities of relevant cytochrome P450 (CYP) enzymes with potential roles in all- trans-RA synthesis and/or degradation (CYP1A1, 1A2, and 2B1/2) were also monitored. A possible role of CYP1A1 in TCDD-induced all- trans-RA synthesis is suggested from the time-course relationship between CYP1A1 activity and all- trans-RA levels in liver and kidney. The significant alteration of the all- trans-RA metabolism has the potential to contribute significantly to the toxicity of TCDD.

Effect of tributyltin (TBT) on ATP levels in human natural killer (NK) cells: relationship to TBT-induced decreases in NK function.

PubMed

Dudimah, Fred D; Odman-Ghazi, Sabah O; Hatcher, Frank; Whalen, Margaret M

2007-01-01

The purpose of this study was to investigate the role that tributyltin (TBT)-induced decreases in ATP levels may play in TBT-induced decreases in the tumor lysing (lytic) function of natural killer (NK) cells. NK cells are a subset of lymphocytes that act as an initial immune defense against tumor cells and virally infected cells. TBT is an environmental contaminant that has been detected in human blood, which has been shown to interfere with ATP synthesis. Previous studies have shown that TBT is able to decrease very significantly the lytic function of NK cells. In this study NK cells were exposed to various concentrations of TBT and to two other compounds that interfere with ATP synthesis (rotenone a complex I inhibitor and oligomycin an ATP synthase inhibitor) for various lengths of time before determining the levels of ATP and lytic function. Exposures of NK cells to 10, 25, 50 and 100 nm TBT did not significantly reduce ATP levels after 24 h. However, these same exposures caused significant decreases in cytotoxic function. Studies of brief 1 h exposures to a range of TBT, rotenone and oligomycin concentrations followed by 24 h, 48 h and 6 day periods in compound-free media prior to assaying for ATP levels or cytotoxic function showed that each of the compounds caused persistent decreases in ATP levels and lytic function of NK cells. Exposures to 0.05-5 microm rotenone or oligomycin for 1 h reduced ATP levels by 20-25% but did not have any measurable effect on the ability of NK cells to lyse tumor cells. ATP levels were also decreased by about 20-25% after 24 h or 48 h exposures to rotenone or oligomycin (0.5 microm ), and the lytic function was decreased by about 50%. The results suggest that TBT-induced decreases in ATP levels were not responsible for the loss of cytotoxic function seen at 1 h and 24 h. However, TBT-induced decreases of NK-ATP levels may be at least in part responsible for losses of NK-cytotoxic function seen after 48 h and 6 day exposures

TCDD AND EGF AFFECT MAPK PATHWAY ACTIVATION IN MURINE EMBRYONIC PALATE

EPA Science Inventory

Palatal fusion occurs on GD 14-15 in the mouse, accompanied by a decrease in EGF receptor (EGFR) at the medial edge of the palatal shelves. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces cleft palate and maintains EGF and EGF receptor (EGFR) expression levels in the medial ed...

Interactive effects of n-TiO2 and 2,3,7,8-TCDD on the marine bivalve Mytilus galloprovincialis.

PubMed

Canesi, Laura; Frenzilli, Giada; Balbi, Teresa; Bernardeschi, Margherita; Ciacci, Caterina; Corsolini, Simonetta; Della Torre, Camilla; Fabbri, Rita; Faleri, Claudia; Focardi, Silvano; Guidi, Patrizia; Kočan, Anton; Marcomini, Antonio; Mariottini, Michela; Nigro, Marco; Pozo-Gallardo, Karla; Rocco, Lucia; Scarcelli, Vittoria; Smerilli, Arianna; Corsi, Ilaria

2014-08-01

Despite the growing concern over the potential biological impact of nanoparticles (NPs) in the aquatic environment, little is known about their interactions with other pollutants. The bivalve Mytilus sp, largely utilized as a sentinel for marine contamination, has been shown to represent a significant target for different types of NP, including n-TiO2, one of the most widespread in use. In this work, the possible interactive effects of n-TiO2 and 2,3,7,8-TCDD, chosen as models of NP and organic contaminant, respectively, were investigated in Mytilus galloprovincialis. In vitro experiments with n-TiO2 and TCDD, alone and in combination, were carried out in different conditions (concentrations and times of exposure), depending on the target (hemocytes, gill cells and biopsies) and the endpoint measured. Mussels were also exposed in vivo to n-TiO2 (100 μg L(-1)) or to TCDD (0.25 μg L(-1)), alone and in combination, for 96 h. A wide range of biomarkers, from molecular to tissue level, were measured: lysosomal membrane stability and phagocytosis in hemocytes, ATP-binding cassette efflux transporters in gills (gene transcription and efflux activity), several biomarkers of genotoxicity in gill and digestive cells (DNA damage, random amplified polymorphic DNA-RAPD changes), lysosomal biomarkers and transcription of selected genes in the digestive gland. The results demonstrate that n-TiO2 and TCDD can exert synergistic or antagonistic effects, depending on experimental condition, cell/tissue and type of measured response. Some of these interactions may result from a significant increase in TCDD accumulation in whole mussel organisms in the presence of n-TiO2, indicating a Trojan horse effect. The results represent the most extensive data obtained so far on the sub-lethal effects of NPs and organic contaminants in aquatic organisms. Moreover, these data extend the knowledge on the molecular and cellular targets of NPs in bivalves. Copyright © 2013 Elsevier B.V. All

An enhanced postnatal autoimmune profile in 24 week-old C57BL/6 mice developmentally exposed to TCDD

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mustafa, A.; Holladay, S.D.; Goff, M.

Developmental exposure of mice to the environmental contaminant and AhR agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), causes persistent postnatal suppression of T cell-mediated immune responses. The extent to which prenatal TCDD may induce or exacerbate postnatal autoimmune disease remains unknown. In the present study, time-pregnant high affinity AhR C57BL/6 mice received a single oral administration of 0, 2.5, or 5 {mu}g/kg TCDD on gestation day (gd) 12. Offspring of these mice (n = 5/gender/treatment) were evaluated at 24 weeks-of-age and showed considerable immune dysregulation that was often gender-specific. Decreased thymic weight and percentages of CD4{sup +}CD8{sup +} thymocytes, and increased CD4{sup +}CD8{sup -}more » thymocytes, were present in the female but not male offspring. Males but not females showed decreased CD4{sup -}CD8{sup +} T cells, and increased V{beta}3{sup +} and V{beta}17a{sup +} T cells, in the spleen. Males but not females also showed increased percentages of bone marrow CD24{sup -}B220{sup +} B cell progenitors. Antibody titers to dsDNA, ssDNA and cardiolipin displayed increasing trends in both male and female mice, reaching significance for anti-dsDNA in both genders and for ssDNA in males at 5 {mu}g/kg TCDD. Immunofluorescent staining of IgG and C3 deposition in kidney glomeruli increased in both genders of prenatal TCDD-exposed mice, suggestive of early stages of autoimmune glomerulonephritis. Collectively, these results show that exposure to TCDD during immune system development causes persistent humoral immune dysregulation as well as altered cell-mediated responses, and induces an adult profile of changes suggestive of increased risk for autoimmune disease.« less

SHP-1 is directly activated by the aryl hydrocarbon receptor and regulates BCL-6 in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phadnis-Moghe, Ashwini S.; Li, Jinpeng

2016-11-01

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a strong AHR agonist, causes significant suppression of human B cell activation and differentiation. The current studies describe the identification of Src homology phosphatase 1 (SHP-1) encoded by the gene PTPN6 as a putative regulator of TCDD-mediated suppression of B cell activation. Shp-1 was initially identified through a genome-wide analysis of AHR binding in mouse B cells in the presence of TCDD. The binding of AHR to the PTPN6 promoter was further confirmed using electrophoretic mobility shift assays in which, specific binding of AHR was detected at four putative DRE sites within PTPN6more » promoter. Time-course measurements performed in human B cells highlighted a significant increase in SHP-1 mRNA and protein levels in the presence of TCDD. The changes in the protein levels of SHP-1 were also observed in a TCDD concentration-dependent manner. The increase in SHP-1 levels was also seen to occur due to a change in early signaling events in the presence of TCDD. We have shown that BCL-6 regulates B cell activation by repressing activation marker CD80 in the presence of TCDD. TCDD-treatment led to a significant increase in the double positive (SHP-1{sup hi} BCL-6{sup hi}) population. Interestingly, treatment of naïve human B cells with SHP-1 inhibitor decreased BCL-6 protein levels suggesting possible regulation of BCL-6 by SHP-1 for the first time. Collectively, these results suggest that SHP-1 is regulated by AHR in the presence of TCDD and may, in part through BCL-6, regulate TCDD-mediated suppression of human B cell activation. - Highlights: • SHP-1 encoded by the gene PTPN6 is directly activated by the AHR. • AHR binds to dioxin response elements within the SHP-1 promoter in a TCDD-inducible manner. • TCDD-mediated increase in SHP-1 levels is observed in primary human B cells. • Higher SHP-1 levels help in maintaining high BCL-6 levels in the presence of TCDD

TCDD alters medial epithelial cell differentiation during palatogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abbott, B.D.; Birnbaum, L.S.

1989-06-15

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widely distributed, persistent environmental contaminant that is teratogenic in mice, where it induces hydronephrosis and cleft palate. The incidence of clefting has been shown to be dose dependent after exposure on either gestation Day (GD) 10 or 12, although the embryo is more susceptible on GD 12. TCDD-exposed palatal shelves meet but do not fuse, and programmed cell death of the medial epithelial cells is inhibited. The mechanism of action through which TCDD alters the program of medial cell development has not been examined in earlier studies, and it is not known whether the mechanism ismore » the same regardless of the dose or developmental stage of exposure. In this study, C57BL/6N mice, a strain sensitive to TCDD, were dosed orally on GD 10 or 12 with 0, 6, 12, 24, or 30 micrograms/kg body wt, in 10 ml corn oil/kg. Embryonic palatal shelves were examined on GD 14, 15, or 16. The degree of palatal closure, epithelial surface morphology, and cellular ultrastructure, the incorporation of (3H)TdR, the expression of EGF receptors, and the binding of 125I-EGF were assessed. After exposure on GD 10 or 12, TCDD altered the differentiation pathway of the medial epithelial cells. The palatal shelves were of normal size and overall morphology, but fusion of the medial epithelia of the opposing shelves did not occur. TCDD prevented programmed cell death of the medial peridermal cells. The expression of EGF receptors by medial cells continued through Day 16 and the receptors were able to bind ligand. The medial cells differentiated into a stratified, squamous, keratinizing epithelium. The shift in phenotype to an oral-like epithelium occurred after exposure on either GD 10 or 12. At the lower dose (6 micrograms/kg), fewer cleft palates were produced, but those shelves which did respond had a fully expressed shift in differentiation.« less

A critical review of the epidemiology of Agent Orange/TCDD and prostate cancer.

PubMed

Chang, Ellen T; Boffetta, Paolo; Adami, Hans-Olov; Cole, Philip; Mandel, Jack S

2014-10-01

To inform risk assessment and regulatory decision-making, the relationship between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and prostate cancer requires clarification. This article systematically and critically reviews the epidemiologic evidence on the association between exposure to TCDD or Agent Orange, a TCDD-contaminated herbicide used during the Vietnam War, and prostate cancer risk. Articles evaluated include 11 studies of three cohorts, four case-control or cross-sectional studies, and three case-only studies of military veterans with information on estimated Agent Orange or TCDD exposure; 13 studies of seven cohorts, one case-control study, and eight proportionate morbidity or mortality studies of Vietnam veterans without information on Agent Orange exposure; 11 cohort studies of workers with occupational exposure to TCDD; and two studies of one community cohort with environmental exposure to TCDD. The most informative studies, including those of Vietnam veterans involved in Agent Orange spraying or other handling, herbicide manufacturing or spraying workers with occupational TCDD exposure, and community members exposed to TCDD through an industrial accident, consistently reported no significant increase in prostate cancer incidence or mortality. Only some potentially confounded studies of Vietnam veterans compared with the general population, studies with unreliable estimates of Agent Orange exposure, and analyses of selected subgroups of Vietnam veterans reported positive associations. Overall, epidemiologic research offers no consistent or convincing evidence of a causal relationship between exposure to Agent Orange or TCDD and prostate cancer. More accurate exposure assessment is needed in large epidemiologic studies to rule out a causal association more conclusively.

TCDD promoted EMT of hFPECs via AhR, which involved the activation of EGFR/ERK signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Zhan; The Fifth Affiliated Hospital, Zhengzhou University, 450052; Bu, Yongjun

2016-05-01

One critical step of second palatal fusion is the newly formed medial epithelia seam (MES) disintegration, which involves apoptosis, epithelial to mesenchymal transition (EMT), and cell migration. Although the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces cleft palate at high rates, little is known about the effects of TCDD exposure on the fate of palatal epithelial cells. By using primary epithelial cells isolated from human fetal palatal shelves (hFPECs), we show that TCDD increased cell proliferation and EMT, as demonstrated by increased the epithelial markers (E-cadherin and cytokeratin14) and enhanced the mesenchymal markers (vimentin and fibronectin), but had no effect on cellmore » migration and apoptosis. TCDD exposure led to a dose-dependent increase in Slug protein expression. Coimmunoprecipitation revealed that TCDD promoted AhR to form a protein complex with Slug. ChIP assay confirmed that TCDD exposure recruited AhR to the xenobiotic responsive element of Slug promoter. Knockdown of AhR by siRNA remarkably weakened TCDD-induced binding of AhR to the XRE promoter of slug, thereby suppressed TCDD-induced vimentin. Further experiment showed that TCDD stimulated EGFR phosphorylation did not influence the TGFβ3/Smad signaling; whereas TCDD increased phosphorylation of ERK1/2 and p38 with no effect on activation of JNK. By using varieties of inhibitors, we confirmed that TCDD promoted proliferation and EMT of hFPECs via activation of EGFR/ERK pathway. These data make a novel contribution to the molecular mechanism of cleft palate by TCDD. - Highlights: • TCDD exposure promoted cell proliferation and EMT of hFPECs; • AhR signaling was activated and required for TCDD-induced EMT; • TCDD-mediated EMT of hFPECs involved the activation of EGFR/ERK signaling; • TCDD exposure had no effect on TGFβ3/Smad pathway.« less

Cellular alterations and enhanced induction of cleft palate after coadministration of retinoic acid and TCDD

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abbott, B.D.; Birnbaum, L.S.

1989-06-15

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and retinoic acid (RA) are both teratogenic in mice. TCDD is a highly toxic, stable environmental contaminant, while RA is a naturally occurring form of vitamin A. Exposure to TCDD induces hydronephrosis and cleft palate, and exposure to RA induces limb defects and cleft palate. Teratology studies previously have shown that the incidence of clefting is higher after exposure to RA + TCDD than would be observed for the same doses of either compound given alone. This study examines the cellular effects which result in cleft palate, after po administration on gestation Day (GD) 10 or 12 ofmore » RA + TCDD in corn oil (10 ml/kg total volume). Exposure on GD 10 to 6 micrograms TCDD + 40 mg RA/kg inhibited early growth of the shelves and clefting was due to a failure of shelves to meet and fuse. This effect on mesenchyme was observed in previous studies to occur after exposure on GD 10 to 40 mg/kg RA alone, but not after TCDD alone. After exposure on GD 12 to 6 micrograms TCDD + 80 mg RA/kg, clefting was due to a failure of shelves to fuse after making contact, because the medial cells differentiated into an oral-like epithelium. This response was observed in previous studies to occur after exposure to TCDD alone, but RA alone on GD 12 resulted in differentiation toward nasal-like cells. The interaction between TCDD and RA results in RA-like clefting after exposure on GD 10 and TCDD-like clefting after exposure on GD 12, and this clefting occurs at higher incidences than would occur after the same levels of either agent alone. After exposure on either GD 10 or 12 to RA + TCDD, the programmed cell death of the medial cells does not occur, and these cells continue to express EGF receptors and to bind 125I-EGF. The effects of RA and TCDD may involve modulation of the cells responses to embryonic growth and differentiation factors.« less

ASSESSING TCDD WASTING SYNDROME IN AN IN VIVO OBESITY MODEL

EPA Science Inventory

TCDD is a by-product of incineration commonly found as a microcontaminant in the food supply. The TCDD wasting syndrome, characterized by prolonged weight loss, has been examined for decades. Much of this work has focused on high dose in vivo and in vitro studies....

DEVELOPMENTAL TOXICITY OF TCDD AND RELATED COMPOUNDS: SENSITIVITIES AND DIFFERENCES

EPA Science Inventory

The issue of the developmental toxicity of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) and related compounds has been the subject of two recent reviews (Morrissey and Schwetz, 1989; Couture et al., 1990a). here is little doubt that TCDD is one of the most potent developmental tox...

Obesity and perinatal TCDD exposure increases mammary tumors in FVB mice

EPA Science Inventory

Risk of breast cancer has been consistently shown to correlate to total lifetime exposure to estrogens. Because both TCDD exposure and the state of obesity interact with the estrogen pathway, we wanted to investigate how TCDD and obesity interact with mammary cancer susceptibili...

Mechanisms of Cellular Membrane Effects of TCDD (2,3,7,8-Tetrachlorodibenzo-p-Dioxin) Selected Perfluorinated Acids and Polyhalogenated Aromatic Hydrocarbons.

DTIC Science & Technology

1986-10-15

tetrachloro dibenzo-p-dioxin (TCDD); dioxin; perfluoro -n-decanoic acid ( PFDA ); perfluoro -n-octanoic acid ( PFOA ); 3,31,4,41,5,51 hexachlorobiphenyl (345PCB) and...analysis of livers from rats treated with perfluoro -n-decanoic acid ( PFDA ) showed a decrease in stearic acid and an increase in 18 carbon unsaturates...effects of TCDD, PHAH and perfluorinated acids ( PFA ) on mammalian cell membrane function by examing the phenomenon of metabolic cooperation. Metabolic

TCDD and cancer: A critical review of epidemiologic studies

PubMed Central

Boffetta, Paolo; Mundt, Kenneth A; Adami, Hans-Olov; Cole, Philip; Mandel, Jack S

2011-01-01

The authors reviewed the epidemiologic studies on exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and cancer risk, published since the last full-scale review made by the International Agency for Research on Cancer Monographs program in 1997. The update of a cohort of US herbicide producers generated negative results overall; the internal analysis provided evidence of an increased “all-cancer” risk in the highest exposure category, with a statistically significant exposure-response association in some of the many analyses performed.The update of a similar Dutch cohort did not confirm the previously observed association with TCDD exposure. The updated surveillance of the Seveso population provided evidence of increased all-cancer mortality 15-20 years after exposure among those living in the most contaminated area but might also reflect random variation, as overall excesses in the most recent follow-up were not observed. Corresponding data on cancer incidence offer little support to the mortality results. Updated results from cohort studies of Vietnam veterans potentially exposed to TCDD did not consistently suggest an increased risk of cancer. Results of additional, smaller studies of other occupational groups potentially exposed to TCDD, and of community-based case-control studies, did not provide consistent evidence of an increased cancer risk. In conclusion, recent epidemiological evidence falls far short of conclusively demonstrating a causal link between TCDD exposure and cancer risk in humans. The emphasis on results for overall cancer risk—rather than risk for specific neoplasms—is notjustified on epidemiologic grounds and is nota reason for ignoring the weaknesses of the available evidence. PMID:21718216

Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering.

PubMed

Plecitá-Hlavatá, Lydie; Engstová, Hana; Alán, Lukáš; Špaček, Tomáš; Dlasková, Andrea; Smolková, Katarína; Špačková, Jitka; Tauber, Jan; Strádalová, Vendula; Malínský, Jan; Lessard, Mark; Bewersdorf, Joerg; Ježek, Petr

2016-05-01

The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/topogenesis of mitochondrial outer membrane β-barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super-resolution biplane fluorescence photoactivation localization microscopy with Eos-conjugated, ICS-located lactamase-β indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos-mitochondrial fission protein-1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MICOS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by ∼20%. ATP synthase dimers vs monomers and state-3/state-4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMM-detached cristae of OPA1- and mitofilin-silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down-regulation. Mitofilin-depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.-Plecitá-Hlavatá, L., Engstová, H., Alán, L., Špaček, T., Dlasková, A., Smolková, K., Špačková, J., Tauber, J., Strádalová, V., Malínský, J

Comparative analysis of TCDD-induced AhR-mediated gene expression in human, mouse and rat primary B cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kovalova, Natalia, E-mail: kovalova@msu.edu

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant that activates the aryl hydrocarbon receptor (AhR) resulting in altered gene expression. In vivo, in vitro, and ex vivo studies have demonstrated that B cells are directly impaired by TCDD, and are a sensitive target as evidenced by suppression of antibody responses. The window of sensitivity to TCDD-induced suppression of IgM secretion among mouse, rat and human B cells is similar. Specifically, TCDD must be present within the initial 12 h post B cell stimulation, indicating that TCDD disrupts early signaling network(s) necessary for B lymphocyte activation and differentiation. Therefore, we hypothesized thatmore » TCDD treatment across three different species (mouse, rat and human) triggers a conserved, B cell-specific mechanism that is involved in TCDD-induced immunosuppression. RNA sequencing (RNA-Seq) was used to identify B cell-specific orthologous genes that are differentially expressed in response to TCDD in primary mouse, rat and human B cells. Time course studies identified TCDD-elicited differential expression of 515 human, 2371 mouse and 712 rat orthologous genes over the 24-h period. 28 orthologs were differentially expressed in response to TCDD in all three species. Overrepresented pathways enriched in all three species included cytokine-cytokine receptor interaction, ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton and pathways in cancer. Differentially expressed genes functionally associated with cell-cell signaling in humans, immune response in mice, and oxidation reduction in rats. Overall, these results suggest that despite the conservation of the AhR and its signaling mechanism, TCDD elicits species-specific gene expression changes. - Highlights: • Kovalova TAAP Highlights Nov. 2016 • RNA-Seq identified TCDD-induced gene expression in PWM-activated primary B cells. • TCDD elicited differential expression of 515 human, 2371 mouse

ANALYSIS OF 2,3,7,8-TCDD TUMOR PROMOTION ACTIVITY AND ITS RELATIONSHIP TO CANCER

EPA Science Inventory

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has a high estimated cancer potency in animals which has been reasoned to imply that TCDD might be carcinogenic to man. The animal cancer data show that TCDD can act in a solitary manner causing tumors without the participation of other ...

Obesity and perinatal TCDD exposure increases mammary tumor incidence in FVB mice

EPA Science Inventory

Breast cancer risk consistently correlates with total lifetime exposure to estrogens. Because both TCDD and adipocytes impact the estrogen pathway, we examined how TCDD and obesity interact to alter mammary cancer susceptibility. At 12.5 days post conception, we exposed FVB fema...

EVIDENCE FOR EGFR PATHWAY MEDIATION OF CLEFT PALATE INDUCTION BY TCDD

EPA Science Inventory

EVIDENCE FOR EGFR PATHWAY MEDIATION OF CLEFT PALATE INDUCTION BY TCDD. B D Abbott, A R Buckalew, and K E Leffler. RTD, NHEERL, ORD,US EPA, RTP, NC, USA.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in C57BL/6J mice, producing cleft palate (CP) after exposure...

A functional 12T-insertion polymorphism in the ATP1A1 promoter confers decreased susceptibility to hypertension in a male Sardinian population.

PubMed

Herrera, Victoria L; Pasion, Khristine A; Moran, Ann Marie; Zaninello, Roberta; Ortu, Maria Francesca; Fresu, Giovanni; Piras, Daniela Antonella; Argiolas, Giuseppe; Troffa, Chiara; Glorioso, Valeria; Masala, Wanda; Glorioso, Nicola; Ruiz-Opazo, Nelson

2015-01-01

Identification of susceptibility genes for essential hypertension in humans has been a challenge due to its multifactorial pathogenesis complicated by gene-gene and gene-environment interactions, developmental programing and sex specific differences. These concurrent features make identification of causal hypertension susceptibility genes with a single approach difficult, thus requiring multiple lines of evidence involving genetic, biochemical and biological experimentation to establish causal functional mutations. Here we report experimental evidence encompassing genetic, biochemical and in vivo modeling that altogether support ATP1A1 as a hypertension susceptibility gene in males in Sardinia, Italy. ATP1A1 encodes the α1Na,K-ATPase isoform, the sole sodium pump in vascular endothelial and renal tubular epithelial cells. DNA-sequencing detected a 12-nucleotide long thymidine (12T) insertion(ins)/deletion(del) polymorphism within a poly-T sequence (38T vs 26T) in the ATP1A1 5'-regulatory region associated with hypertension in a male Sardinian population. The 12T-insertion allele confers decreased susceptibility to hypertension (P = 0.035; OR = 0.50 [0.28-0.93]) accounting for 12.1 mmHg decrease in systolic BP (P = 0.02) and 6.6 mmHg in diastolic BP (P = 0.046). The ATP1A1 promoter containing the 12T-insertion exhibited decreased transcriptional activity in in vitro reporter-assay systems, indicating decreased α1Na,K-ATPase expression with the 12T-insertion, compared with the 12T-deletion ATP1A1 promoter. To test the effects of decreased α1Na,K-ATPase expression on blood pressure, we measured blood pressure by radiotelemetry in three month-old, highly inbred heterozygous knockout ATP1A1+/- male mice with resultant 58% reduction in ATP1A1 protein levels. Male ATP1A1+/- mice showed significantly lower blood pressure (P < 0.03) than age-matched male wild-type littermate controls. Concordantly, lower ATP1A1 expression is expected to lower Na-reabsorption in the

A Functional 12T-Insertion Polymorphism in the ATP1A1 Promoter Confers Decreased Susceptibility to Hypertension in a Male Sardinian Population

PubMed Central

Herrera, Victoria L.; Pasion, Khristine A.; Moran, Ann Marie; Zaninello, Roberta; Ortu, Maria Francesca; Fresu, Giovanni; Piras, Daniela Antonella; Argiolas, Giuseppe; Troffa, Chiara; Glorioso, Valeria; Masala, Wanda; Glorioso, Nicola; Ruiz-Opazo, Nelson

2015-01-01

Identification of susceptibility genes for essential hypertension in humans has been a challenge due to its multifactorial pathogenesis complicated by gene-gene and gene-environment interactions, developmental programing and sex specific differences. These concurrent features make identification of causal hypertension susceptibility genes with a single approach difficult, thus requiring multiple lines of evidence involving genetic, biochemical and biological experimentation to establish causal functional mutations. Here we report experimental evidence encompassing genetic, biochemical and in vivo modeling that altogether support ATP1A1 as a hypertension susceptibility gene in males in Sardinia, Italy. ATP1A1 encodes the α1Na,K-ATPase isoform, the sole sodium pump in vascular endothelial and renal tubular epithelial cells. DNA-sequencing detected a 12-nucleotide long thymidine (12T) insertion(ins)/deletion(del) polymorphism within a poly-T sequence (38T vs 26T) in the ATP1A1 5’-regulatory region associated with hypertension in a male Sardinian population. The 12T-insertion allele confers decreased susceptibility to hypertension (P = 0.035; OR = 0.50 [0.28–0.93]) accounting for 12.1 mmHg decrease in systolic BP (P = 0.02) and 6.6 mmHg in diastolic BP (P = 0.046). The ATP1A1 promoter containing the 12T-insertion exhibited decreased transcriptional activity in in vitro reporter-assay systems, indicating decreased α1Na,K-ATPase expression with the 12T-insertion, compared with the 12T-deletion ATP1A1 promoter. To test the effects of decreased α1Na,K-ATPase expression on blood pressure, we measured blood pressure by radiotelemetry in three month-old, highly inbred heterozygous knockout ATP1A1+/− male mice with resultant 58% reduction in ATP1A1 protein levels. Male ATP1A1+/− mice showed significantly lower blood pressure (P < 0.03) than age-matched male wild-type littermate controls. Concordantly, lower ATP1A1 expression is expected to lower Na-reabsorption in

Sex-related differences in murine hepatic transcriptional and proteomic responses to TCDD

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prokopec, Stephenie D.; Watson, John D.; Lee, Jamie

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that produces myriad toxicities in most mammals. In rodents alone, there is a huge divergence in the toxicological response across species, as well as among different strains within a species. But there are also significant differences between males and females animals of a single strain. These differences are inconsistent across model systems: the severity of toxicity is greater in female rats than males, while male mice and guinea pigs are more sensitive than females. Because the specific events that underlie this difference remain unclear, we characterized the hepatic transcriptional response of adult male andmore » female C57BL/6 mice to 500 μg/kg TCDD at multiple time-points. The transcriptional profile diverged significantly between the sexes. Female mice demonstrated a large number of altered transcripts as early as 6 h following treatment, suggesting a large primary response. Conversely, male animals showed the greatest TCDD-mediated response 144 h following exposure, potentially implicating significant secondary responses. Nr1i3 was statistically significantly induced at all time-points in the sensitive male animals. This mRNA encodes the constitutive androstane receptor (CAR), a transcription factor involved in the regulation of xenobiotic metabolism, lipid metabolism, cell cycle and apoptosis. Surprisingly though, changes at the protein level (aside from the positive control, CYP1A1) were modest, with only FMO3 showing clear induction, and no genes with sex-differences. Thus, while male and female mice show transcriptional differences in their response to TCDD, their association with TCDD-induced toxicities remains unclear. - Highlights: • Differences exist between the toxicity phenotypes to TCDD in male and female mice. • TCDD-mediated transcriptomic differences were identified between the sexes. • Resistant female mice displayed a large, early-onset, transcriptomic

THE INFLUENCE OF VARIABLE ELIMINATION RATE AND BODY FAT MASS IN A PBPK MODEL FOR TCDD IN PREDICTING THE SERUM TCDD CONCENTRATIONS FROM VETERANS OF OPERATION RANCH HAND

EPA Science Inventory

The Influence of Variable Elimination Rate and Body Fat Mass in a PBPK Model for TCDD in Predicting the Serum TCDD Concentrations from Veterans of Operation Ranch Hand.
C Emond1,2, LS Birnbaum2, JE Michalek3, MJ DeVito2
1 National Research Council, National Academy of Scien...

Agent Orange Exposure and 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) in Human Milk.

PubMed

Scialli, Anthony R; Watkins, Deborah K; Ginevan, Michael E

2015-06-01

Agent Orange was sprayed in parts of southern Vietnam during the U.S.-Vietnam war and was a mixture of two chlorophenoxy herbicides. The mixture was contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD and other dioxins and furans are measurable in the milk of Vietnamese women. We explored whether the TCDD in milk from these women was from Agent Orange and whether lactational exposure can be a mode of transgenerational effects of TCDD from Agent Orange. A review of the world's literature on milk concentrations of polychlorinated compounds showed the presence of TCDD and other dioxins and furans in all countries that have been assessed. The congener profile of these chemicals, that is, the proportion of different congeners in the sample, can be used to assess the source of milk contamination. Measurements in most countries, including contemporary measurements in Vietnam, are consistent with non-Agent Orange exposure sources, including industrial activities and incineration of waste. Models and supporting human data suggest that TCDD from breastfeeding does not persist in a child past adolescence and that the adult body burden of TCDD is independent of whether the individual was breast- or bottle-fed as a child. These findings suggest that exposure to Agent Orange in Vietnam did not result in persistent transgenerational exposure through human milk. © 2015 Wiley Periodicals, Inc.

Morbidity in New Zealand pesticide producers exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

PubMed

't Mannetje, Andrea; Eng, Amanda; Walls, Chris; Dryson, Evan; Douwes, Jeroen; Bertazzi, Pier; Ryder-Lewis, Simon; Scott, David; Brooks, Collin; McLean, Dave; Cheng, Soo; Pearce, Neil

2018-01-01

To conduct a cross-sectional morbidity survey among 245 former employees of a pesticide production plant exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in New Zealand. Demographic factors and health information were collected in face-to-face interviews. TCDD, lipids, thyroid hormones, glucose and immunoglobulin G (IgG) were determined in non-fasting blood. For 111 participants, a neurological examination was conducted. Associations between health outcomes and working in a TCDD exposed job (prevalence 49%) and serum TCDD concentration≥10pg/g lipid (18%) were assessed using logistic regression whilst controlling for age, gender, smoking, body mass index and ethnicity. Diabetes was more common in those who had worked in TCDD exposed jobs (OR 4.0, 95%CI 1.0-15.4) and in those with serum TCDD ≥10pg/g (OR 3.1, 95%CI 0.9-10.7). Non-fasting glucose levels >6.6mmol/l were more common in those with TCDD exposed jobs (OR 3.6, 95%CI 1.0-12.9), as were serum free thyroxine 4<12.8pmol/l (OR 4.5, 95%CI 1.4-14.4), triglycerides >1.7mmol/l (OR 2.5, 95%CI 1.1-5.7) and high density lipoprotein cholesterol (HDL) <1mmol/l (OR 4.0, 95%CI 1.2-13.2). IgG was negatively associated with TCDD (linear regression p=0.05). The neurological examination revealed a higher frequency of abnormal reflexes in those with serum TCDD ≥10pg/g (OR 4.8, 95%CI 1.1-21.0). In this occupationally exposed population, TCDD was associated with an increased risk of diabetes and a range of subclinical responses in multiple systems (peripheral nervous system, immune system, thyroid hormones and lipid metabolism), several decades after last exposure. These results need to be interpreted with caution due to the small study size and the cross-sectional nature of the study. Copyright © 2017 Elsevier Ltd. All rights reserved.

The AhR Ligand, TCDD, Regulates Androgen Receptor Activity Differently in Androgen-Sensitive versus Castration-Resistant Human Prostate Cancer Cells.

PubMed

Ghotbaddini, Maryam; Powell, Joann B

2015-07-06

The reported biological effects of TCDD include induction of drug metabolizing enzymes, wasting syndrome and tumor promotion. TCDD elicits most of its effects through binding the aryl hydrocarbon receptor (AhR). TCDD induced degradation of AhR has been widely reported and requires ubiquitination of the protein. The rapid depletion of AhR following TCDD activation serves as a mechanism to modulate AhR mediated gene induction. In addition to inducing AhR degradation, TCDD has been reported to induce degradation of hormone receptors. The studies reported here, evaluate the effect of TCDD exposure on androgen receptor (AR) expression and activity in androgen-sensitive LNCaP and castration-resistant C4-2 prostate cancer cells. Our results show that TCDD exposure does not induce AhR or AR degradation in C4-2 cells. However, both AhR and AR are degraded in LNCaP cells following TCDD exposure. In addition, TCDD enhances AR phosphorylation and induces expression of AR responsive genes in LNCaP cells. Our data reveals that TCDD effect on AR expression and activity differs in androgen-sensitive and castration-resistant prostate cancer cell models.

DISPOSITION OF TCDD IN A MOUSE MODEL OF OBESITY AND TYPE II DIABETESE

EPA Science Inventory

Recent epidemiology studies have shown an association between type II diabetes and exposure to TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin). A possible explanation is that diabetics have a slower elimination of TCDD than non-diabetics. The objective of the present study was to ex...

Comparison of the in vitro effects of TCDD, PCB 126 and PCB 153 on thyroid-restricted gene expression and thyroid hormone secretion by the chicken thyroid gland.

PubMed

Katarzyńska, Dorota; Hrabia, Anna; Kowalik, Kinga; Sechman, Andrzej

2015-03-01

The aim of this study was to compare the in vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4',5-pentachlorobiphenyl (PCB 126; a coplanar PCB congener) and 2,2'4,4',5,5'-hexachlorobiphenyl (PCB153; non-coplanar PCB) on mRNA expression of thyroid-restricted genes, i.e. sodium iodide symporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (TG), and thyroid hormone secretion from the thyroid gland of the laying chicken. Relative expression levels of NIS, TG and TPO genes and thyroxine (T4) and triiodothyronine (T3) secretion from the thyroidal explants were quantified by the real-time qPCR and RIA methods, respectively. In comparison with the control group, TCDD and PCB 126 significantly increased mRNA expression of TPO and TG genes. TCDD did not affect NIS mRNA levels, but PCB 126 decreased its expression. No effect of PCB 153 on the expression of these genes was observed. TCDD and PCB 126 significantly decreased T4 and T3 secretion. There was no significant effect of PCB 153 on these hormone secretions. In conclusion, the results obtained show that in comparison with non-coplanar PCB 153, TCDD and coplanar PCB 126 can directly affect thyroid hormone synthesis and secretion, and in consequence, they may disrupt the endocrine function of the thyroid gland of the laying chicken. Copyright © 2015 Elsevier B.V. All rights reserved.

TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Abrew, K. Nadira; Thomas-Virnig, Christina L.; Rasmussen, Cathy A.

2014-05-01

The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highlymore » induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial–stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin. - Highlights: • TCDD causes hyperkeratosis and basement membrane changes in a model of human skin. • TCDD induces MMP-10 expression in organotypic

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) persistence in the Seveso (Milan, Italy) soil.

PubMed

Cerlesi, S; Di Domenico, A; Ratti, S

1989-10-01

Preliminary results of a new study on TCDD environmental persistence at Seveso (Milan, Italy) are presented. For this study, the most contaminated territory, Zone A, was divided into areas to fractionate the available TCDD levels in soil into data sets with reduced value spreads. In addition, various time subsets were defined for each area. Selected data were fitted with the exponential model y = y0.e-k.1. It was estimated that at least 1.2 kg TCDD was present in Zone A shortly after the accident. On average, a considerable portion (23%) of this amount lay on vegetation; TCDD which was not photodegraded or volatilized before the heavy rains of fall 1976, was later washed off and transferred to ground by water action. From this study, mean analytical underestimations affecting January 1977 and March 1978 contamination map data were on the order of 30 and 24%. All the above figures are considered optimistic. A few years after the accident, mean TCDD half-life in soil appeared to be 9.1y (t1/2-95% CLs, 6.2-17y).

Endocrine and metabolic aspects of the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gorski, J.R.

1988-01-01

Toxic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were characterized in male Sprague-Dawley rats in order to elucidate the mechanism of acute toxicity of this potent halogenated hydrocarbon. Studies in TCDD-treated, pair-fed control and ad libitum-fed control rates, as well as in thyroidectomized, adrenalectomized and hypophysectomized, revealed differential hormonal, toxicologic and histophathologic responses suggesting that these manifestations of TCDD exposure are the results of an insult to intermediary metabolism. Tissue specific alterations in de novo fatty acid synthesis were directly related to differential changes observed in thyroid hormone homeostasis. The increased hepatic de novo fatty acid synthesis provided a likely mechanism for themore » documented fact that TCDD-treated rats lose more body weight than corresponding pair-fed controls because de novo fatty acid synthesis represents an energy inefficient metabolic process. Experiments in adrenalectomized and hypophysectomized rats led to the hypothesis that severe hypoglycemia due to inhibition of gluconeogenesis is the cause of TCDD-induced death. A subsequent characterization of gluconeogenesis in TCDD-treated rats confirmed this hypothesis.« less

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on development of anuran amphibians

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jung, R.E.; Walker, M.K.

1997-02-01

The authors exposed anuran eggs and tadpoles to vehicle control (0.7% acetone) or waterborne [{sup 3}H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h and subsequently raised them in clean water. Neither American toads nor green frogs exhibited TCDD-related mortality, but leopard frogs showed significantly increased mortality over controls. Eggs and tadpoles eliminated TCDD relatively quickly compared with published data for other vertebrates, with t{sub {1/2}} of 1 to 5 d (American toad), 2 to 7 d (leopard frog), and 4 to 6 d (green frog). Elimination rates were slowest for tadpoles fed nothing, fastest for those fed a low-fat diet, and intermediate formore » those fed a high-fat diet. Although not significant, American toads exposed to {ge}0.03 {micro}g TCDD/L appeared to metamorphose earlier, and those exposed to higher TCDD treatments appeared to metamorphose at a larger body mass than controls. Comparisons of these results with studies of fish early life stages suggest that anuran eggs and tadpoles eliminate TCDD more rapidly and are 100- to 1,000-fold less sensitive to its deleterious effects during development. These differences may be related to differences in metabolic rate, patterns of lipid storage and utilization, and aryl hydrocarbon receptor binding and signal transduction.« less

SEVESO WOMEN'S HEALTH STUDY: DOES ZONE OF RESIDENCE PREDICT INDIVIDUAL TCDD EXPOSURE? (R824761)

EPA Science Inventory

The compound, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is produced as an unwanted by-product of various chemical reactions and combustion processes, including the manufacture of chlorinated phenols and derivatives. In animals, TCDD exposure is associated with toxic, car...

TCDD influences reservoir of antibiotic resistance genes in murine gut microbiome.

PubMed

Stedtfeld, Robert D; Stedtfeld, Tiffany M; Fader, Kelly A; Williams, Maggie R; Bhaduri, Prianca; Quensen, John; Zacharewski, Timothy R; Tiedje, James M; Hashsham, Syed A

2017-05-01

Dysbiosis of the gut microbiome via antibiotics, changes in diet and infection can select for bacterial groups that more frequently harbor antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs). However, the impact of environmental toxicants on the reservoir of ARGs in the gut microbiome has received less attention. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent aryl hydrocarbon receptor (AhR) agonist with multiple toxic health effects including immune dysfunction. The selective pressure of TCDD on the abundance of ARG and MGE-harboring gut populations was examined using C57BL/6 mice exposed to 0-30 μg/kg TCDD for 28 and 92 days with the latter having a 30-day recovery period. DNA extracted from temporally collected fecal pellets was characterized using a qPCR array with 384 assays targeting ARGs and MGEs. Fourteen genes, typically observed in Enterobacteriaceae, increased significantly within 8 days of initial dosing, persisted throughout the treatment period, and remained induced 30 days post dosing. A qPCR primer set targeting Enterobacteriaceae also showed 10- to 100-fold higher abundance in TCDD-treated groups, which was further verified using metagenomics. Results show a bloom of ARG-harboring bacterial groups in the gut due to a xenobiotic compound that is not a metal, biocide or antimicrobial. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Adsorption of TCDD molecule onto CNTs and BNNTs: Ab initio van der Waals density-functional study

NASA Astrophysics Data System (ADS)

Darvish Ganji, M.; Alinezhad, H.; Soleymani, E.; Tajbakhsh, M.

2015-03-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCCD) is one of the most dangerous compounds that infect the environment and hence its removal is crucial for safety in human life. In this work, we have investigated the interaction of TCDD with boron nitride nanotubes (BNNTs) and carbon nanotubes (CNTs) by using the density functional theory (DFT) calculations. Our first-principles results have been validated by experiment and also other theoretical values for the similar system. The adsorption energies for TCDD molecule on the BNNTs and CNT are calculated. It was found that TCDD adsorption ability of BNNT is slightly stronger than that of CNT and TCDD molecule prefers to be adsorbed on BNNTs with molecular axis parallel to the tube axis. The results obtained indicate that TCDD is weakly bound to the outer surface of all the considered nanotubes and the obtained adsorption energy values and binding distance are typical for the physisorption. We also evaluated the influence of curvature and introduced defects on the TCDD adsorption ability of BNNTs. Furthermore, we have analyzed the electronic structure and charge population for the energetically most favorable complexes and the results indicate that no significant hybridization between the respective orbitals of the two entities was accomplished.

Sox9b Is Required for Epicardium Formation and Plays a Role in TCDD-Induced Heart Malformation in Zebrafish

PubMed Central

Hofsteen, Peter; Plavicki, Jessica; Johnson, Shaina D.; Peterson, Richard E.

2013-01-01

Activation of the transcription factor aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prevents the formation of the epicardium and leads to severe heart malformations in developing zebrafish (Danio rerio). The downstream genes that cause heart malformation are not known. Because TCDD causes craniofacial malformations in zebrafish by downregulating the sox9b gene, we hypothesized that cardiotoxicity might also result from sox9b downregulation. We found that sox9b is expressed in the developing zebrafish heart ventricle and that TCDD exposure markedly reduces this expression. Furthermore, we found that manipulation of sox9b expression could phenocopy many but not all of the effects of TCDD at the heart. Loss of sox9b prevented the formation of epicardium progenitors comprising the proepicardium on the pericardial wall, and prevented the formation and migration of the epicardial layer around the heart. Zebrafish lacking sox9b showed pericardial edema, an elongated heart, and reduced blood circulation. Fish lacking sox9b failed to form valve cushions and leaflets. Sox9b is one of two mammalian Sox9 homologs, sox9b and sox9a. Knock down of sox9a expression did not cause cardiac malformations, or defects in epicardium development. We conclude that the decrease in sox9b expression in the heart caused by TCDD plays a role in many of the observed signs of cardiotoxicity. We find that while sox9b is expressed in myocardial cells, it is not normally expressed in the affected epicardial cells or progenitors. We therefore speculate that sox9b is involved in signals between the cardiomyocytes and the nascent epicardial cells. PMID:23775563

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) persistence in the Seveso (Milan, Italy) soil

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cerlesi, S.; Di Domenico, A.; Ratti, S.

1989-10-01

Preliminary results of a new study on TCDD environmental persistence at Seveso (Milan, Italy) are presented. For this study, the most contaminated territory, Zone A, was divided into areas to fractionate the available TCDD levels in soil into data sets with reduced value spreads. In addition, various time subsets were defined for each area. Selected data were fitted with the exponential model y = y0.e-k.1. It was estimated that at least 1.2 kg TCDD was present in Zone A shortly after the accident. On average, a considerable portion (23%) of this amount lay on vegetation; TCDD which was not photodegradedmore » or volatilized before the heavy rains of fall 1976, was later washed off and transferred to ground by water action. From this study, mean analytical underestimations affecting January 1977 and March 1978 contamination map data were on the order of 30 and 24%. All the above figures are considered optimistic. A few years after the accident, mean TCDD half-life in soil appeared to be 9.1y (t1/2-95% CLs, 6.2-17y).« less

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and long term immunologic memory.

PubMed

Sherr, David H

2004-06-01

The highlighted article by B. Paige Lawrence and Beth Vorderstrasse addresses an oft forgotten aspect of immunotoxicity, the effects of environmental toxins on immunologic memory. Here, the authors take a step towards filling that information gap by evaluating the effects of a prototypic environmental toxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on memory responses to a real-world pathogen, influenza A virus, presented to an animal model in a physiologically relevant manner. Multiple outcomes are evaluated, the vast majority of which suggest important and long-term TCDD-induced changes in the immune system after both primary and secondary exposure to this pathogen. The implications of these studies with regard to the immuno-competence of TCDD-exposed individuals are far reaching.

Sex ratio of the offspring of Sprague-Dawley rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in utero and lactationally in a three-generation study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rowlands, J.C.; Budinsky, R.A.; Aylward, L.L.

Reports of a decreased male/female sex ratio in children born to males exposed to TCDD in Seveso, Italy, at a young age have sparked examinations of this endpoint in other populations exposed to TCDD or related compounds. Overall, the male/female sex ratio results reported in these studies, with slightly different age-exposed male populations, have shown mixed results. Experimental studies of the effects of in utero exposure to TCDD in laboratory animals have reported no effect on the f{sub 1} sex ratio and mixed results for the sex ratio of the f{sub 2} generation. In order to better understand the potentialmore » effects of TCDD on second generation sex ratio, we retrieved archived data from a comprehensive three-generation feeding study of TCDD in rats that was conducted and published in the 1970s, but which did not publish data on sex ratio of the offspring [Murray, F.J., Smith, F.A., Nitschke, K.D., Humiston, C.G., Kociba, R.J., Schwetz, B.A., 1979. Three-generation reproduction study of rats given 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the diet. Toxicol. Appl. Pharmacol. 50, 241-252]. A re-examination of the original Murray et al. data found no statistically significant treatment-related changes in postnatal day 1 sex ratio in any generation of treated animals, consistent with one other relatively large study reporting on this endpoint. We discuss mechanistic data underlying a potential effect of TCDD on this endpoint. We conclude that the inconsistency in findings on sex ratio of the offspring of male rats exposed to TCDD in utero is likely due to random variation associated with a relatively small sample size, although differences between studies in strain of rat, dose regimen, and day of ascertainment of sex ratio cannot be ruled out.« less

2, 3, 7, 8-Tetrachlorodibenzo-P-dioxin (TCDD) induces premature senescence in human and rodent neuronal cells via ROS-dependent mechanisms.

PubMed

Wan, Chunhua; Liu, Jiao; Nie, Xiaoke; Zhao, Jianya; Zhou, Songlin; Duan, Zhiqing; Tang, Cuiying; Liang, Lingwei; Xu, Guangfei

2014-01-01

The widespread environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent toxicant that causes significant neurotoxicity. However, the biological events that participate in this process remain largely elusive. In the present study, we demonstrated that TCDD exposure triggered apparent premature senescence in rat pheochromocytoma (PC12) and human neuroblastoma SH-SY5Y cells. Senescence-associated β-galactosidase (SA-β-Gal) assay revealed that TCDD induced senescence in PC12 neuronal cells at doses as low as 10 nM. TCDD led to F-actin reorganization and the appearance of an alternative senescence marker, γ-H2AX foci, both of which are important features of cellular senescence. In addition, TCDD exposure altered the expression of senescence marker proteins, such as p16, p21 and p-Rb, in both dose- and time-dependent manners. Furthermore, we demonstrated that TCDD promotes mitochondrial dysfunction and the accumulation of cellular reactive oxygen species (ROS) in PC12 cells, leading to the activation of signaling pathways that are involved in ROS metabolism and senescence. TCDD-induced ROS generation promoted significant oxidative DNA damage and lipid peroxidation. Notably, treatment with the ROS scavenger N-acetylcysteine (NAC) markedly attenuated TCDD-induced ROS production, cellular oxidative damage and neuronal senescence. Moreover, we found that TCDD induced a similar ROS-mediated senescence response in human neuroblastoma SH-SY5Y cells. In sum, these results demonstrate for the first time that TCDD induces premature senescence in neuronal cells by promoting intracellular ROS production, supporting the idea that accelerating the onset of neuronal senescence may be an important mechanism underlying TCDD-induced neurotoxic effects.

Comparative Study of Folic Acid and α-Naphthoflavone on Reducing TCDD-Induced Cleft Palate in Fetal Mice.

PubMed

Yuan, Xingang; He, Xiaomeng; Zhang, Xuan; Liu, Cuiping; Wang, Chen; Qiu, Lin; Pu, Wei; Fu, Yuexian

2017-03-01

  Tocompare the effect of folic acid (FA) and α-naphthoflavone on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cleft palate in fetal mice.   Pregnant mice were randomly divided into seven groups. The mice treated with corn oil were used as a negative control. The mice in the other six groups were given a single dose of 28 μg/kg TCDD on GD 10 by gavage. For FA treatment, TCDD-treated mice were also dosed with 5, 10, and 15 mg/kg FA on GD 10, while for α-naphthoflavone treatment, the mice received a single dose of 50 μg/kg or 5 mg/kg α-naphthoflavone on GD 10.   Fetal mice palates were imaged using light and scanning electron microscopy on GD 13.5, GD 14.5, and GD 15.5, and cleft palate were recorded on GD 17.5. The expression of guanosine diphosphate dissociation inhibitor (GDI) in fetal mice palate on GD 15.5 was examined by immunohistochemistry.   TCDD successfully induced cleft palate. Ten mg/ml FA and 5 mg/ml α-naphthoflavone significantly reduced TCDD-induced cleft palate. FA and α-naphthoflavone partly reduced TCDD-induced cleft palate but did not affect the expression of Rho GDI.   FA and α-naphthoflavone may reduce the generation of reactive oxygen species, inhibit MEE apoptosis through anti-oxidation, and increase filopodia and MEE movement. This may result in restoration of the ultrastructure of the palatal surface to a normal state, leading to the fusion and formation of complete palate in TCDD-treated fetal mice.

Tissue disposition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in maternal and developing long-evans rats following subchronic exposure.

PubMed

Hurst, C H; DeVito, M J; Birnbaum, L S

2000-10-01

Prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces alterations in the reproductive system of the developing pups. The objective of this study was to determine the disposition of TCDD in maternal and fetal Long-Evans (LE) rats following subchronic exposure, since the adverse reproductive and developmental effects have been extensively characterized in this strain of rat. LE rats were dosed by gavage with 1, 10, or 30 ng [(3)H]TCDD/kg in corn oil, 5 days/week for 13 weeks. At the end of 13 weeks, females were mated and dosing continued every day throughout gestation. Dams were sacrificed on gestation day (GD) 9, GD16, GD21, and post-natal day 4 and analyzed for [(3)H]TCDD-derived activity in maternal and fetal tissues. Maternal body burdens were equivalent at different time points, indicating that the dams were at steady state. Maternal body burdens were approximately 19, 120, and 300 ng TCDD/kg following doses of 1, 10, and 30 ng TCDD/kg, respectively. Individual embryo concentrations on GD9 were 1.6, 7, and 16 pg TCDD/g after maternal exposure of 1, 10, and 30 ng/kg/d, respectively. On GD 16, fetal liver, urogenital tract, head, and body concentrations were similar and averaged 1.4, 7.8, and 16.4 pg TCDD/g after administration of 1, 10, or 30 ng TCDD/kg/d, respectively, indicating no preferential sequestration within the different fetal tissues. These concentrations of TCDD within fetal tissues after subchronic exposure are comparable to those seen after a single dose of 50, 200, or 1000 ng TCDD/kg administered on GD15, a critical period of gestation.

Severe 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) intoxication: kinetics and trials to enhance elimination in two patients.

PubMed

Geusau, Alexandra; Schmaldienst, Sabine; Derfler, Kurt; Päpke, Olaf; Abraham, Klaus

2002-06-01

In spring 1998, two women were diagnosed with severe 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) intoxication. Over the following 3 years, TCDD levels were monitored under various attempts to enhance its elimination, and the half-lives were evaluated. Olestra, a non-digestible, non-absorbable dietary fat substitute, was continuously administered to the patients either as pure substance or in potato-chips. Additionally, in the more severely contaminated patient, we studied whether low-density lipoprotein (LDL)-apheresis, an extracorporeal means of blood lipid elimination, was effective in reducing the TCDD body burden. The blood concentrations initially measured in spring 1998 were 144,000 pg/g blood fat in patient 1 and 26,000 pg/g in patient 2, the highest levels ever measured in adults. In March 2001, concentrations in blood fat were 35,900 and 9,500 pg/g, corresponding to overall elimination half-lives of 560 days (1.5 years) in patient 1 and 1050 days (2.9 years) in patient 2, which are considerably shorter than median values of 7-9 years reported for background and moderate exposure levels. Calculations of the TCDD half-lives and measurements of TCDD elimination via different routes allowed the calculation of an unidentified route of elimination, representing 78 and 62% of the overall elimination in patient 1 and 2, respectively, probably due to an induced hepatic metabolism caused by the high TCDD exposure. As previously reported, administration of olestra was found to be effective in increasing the fecal excretion of TCDD. Due to the short half-lives in our patients, the effect of olestra on the overall elimination was relatively small, but is expected to be much greater for 'normal' half-lives. LDL-apheresis was shown to eliminate TCDD, corresponding to the eliminated blood fat. When employed twice a week, the amount of TCDD excreted by this method was comparable to fecal excretion. In view of costs and time involved, LDL-apheresis does not seem to be

Compound- and sex-specific effects on programming of energy and immune homeostasis in adult C57BL/6JxFVB mice after perinatal TCDD and PCB 153

DOE Office of Scientific and Technical Information (OSTI.GOV)

Esterik, J.C.J. van, E-mail: joantine.van.esterik@rivm.nl; Department of Chemistry and Biology, Institute for Environmental Studies; Verharen, H.W., E-mail: henny.verharen@rivm.nl

Early life exposure to endocrine disrupting compounds has been linked to chronic diseases later in life, like obesity and related metabolic disorders. We exposed C57BL/6JxFVB hybrid mice to the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the constitutive androstane receptor/pregnane X receptor agonist polychlorinated biphenyl 153 (PCB 153) in an experimental design relevant for human exposure. Exposure occurred during gestation and lactation via maternal feed to a wide dose range (TCDD: 10–10,000 pg/kg body weight/day; PCB 153: 0.09–1406 μg/kg body weight/d). Then exposure was ceased and offspring were followed up to 1 year of age. Metabolic parameters like body weight,more » fat pad weights, glucose tolerance, endocrine serum profile, and neurobehavioral and immunological parameters were determined. Body weight was transiently affected by both compounds throughout the follow-up. TCDD-exposed males showed decreased fat pad and spleen weights and an increase in IL-4 production of splenic immune cells. In contrast, females showed increased fat pad weights and production of IFNγ. PCB 153-exposed males showed an increase in glucose, whereas females showed an increase in glucagon, a decrease in pancreas weight, and an increase in thymus weight. In conclusion, early life exposure to TCDD appears to affect programming of energy and immune homeostasis in offspring, whereas the effects of perinatal PCB 153 were mainly on programming of glucose homeostasis. Both compounds act sex-specifically. Lowest derived BMDLs (lower bounds of the (two sided) 90%-confidence interval for the benchmark dose) for both compounds are not lower than current tolerable daily intakes. - Highlights: • Early life exposure to TCDD affects programming of energy and immune homeostasis. • Early life exposure to PCB 153 affects programming of energy homeostasis. • Both compounds act sex-specifically. • Lowest derived BMDLs for both TCDD and PCB 153

TCDD causes stimulation of c-ras expression in the hepatic plasma membranes in vivo and in vitro.

PubMed

Tullis, K; Olsen, H; Bombick, D W; Matsumura, F; Jankun, J

1992-01-01

A series of in vivo and in vitro experiments were conducted to determine the effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) administered on the expression of c-ras. Differences in c-ras expression between control and TCDD treated groups were determined by immunoassay of p21ras protein, or indirectly measured by the specific binding of 3H-GTP to hepatic plasma membrane preparations. Intraperitoneal injection of sublethal doses of TCDD significantly elevated (P less than 0.05, Student t test) levels of hepatic p21ras protein in Sprague-Dawley rats and TCDD sensitive C57BL/6J mice. Such an increase occurred at an early stage of poisoning in the C57BL/6J mice. The earliest increase was detectable 6 hr after dosing, and the difference became statistically significant by 12 and 24 hr after dosing. In contrast, TCDD tolerant DBA/2J mice had only a marginal increase in hepatic p21ras protein which did not become statistically significant even at 24 hr host-dosing. TCDD evoked increases in hepatic p21ras protein of C57BL/6J mice were accompanied by the increase in the specific binding of GTP to hepatic plasma membranes. Column chromatography of solubilized rat hepatic membrane proteins on sephadex G-50 showed TCDD administration increased levels of a 3H-GTP binding protein with MW of approximately 21 Kd. 3H-GTP binding in total hepatic membranes was also elevated (P less than 0.05, Fisher PLSD multiple comparison test) 6 hr and 24 hr after dosing of C57BL/6J mice, but as expected the effect of TCDD was not as conspicuous as that found in the plasma membrane. TCDD treatment increased levels of a 21 Kd protein found in the in vitro translation products of RNA purified from guinea pig liver. This protein was identified as a c-ras protein based upon its ability to bind GTP, precipitation by a polyclonal antibody against the rasHa and Ki proteins and subsequent SDS-PAGE which showed a single protein band of approximately 21 Kd.

Mutations in the Atp1p and Atp3p subunits of yeast ATP synthase differentially affect respiration and fermentation in Saccharomyces cerevisiae.

PubMed

Francis, Brian R; White, Karen H; Thorsness, Peter E

2007-04-01

ATP1-111, a suppressor of the slow-growth phenotype of yme1Delta lacking mitochondrial DNA is due to the substitution of phenylalanine for valine at position 111 of the alpha-subunit of mitochondrial ATP synthase (Atp1p in yeast). The suppressing activity of ATP1-111 requires intact beta (Atp2p) and gamma (Atp3p) subunits of mitochondrial ATP synthase, but not the stator stalk subunits b (Atp4p) and OSCP (Atp5p). ATP1-111 and other similarly suppressing mutations in ATP1 and ATP3 increase the growth rate of wild-type strains lacking mitochondrial DNA. These suppressing mutations decrease the growth rate of yeast containing an intact mitochondrial chromosome on media requiring oxidative phosphorylation, but not when grown on fermentable media. Measurement of chronological aging of yeast in culture reveals that ATP1 and ATP3 suppressor alleles in strains that contain mitochondrial DNA are longer lived than the isogenic wild-type strain. In contrast, the chronological life span of yeast cells lacking mitochondrial DNA and containing these mutations is shorter than that of the isogenic wild-type strain. Spore viability of strains bearing ATP1-111 is reduced compared to wild type, although ATP1-111 enhances the survival of spores that lacked mitochondrial DNA.

TERATOGENIC RESPONSES OF TGFALPHA KNOCKOUT FETUSES TO 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD)

EPA Science Inventory

ABBOTT1, B.D., A.R. BUCKALEW1, and P.L. BRYANT2. 1Reproductive Toxicology Division, EPA, RAP, NC; 2Dept. Environ. Sciences & Engineering, UNC, Chapel Hill, NC. Teratogenic responses of TGF knockout fetuses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

TCDD induces cl...

CHANGES IN EXPRESSION OF PHOSPHORYLATED AND TOTAL ERK 1/2 IN TCDD-EXPOSED EMBRYONIC MOUSE PALATES

EPA Science Inventory

CHANGES IN EXPRESSION OF PHOSPHORYLATED AND TOTAL ERK1/2 IN TCDD-EXPOSED EMBRYONIC MOUSE PALATES.
C Wolf and B Abbott, USEPA, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle Park, NC 27711

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces cleft palate...

Comparison of mathematically-predicted toxic equivalents (TEQs) and bioassay-derived dioxin-equivalents (TCDD-EQs) in heron embryos

USGS Publications Warehouse

Rattner, B.; Hatfield, J.; Melancon, M.; Custer, T.; Tillitt, D.

1995-01-01

Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from an uncontaminated site (Chincoteague National Wildlife Refuge,VA) and three polluted sites (Cat Island, Green Bay, WI; Bair and West Marin Islands, San Francisco Bay, CA). Hepatic microsomal monooxygenases were induced up to 85-fold relative to the reference site, and was associated with concentrations of total PCBs and 11 PCB congeners that are presumed to concern.to express toxicity through the Ah receptor. TEQs [mathematically predicted; summed product of PCB congener concentrations using 5 different sets of toxic equivalency factors (TEFs)] were compared to TCDD-EQs [derived by bioassay; ethoxyresorufin-O-dealkylase activity of treated H411E rat hepatoma cells]. Although TEQs were up to 15-fold greater than TCDD-EQs, the pattern among sites was consistent and TEQs were highly correlated with TCDD-EQs. TEFs based on single congener mammalian studies yielded TEQs that greatly exceeded values from the H411E bioassay of field sample. TEFs generated from avian egg injection studies yielded TEQs that most closely approximated bioassay-derived TCDD-EQs. Cytochrome P450 parameters were related to TEQs and TCDD-EQs; adjusted r2 often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data document the general predictive value of TEQs and TCDD-EQs for P450 induction in field collected samples, but also indicate the need for development of TEFs for the species and biological end point of concern.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) increases necroinflammation and hepatic stellate cell activation but does not exacerbate experimental liver fibrosis in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lamb, Cheri L.; Cholico, Giovan N.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high-affinity ligand for the aryl hydrocarbon receptor (AhR). Increasing evidence indicates that AhR signaling contributes to wound healing, which involves the coordinated deposition and remodeling of the extracellular matrix. In the liver, wound healing is attributed to the activation of hepatic stellate cells (HSCs), which mediate fibrogenesis through the production of soluble mediators and collagen type I. We recently reported that TCDD treatment increases the activation of human HSCs in vitro. The goal of this study was to determine how TCDD impacts HSC activation in vivo using a mouse model of experimentalmore » liver fibrosis. To elicit fibrosis, C57BL6/male mice were treated twice weekly for 8 weeks with 0.5 ml/kg carbon tetrachloride (CCl{sub 4}). TCDD (20 μg/kg) or peanut oil (vehicle) was administered once a week during the last 2 weeks. Results indicate that TCDD increased liver-body-weight ratios, serum alanine aminotransferase activity, and hepatic necroinflammation in CCl{sub 4}-treated mice. Likewise, TCDD treatment increased mRNA expression of HSC activation and fibrogenesis genes, namely α-smooth muscle actin, desmin, delta-like homolog-1, TGF-β1, and collagen type I. However, TCDD treatment did not exacerbate fibrosis, nor did it increase the collagen content of the liver. Instead, TCDD increased hepatic collagenase activity and increased expression of matrix metalloproteinase (MMP)-13 and the matrix regulatory proteins, TIMP-1 and PAI-1. These results support the conclusion that TCDD increases CCl{sub 4}-induced liver damage and exacerbates HSC activation, yet collagen deposition and the development of fibrosis may be limited by TCDD-mediated changes in extracellular matrix remodeling. - Highlights: • TCDD increased liver damage and inflammation in mice treated with CCl{sub 4}. • TCDD treatment enhanced markers of hepatic stellate cell activation and

ITE and TCDD differentially regulate the vascular remodeling of rat placenta via the activation of AhR.

PubMed

Wu, Yanming; Chen, Xiao; Zhou, Qian; He, Qizhi; Kang, Jiuhong; Zheng, Jing; Wang, Kai; Duan, Tao

2014-01-01

Vascular remodeling in the placenta is essential for normal fetal development. The previous studies have demonstrated that in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, an environmental toxicant) induces the intrauterine fetal death in many species via the activation of aryl hydrocarbon receptor (AhR). In the current study, we compared the effects of 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) and TCDD on the vascular remodeling of rat placentas. Pregnant rats on gestational day (GD) 15 were randomly assigned into 5 groups, and were exposed to a single dose of 1.6 and 8.0 mg/kg body weight (bw) ITE, 1.6 and 8.0 µg/kg bw TCDD, or an equivalent volume of the vehicle, respectively. The dams were sacrificed on GD20 and the placental tissues were gathered. The intrauterine fetal death was observed only in 8.0 µg/kg bw TCDD-exposed group and no significant difference was seen in either the placental weight or the fetal weight among all these groups. The immunohistochemical and histological analyses revealed that as compared with the vehicle-control, TCDD, but not ITE, suppressed the placental vascular remodeling, including reduced the ratio of the placental labyrinth zone to the basal zone thickness (at least 0.71 fold of control), inhibited the maternal sinusoids dilation and thickened the trophoblastic septa. However, no marked difference was observed in the density of fetal capillaries in the labyrinth zone among these groups, although significant differences were detected in the expression of angiogenic growth factors between ITE and TCDD-exposed groups, especially Angiopoietin-2 (Ang-2), Endoglin, Interferon-γ (IFN-γ) and placenta growth factor (PIGF). These results suggest ITE and TCDD differentially regulate the vascular remodeling of rat placentas, as well as the expression of angiogenic factors and their receptors, which in turn may alter the blood flow in the late gestation and partially resulted in

ITE and TCDD Differentially Regulate the Vascular Remodeling of Rat Placenta via the Activation of AhR

PubMed Central

Zhou, Qian; He, Qizhi; Kang, Jiuhong; Zheng, Jing; Wang, Kai; Duan, Tao

2014-01-01

Vascular remodeling in the placenta is essential for normal fetal development. The previous studies have demonstrated that in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, an environmental toxicant) induces the intrauterine fetal death in many species via the activation of aryl hydrocarbon receptor (AhR). In the current study, we compared the effects of 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) and TCDD on the vascular remodeling of rat placentas. Pregnant rats on gestational day (GD) 15 were randomly assigned into 5 groups, and were exposed to a single dose of 1.6 and 8.0 mg/kg body weight (bw) ITE, 1.6 and 8.0 µg/kg bw TCDD, or an equivalent volume of the vehicle, respectively. The dams were sacrificed on GD20 and the placental tissues were gathered. The intrauterine fetal death was observed only in 8.0 µg/kg bw TCDD-exposed group and no significant difference was seen in either the placental weight or the fetal weight among all these groups. The immunohistochemical and histological analyses revealed that as compared with the vehicle-control, TCDD, but not ITE, suppressed the placental vascular remodeling, including reduced the ratio of the placental labyrinth zone to the basal zone thickness (at least 0.71 fold of control), inhibited the maternal sinusoids dilation and thickened the trophoblastic septa. However, no marked difference was observed in the density of fetal capillaries in the labyrinth zone among these groups, although significant differences were detected in the expression of angiogenic growth factors between ITE and TCDD-exposed groups, especially Angiopoietin-2 (Ang-2), Endoglin, Interferon-γ (IFN-γ) and placenta growth factor (PIGF). These results suggest ITE and TCDD differentially regulate the vascular remodeling of rat placentas, as well as the expression of angiogenic factors and their receptors, which in turn may alter the blood flow in the late gestation and partially resulted in

COMPARATIVE SENSITIVITY OF DIFFERENT LIFE-STAGES OF MEDAKA AND SALMONID FISHES TO 2,3,7,8-TCDD

EPA Science Inventory

The early life stages of fish are known to be more sensitive than the adults to the toxicological effects of 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD). Embryo larval stages of medake, for example, experience 50% lethality when TCDD residues in the eggs are 1396 pg/g. By contrast...

Hepatic transcriptomic responses to TCDD in dioxin-sensitive and dioxin-resistant rats during the onset of toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boutros, Paul C.; Yao, Cindy Q.; Watson, John D.

2011-03-01

The dioxin congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a wide range of toxic effects in rodent species, all of which are mediated by a ligand-dependent transcription-factor, the aryl hydrocarbon receptor (AHR). The Han/Wistar (Kuopio) (H/W) strain shows exceptional resistance to many TCDD-induced toxicities; the LD{sub 50} of > 9600 {mu}g/kg for H/W rats is higher than for any other wild-type mammal known. We previously showed that this resistance primarily results from H/W rats expressing a variant AHR isoform that has a substantial portion of the AHR transactivation domain deleted. Despite this large deletion, H/W rats are not entirely refractory to the effectsmore » of TCDD; the variant AHR in these animals remains fully competent to up-regulate well-known dioxin-inducible genes. TCDD-sensitive (Long-Evans, L-E) and resistant (H/W) rats were treated with either corn-oil (with or without feed-restriction) or 100 {mu}g/kg TCDD for either four or ten days. Hepatic transcriptional profiling was done using microarrays, and was validated by RT-PCR analysis of 41 genes. A core set of genes was altered in both strains at all time points tested, including CYP1A1, CYP1A2, CYP1B1, Nqo1, Aldh3a1, Tiparp, Exoc3, and Inmt. Outside this core, the strains differed significantly in the breadth of response: three-fold more genes were altered in L-E than H/W rats. At ten days almost all expressed genes were dysregulated in L-E rats, likely reflecting emerging toxic responses. Far fewer genes were affected by feed-restriction, suggesting that only a minority of the TCDD-induced changes are secondary to the wasting syndrome.« less

EFFECTS OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) ON FETAL MOUSE URINARY TRACT EPITHELIUM IN VITRO

EPA Science Inventory

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produces hydronephrosis by altering the differentiation and proliferation of ureteric epithelial cells in the embryonic C57BL/6N mouse urinary tract. This study examines the effects of TCDD on late gestation fetal urinary tract cells u...

Toxicogenomic investigation of Tetrahymena thermophila exposed to dichlorodiphenyltrichloroethane (DDT), tributyltin (TBT), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

PubMed

Chang, Yue; Feng, LiFang; Miao, Wei

2011-07-01

Dichlorodiphenyltrichloroethane (DDT), tributyltin (TBT), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are persistent in the environment and cause continuous toxic effects in humans and aquatic life. Tetrahymena thermophila has the potential for use as a model for research regarding toxicants. In this study, this organism was used to analyze a genome-wide microarray generated from cells exposed to DDT, TBT and TCDD. To accomplish this, genes differentially expressed when treated with each toxicant were identified, after which their functions were categorized using GO enrichment analysis. The results suggested that the responses of T. thermophila were similar to those of multicellular organisms. Additionally, the context likelihood of relatedness method (CLR) was applied to construct a TCDD-relevant network. The T-shaped network obtained could be functionally divided into two subnetworks. The general functions of both subnetworks were related to the epigenetic mechanism of TCDD. Based on analysis of the networks, a model of the TCDD effect on T. thermophila was inferred. Thus, Tetrahymena has the potential to be a good unicellular eukaryotic model for toxic mechanism research at the genome level.

Effects of selected food phytochemicals in reducing the toxic actions of TCDD and p,p′-DDT in U937 macrophages

PubMed Central

Sciullo, Eric M.; Vogel, Christoph F.; Wu, Dalei; Murakami, Akira; Ohigashi, Hajime

2010-01-01

To assess the effectiveness of selected food phytochemicals in reducing the toxic effects of the environmental toxicants, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and p,p′-DDT (DDT), we tested the potencies of auraptene, nobiletin, zerumbone, and (±)-13-hydroxy-10-oxo-trans-11-octadecenoic acid (13-HOA) in reversing the inflammatory action of these toxicants in U937 human macrophages. Using quantitative RT–PCR as the initial screening assay, we identified antagonistic actions of zerumbone and auraptene against the action of TCDD and DDT in up-regulating the mRNA expressions of COX-2 and VEGF. The functional significance of the inhibitory action of zerumbone on COX-2 expression was confirmed by demonstrating its suppression of TCDD-induced activation of COX-2 gene expression in mouse MMDD1 cells. We tested auraptene on DDT-induced reactive oxygen species (ROS) formation in U937 macrophages and found that auraptene is a powerful agent antagonizing this action of DDT. To confirm the significance of these actions of zerumbone and auraptene at the cellular level, we assessed their influence on TCDD-induced apoptosis resistance in intact U937 macrophages and found that they are capable of reversing this action of TCDD. In conclusion, zerumbone and auraptene were identified to be the most effective agents in protecting U937 macrophages from developing these cell toxic effects of TCDD and DDT. PMID:20865247

Compound- and sex-specific effects on programming of energy and immune homeostasis in adult C57BL/6JxFVB mice after perinatal TCDD and PCB 153.

PubMed

van Esterik, J C J; Verharen, H W; Hodemaekers, H M; Gremmer, E R; Nagarajah, B; Kamstra, J H; Dollé, M E T; Legler, J; van der Ven, L T M

2015-12-01

Early life exposure to endocrine disrupting compounds has been linked to chronic diseases later in life, like obesity and related metabolic disorders. We exposed C57BL/6JxFVB hybrid mice to the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the constitutive androstane receptor/pregnane X receptor agonist polychlorinated biphenyl 153 (PCB 153) in an experimental design relevant for human exposure. Exposure occurred during gestation and lactation via maternal feed to a wide dose range (TCDD: 10-10,000 pg/kg body weight/day; PCB 153: 0.09-1406 μg/kg body weight/d). Then exposure was ceased and offspring were followed up to 1 year of age. Metabolic parameters like body weight, fat pad weights, glucose tolerance, endocrine serum profile, and neurobehavioral and immunological parameters were determined. Body weight was transiently affected by both compounds throughout the follow-up. TCDD-exposed males showed decreased fat pad and spleen weights and an increase in IL-4 production of splenic immune cells. In contrast, females showed increased fat pad weights and production of IFNγ. PCB 153-exposed males showed an increase in glucose, whereas females showed an increase in glucagon, a decrease in pancreas weight, and an increase in thymus weight. In conclusion, early life exposure to TCDD appears to affect programming of energy and immune homeostasis in offspring, whereas the effects of perinatal PCB 153 were mainly on programming of glucose homeostasis. Both compounds act sex-specifically. Lowest derived BMDLs (lower bounds of the (two sided) 90%-confidence interval for the benchmark dose) for both compounds are not lower than current tolerable daily intakes.

Comparison of TCDD-elicited genome-wide hepatic gene expression in Sprague–Dawley rats and C57BL/6 mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nault, Rance; Kim, Suntae; Zacharewski, Timothy R., E-mail: tzachare@msu.edu

2013-03-01

Although the structure and function of the AhR are conserved, emerging evidence suggests that downstream effects are species-specific. In this study, rat hepatic gene expression data from the DrugMatrix database (National Toxicology Program) were compared to mouse hepatic whole-genome gene expression data following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For the DrugMatrix study, male Sprague–Dawley rats were gavaged daily with 20 μg/kg TCDD for 1, 3 and 5 days, while female C57BL/6 ovariectomized mice were examined 1, 3 and 7 days after a single oral gavage of 30 μg/kg TCDD. A total of 649 rat and 1386 mouse genes (|fold change| ≥more » 1.5, P1(t) ≥ 0.99) were differentially expressed following treatment. HomoloGene identified 11,708 orthologs represented across the rat Affymetrix 230 2.0 GeneChip (12,310 total orthologs), and the mouse 4 × 44K v.1 Agilent oligonucleotide array (17,578 total orthologs). Comparative analysis found 563 and 922 orthologs differentially expressed in response to TCDD in the rat and mouse, respectively, with 70 responses associated with immune function and lipid metabolism in common to both. Moreover, QRTPCR analysis of Ceacam1, showed divergent expression (induced in rat; repressed in mouse) functionally consistent with TCDD-elicited hepatic steatosis in the mouse but not the rat. Functional analysis identified orthologs involved in nucleotide binding and acetyltransferase activity in rat, while mouse-specific responses were associated with steroid, phospholipid, fatty acid, and carbohydrate metabolism. These results provide further evidence that TCDD elicits species-specific regulation of distinct gene networks, and outlines considerations for future comparisons of publicly available microarray datasets. - Highlights: ► We performed a whole-genome comparison of TCDD-regulated genes in mice and rats. ► Previous species comparisons were extended using data from the DrugMatrix database. ► Less than 15

LncRNA H19 and Target Gene-mediated Cleft Palate Induced by TCDD.

PubMed

Gao, Li Yun; Zhang, Feng Quan; Zhao, Wei Hui; Han, Guang Liang; Wang, Xiao; Li, Qiang; Gao, Shan Shan; Wu, Wei Dong

2017-09-01

This study investigated the role of long non-coding RNAs (lncRNAs) in the development of the palatal tissues. Cleft palates in mice were induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Expression levels of long non-coding RNA H19 (lncRNA H19) and insulin-like growth factor 2 (IGF2) gene were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The rate of occurrence of cleft palate was found to be 100% by TCDD exposure, and TCDD could cause short upper limb, cerebral fissure, webbed neck, and short neck. The expression levels of lncRNA H19 and IGF2 gene specifically showed embryo age-related differences on E13, E14, and E15 in the palatal tissues. The expression levels of lncRNA H19 and IGF2 gene showed an inverse relationship on E13, E14, and E15. These findings demonstrated that lncRNA H19 and IGF2 can mediate the development of mouse cleft palate. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

The tertiary structures of porcine AhR and ARNT proteins and molecular interactions within the TCDD/AhR/ARNT complex.

PubMed

Orlowska, Karina; Molcan, Tomasz; Swigonska, Sylwia; Sadowska, Agnieszka; Jablonska, Monika; Nynca, Anna; Jastrzebski, Jan P; Ciereszko, Renata E

2016-06-01

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse synthetic and natural chemicals, including toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In the present study, homology models of the porcine AhR-ligand binding domain (LBD) and the porcine aryl hydrocarbon receptor nuclear translocator-ligand binding domain (ARNT-LBD) were created on the basis of structures of closely related respective proteins i.e., human Hif-2α and ARNT. Molecular docking of TCDD to the porcine AhR-LBD model revealed high binding affinity (-8.8kcal/mol) between TCDD and the receptor. Moreover, formation of the TCDD/AhR-LBD complex was confirmed experimentally with the use of electrophoretic mobility shift assay (EMSA). It was found that TCDD (10nM, 2h of incubation) not only bound to the AhR in the porcine granulosa cells but also activated the receptor. The current study provides a framework for examining the key events involved in the ligand-dependent activation of the AhR. Copyright © 2016 Elsevier Inc. All rights reserved.

TCDD, FICZ, and Other High Affinity AhR Ligands Dose-Dependently Determine the Fate of CD4+ T Cell Differentiation.

PubMed

Ehrlich, Allison K; Pennington, Jamie M; Bisson, William H; Kolluri, Siva K; Kerkvliet, Nancy I

2018-02-01

FICZ and TCDD, two high-affinity AhR ligands, are reported to have opposite effects on T cell differentiation with TCDD inducing regulatory T cells and FICZ inducing Th17 cells. This dichotomy has been attributed to ligand-intrinsic differences in AhR activation, although differences in sensitivity to metabolism complicate the issue. TCDD is resistant to AhR-induced metabolism and produces sustained AhR activation following a single dose in the μg/kg range, whereas FICZ is rapidly metabolized and AhR activation is transient. Nonetheless, prior studies comparing FICZ with TCDD have generally used the same 10-50 μg/kg dose range, and thus the two ligands would not equivalently activate AhR. We hypothesized that high-affinity AhR ligands can promote CD4+ T cell differentiation into both Th17 cells and Tregs, with fate depending on the extent and duration of AhR activation. We compared the immunosuppressive effects of TCDD and FICZ, along with two other rapidly metabolized ligands (ITE and 11-Cl-BBQ) in an acute alloresponse mouse model. The dose and timing of administration of each ligand was optimized for TCDD-equivalent Cyp1a1 induction. When optimized, all of the ligands suppressed the alloresponse in conjunction with the induction of Foxp3- Tr1 cells on day 2 and the expansion of natural Foxp3+ Tregs on day 10. In contrast, a low dose of FICZ induced transient expression of Cyp1a1 and did not induce Tregs or suppress the alloresponse but enhanced IL-17 production. Interestingly, low doses of the other ligands, including TCDD, also increased IL-17 production on day 10. These findings support the conclusion that the dose and the duration of AhR activation by high-affinity AhR ligands are the primary factors driving the fate of T cell differentiation. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-mediated deregulation of myeloid and sebaceous gland stem/progenitor cell homeostasis.

PubMed

Bock, Karl Walter

2017-06-01

Studies of TCDD toxicity stimulated identification of the responsible aryl hydrocarbon receptor (AHR), a multifunctional, ligand-activated transcription factor of the basic helix-loop-helix/Per-Arnt-Sim family. Accumulating evidence suggests a role of this receptor in homeostasis of stem/progenitor cells, in addition to its known role in xenobiotic metabolism. (1) Regulation of myelopoiesis is complex. As one example, AHR-mediated downregulation of human CD34+ progenitor differentiation to monocytes/macrophages is discussed. (2) Accumulation of TCDD in sebum leads to deregulation of sebocyte differentiation via Blimp1-mediated inhibition of c-Myc signaling and stimulation of Wnt-mediated proliferation of interfollicular epidermis. The resulting sebaceous gland atrophy and formation of dermal cysts may explain the pathogenesis of chloracne, the hallmark of TCDD toxicity. (3) TCDD treatment of confluent liver stem cell-like rat WB-F344 cells leads to release from cell-cell contact inhibition via AHR-mediated crosstalk with multiple signaling pathways. Further work is needed to delineate AHR function in crosstalk with other signaling pathways.

Immunological characterization of the aryl hydrocarbon receptor (AHR) knockout rat in the presence and absence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

PubMed

Phadnis-Moghe, Ashwini S; Chen, Weimin; Li, Jinpeng; Crawford, Robert B; Bach, Anthony; D'Ingillo, Shawna; Kovalova, Natalia; Suarez-Martinez, Jose E; Kaplan, Barbara L F; Harrill, Joshua A; Budinsky, Robert; Rowlands, J Craig; Thomas, Russell S; Kaminski, Norbert E

2016-08-10

The aryl hydrocarbon receptor (AHR) has been extensively characterized for the essential role it plays in mediating the toxic responses elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Despite similarities across animal species, species-specific differences exist in the profile of toxicity and sensitivity to TCDD owing, in part, to differences in the AHR. Newer reports have implicated the importance of AHR in the development and regulation of the immune system. Our present studies seek to further explore the essential role of AHR in lymphoid tissue composition, B cell function and the immunological responses after TCDD administration using the recently established AHR KO rats. Comprehensive immune cell phenotyping showed a decrease in the CD8 + T cell, CD11c + populations and an increase in NKT cells in 3-week-old AHR KO rats compared to the WT controls. The lipopolysaccharide-induced IgM response and proliferation was markedly suppressed in the WT but not in the AHR KO B cells in the presence of TCDD. However, the percentage of LPS-activated IgM + B cells was significantly higher in the AHR KO B cells as compared to that of WT suggesting the role of AHR in regulating the IgM response. The use of an AHR antagonist further alluded to the endogenous role of AHR in regulating B cell responses in the rat. Overall, the studies report for the first time, comprehensive immune cell phenotyping of the AHR KO rat and the endogenous role of AHR in the regulation of B cell function in the rat. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

HYDRONEPHROSIS IN MICE EXPOSED TO TCDD-CONTAMINATED BREAST MILK: IDENTIFICATION OF THE PEAK PERIOD OF SENSITIVITY AND ASSESSMENT OF POTENTIAL RECOVERY

EPA Science Inventory

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent inducer of hydronephrosis in mice both pre- and post-natally. ritical period of sensitivity to TCDD could not be identified for the hydronephrotic response induced prenatally since the urinary tract appeared equally sensitive...

Comparisons of the effects of TCDD and hydrocortisone on growth factor expression provide insight into their interaction in the embryonic mouse palate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abbott, B.D.; Harris, M.W.; Birnbaum, L.S.

Cleft palate (CP) can be induced in embryonic mice by a wide range of compounds, including glucocorticoids and 2,3,7,8-tyetrachlorodibenzo-p-dioxin (TCDD). Hydrocortisone (HC), a glucocorticoid, retards embryonic growth producing small palatal shelves, while TCDD exposure blocks the fusion of normally sized shelves. TCDD induction of CP involves altered differentiation of the medial epithelial cells. Recent studies indicate that growth factors such as EGF, TGF-alpha, TGF-beta1, and TGF-beta2 are involved in palatogenesis, regulating proliferation, differentiation, and extracellular matrix production. A synergism has been observed between HC and TCDD in which doses too low to induce CP alone are able to produce >90%more » incidence when coadministered. In the present study a standard teratology protocol was performed in C57BL/6N mice to examine the synergism at doses lower than those previously published. Data from the study indicate synergistic interactions at doses as low as 3 micrograms TCDD/kg + 1 mg HC/kg. This extreme sensitivity suggests the involvement of a receptor-mediated mechanism possibly resulting in altered regulation of gene expression. (Copyright (c) 1992 Wiley-Liss, Inc.)« less

Behavior and stability of adenosine triphosphate (ATP) during chlorine disinfection.

PubMed

Nescerecka, Alina; Juhna, Talis; Hammes, Frederik

2016-09-15

Adenosine triphosphate (ATP) analysis is a cultivation-independent alternative method for the determination of bacterial viability in both chlorinated and non-chlorinated water. Here we investigated the behavior and stability of ATP during chlorination in detail. Different sodium hypochlorite doses (0-22.4 mg-Cl2 L(-1); 5 min exposure) were applied to an Escherichia coli pure culture suspended in filtered river water. We observed decreasing intracellular ATP with increasing chlorine concentrations, but extracellular ATP concentrations only increased when the chlorine dose exceeded 0.35 mg L(-1). The release of ATP from chlorine-damaged bacteria coincided with severe membrane damage detected with flow cytometry (FCM). The stability of extracellular ATP was subsequently studied in different water matrixes, and we found that extracellular ATP was stable in sterile deionized water and also in chlorinated water until extremely high chlorine doses (≤11.2 mg-Cl2 L(-1); 5 min exposure). In contrast, ATP decreased relatively slowly (k = 0.145 h(-1)) in 0.1 μm filtered river water, presumably due to degradation by either extracellular enzymes or the fraction of bacteria that were able to pass through the filter. Extracellular ATP decreased considerably faster (k = 0.368 h(-1)) during batch growth of a river water bacterial community. A series of growth potential tests showed that extracellular ATP molecules were utilized as a phosphorus source during bacteria proliferation. From the combined data we conclude that ATP released from bacteria at high chlorine doses could promote bacteria regrowth, contributing to biological instability in drinking water distribution systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

TCDD-MEDIATED OXIDATIVE STRESS IN MALE RAT PUPS FOLLOWING PERINATAL EXPOSURE

EPA Science Inventory

TCDD is a highly persistent trace environmental contaminant and is one of the most potent toxicants known to man. Our laboratory has previously reported an increase in the production of reactive oxygen species (ROS) in the brain of female B6C3F1 mice following subchronic exposur...

Clusterin and COMMD1 Independently Regulate Degradation of the Mammalian Copper ATPases ATP7A and ATP7B*

PubMed Central

Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

2012-01-01

ATP7A and ATP7B are copper-transporting P1B-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and COMMD1 were previously identified as interacting partners of these Cu-ATPases. In this study, we confirmed that clusterin and COMMD1 interact to down-regulate both ATP7A and ATP7B. Overexpression and knockdown of clusterin/COMMD1 decreased and increased, respectively, endogenous levels of ATP7A and ATP7B, consistent with a role in facilitating Cu-ATPase degradation. We demonstrate that whereas the clusterin/ATP7B interaction was enhanced by oxidative stress or mutation of ATP7B, the COMMD1/ATP7B interaction did not change under oxidative stress conditions, and only increased with ATP7B mutations that led to its misfolding. Clusterin and COMMD1 facilitated the degradation of ATP7B containing the same Wilson disease-causing C-terminal mutations via different degradation pathways, clusterin via the lysosomal pathway and COMMD1 via the proteasomal pathway. Furthermore, endogenous ATP7B existed in a complex with clusterin and COMMD1, but these interactions were neither competitive nor cooperative and occurred independently of each other. Together these data indicate that clusterin and COMMD1 represent alternative and independent systems regulating Cu-ATPase quality control, and consequently contributing to the maintenance of copper homeostasis. PMID:22130675

COMPARATIVE SENSITIVITY OF DIFFERENT LIFE-STAGES OF MEDAKA (ORYZIAS LATIPES) AND BROOK TROUT (SALVELINUS FONTINALIS) TO 2,3,7,8-TCDD

EPA Science Inventory

The early life stages of fish are known to be more sensitive than the adults to the toxicological effects of 2,3,7,8-tetrachlorodibenzo(p)dioxide (TCDD). TCDD concentrations in surface waters are sufficiently low that direct exposure of the developing embryo is unlikely to be o...

Magnetic field affects enzymatic ATP synthesis.

PubMed

Buchachenko, Anatoly L; Kuznetsov, Dmitry A

2008-10-01

The rate of ATP synthesis by creatine kinase extracted from V. xanthia venom was shown to depend on the magnetic field. The yield of ATP produced by enzymes with 24Mg2+ and 26Mg2+ ions in catalytic sites increases by 7-8% at 55 mT and then decreases at 80 mT. For enzyme with 25Mg2+ ion in a catalytic site, the ATP yield increases by 50% and 70% in the fields 55 and 80 mT, respectively. In the Earth field the rate of ATP synthesis by enzyme, in which Mg2+ ion has magnetic nucleus 25Mg, is 2.5 times higher than that by enzymes, in which Mg2+ ion has nonmagnetic, spinless nuclei 24Mg or 26Mg. Both magnetic field effect and magnetic isotope effect demonstrate that the ATP synthesis is an ion-radical process, affected by Zeeman interaction and hyperfine coupling in the intermediate ion-radical pair.

Treatment of heterotopic ossification through remote ATP hydrolysis.

PubMed

Peterson, Jonathan R; De La Rosa, Sara; Eboda, Oluwatobi; Cilwa, Katherine E; Agarwal, Shailesh; Buchman, Steven R; Cederna, Paul S; Xi, Chuanwu; Morris, Michael D; Herndon, David N; Xiao, Wenzhong; Tompkins, Ronald G; Krebsbach, Paul H; Wang, Stewart C; Levi, Benjamin

2014-09-24

Heterotopic ossification (HO) is the pathologic development of ectopic bone in soft tissues because of a local or systemic inflammatory insult, such as burn injury or trauma. In HO, mesenchymal stem cells (MSCs) are inappropriately activated to undergo osteogenic differentiation. Through the correlation of in vitro assays and in vivo studies (dorsal scald burn with Achilles tenotomy), we have shown that burn injury enhances the osteogenic potential of MSCs and causes ectopic endochondral heterotopic bone formation and functional contractures through bone morphogenetic protein-mediated canonical SMAD signaling. We further demonstrated a prevention strategy for HO through adenosine triphosphate (ATP) hydrolysis at the burn site using apyrase. Burn site apyrase treatment decreased ATP, increased adenosine 3',5'-monophosphate, and decreased phosphorylation of SMAD1/5/8 in MSCs in vitro. This ATP hydrolysis also decreased HO formation and mitigated functional impairment in vivo. Similarly, selective inhibition of SMAD1/5/8 phosphorylation with LDN-193189 decreased HO formation and increased range of motion at the injury site in our burn model in vivo. Our results suggest that burn injury-exacerbated HO formation can be treated through therapeutics that target burn site ATP hydrolysis and modulation of SMAD1/5/8 phosphorylation. Copyright © 2014, American Association for the Advancement of Science.

POSTNATAL DISPOSITION OF TCDD IN LONG EVANS RATS FOLLOWING GESTATIONAL EXPOSURE

EPA Science Inventory

POSTNATAL DISPOSITION OF TCDD IN LONG EVANS RATS FOLLOWING GESTATIONAL EXPOSURE.
J J Diliberto', J T Hamm'.2, F McQuaid', and L S Birnbaum'. 'US EPA, ORD/NHEERL/ETD, RTP, NC; 2Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC.
2,3,7,8-Tetrachlorodibenz...

Exposure to the environmental endocrine disruptor TCDD and human reproductive dysfunction: Translating lessons from murine models.

PubMed

Bruner-Tran, Kaylon L; Gnecco, Juan; Ding, Tianbing; Glore, Dana R; Pensabene, Virginia; Osteen, Kevin G

2017-03-01

Humans and other animals are exposed to a wide array of man-made toxicants, many of which act as endocrine disruptors that exhibit differential effects across the lifespan. In humans, while the impact of adult exposure is known for some compounds, the potential consequences of developmental exposure to endocrine disrupting chemicals (EDCs) is more difficult to ascertain. Animal studies have revealed that exposure to EDCs prior to puberty can lead to adult reproductive disease and dysfunction. Specifically, in adult female mice with an early life exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we demonstrated a transgenerational occurrence of several reproductive diseases that have been linked to endometriosis in women. Herein, we review the evidence for TCDD-associated development of adult reproductive disease as well as known epigenetic alterations associated with TCDD and/or endometriosis. We will also introduce new "Organ-on-Chip" models which, combined with our established murine model, are expected to further enhance our ability to examine alterations in gene-environment interactions that lead to heritable disease. Copyright © 2016 Elsevier Inc. All rights reserved.

Exposure to the Environmental Endocrine Disruptor TCDD and Human Reproductive Dysfunction: Translating Lessons from Murine Models

PubMed Central

Bruner-Tran, Kaylon L.; Gnecco, Juan; Ding, Tianbing; Glore, Dana R.; Pensabene, Virginia; Osteen, Kevin G.

2016-01-01

Humans and other animals are exposed to a wide array of man-made toxicants, many of which act as endocrine disruptors that exhibit differential effects across the lifespan. In humans, while the impact of adult exposure is known for some compounds, the potential consequences of developmental exposure to endocrine disrupting chemicals (EDCs) is more difficult to ascertain. Animal studies have revealed that exposure to EDCs prior to puberty can lead to adult reproductive disease and dysfunction. Specifically, in adult female mice with an early life exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we demonstrated a transgenerational occurrence of several reproductive diseases that have been linked to endometriosis in women. Herein, we review the evidence for TCDD-associated development of adult reproductive disease as well as known epigenetic alterations associated with TCDD and/or endometriosis. We will also introduce new “Organ-on-Chip” models which, combined with our established murine model, are expected to further enhance our ability to examine alterations in gene-environment interactions that lead to heritable disease. PMID:27423904

General anesthetics cause mitochondrial dysfunction and reduction of intracellular ATP levels

PubMed Central

Kishikawa, Jun-ichi; Inoue, Yuki; Fujikawa, Makoto; Nishimura, Kenji; Nakanishi, Atsuko; Tanabe, Tsutomu; Imamura, Hiromi

2018-01-01

General anesthetics are indispensable for effective clinical care. Although, the mechanism of action of general anesthetics remains controversial, lipid bilayers and proteins have been discussed as their targets. In this study, we focused on the relationship between cellular ATP levels and general anesthetics. The ATP levels of nematodes and cultured mammalian cells were decreased by exposure to three general anesthetics: isoflurane, pentobarbital, and 1-phenoxy-2-propanol. Furthermore, these general anesthetics abolished mitochondrial membrane potential, resulting in the inhibition of mitochondrial ATP synthesis. These results suggest that the observed decrease of cellular ATP level is a common phenomenon of general anesthetics. PMID:29298324

Induction of a chloracne phenotype in an epidermal equivalent model by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on aryl hydrocarbon receptor activation and is not reproduced by aryl hydrocarbon receptor knock down.

PubMed

Forrester, Alison R; Elias, Martina S; Woodward, Emma L; Graham, Mark; Williams, Faith M; Reynolds, Nick J

2014-01-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent activator of the aryl hydrocarbon receptor (AhR) and causes chloracne in humans. The pathogenesis and role of AhR in chloracne remains incompletely understood. To elucidate the mechanisms contributing to the development of the chloracne-like phenotype in a human epidermal equivalent model and identify potential biomarkers. Using primary normal human epidermal keratinocytes (NHEK), we studied AhR activation by XRE-luciferase, AhR degradation and CYP1A1 induction. We treated epidermal equivalents with high affinity TCDD or two non-chloracnegens: β-naphthoflavone (β-NF) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Using Western blotting and immunochemistry for filaggrin (FLG), involucrin (INV) and transglutaminase-1 (TGM-1), we compared the effects of the ligands on keratinocyte differentiation and development of the chloracne-like phenotype by H&E. In NHEKs, activation of an XRE-luciferase and CYP1A1 protein induction correlated with ligand binding affinity: TCDD>β-NF>ITE. AhR degradation was induced by all ligands. In epidermal equivalents, TCDD induced a chloracne-like phenotype, whereas β-NF or ITE did not. All three ligands induced involucrin and TGM-1 protein expression in epidermal equivalents whereas FLG protein expression decreased following treatment with TCDD and β-NF. Inhibition of AhR by α-NF blocked TCDD-induced AhR activation in NHEKs and blocked phenotypic changes in epidermal equivalents; however, AhR knock down did not reproduce the phenotype. Ligand-induced CYP1A1 and AhR degradation did not correlate with their chloracnegenic potential, indicating that neither CYP1A1 nor AhR are suitable biomarkers. Mechanistic studies showed that the TCDD-induced chloracne-like phenotype depends on AhR activation whereas AhR knock down did not appear sufficient to induce the phenotype. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier

TCDD and a putative endogenous AhR ligand, ITE, elicit the same immediate changes in gene expression in mouse lung fibroblasts.

PubMed

Henry, Ellen C; Welle, Stephen L; Gasiewicz, Thomas A

2010-03-01

The aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, mediates toxicity of several classes of xenobiotics and also has important physiological roles in differentiation, reproduction, and immunity, although the endogenous ligand(s) mediating these functions is/are as yet unidentified. One candidate endogenous ligand, 2-(1'H-indolo-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), is a potent AhR agonist in vitro, activates the murine AhR in vivo, but does not induce toxicity. We hypothesized that ITE and the toxic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), may modify transcription of different sets of genes to account for their different toxicity. To test this hypothesis, primary mouse lung fibroblasts were exposed to 0.5muM ITE, 0.2nM TCDD, or vehicle for 4 h, and total gene expression was evaluated using microarrays. After this short-term and low-dose treatment, several hundred genes were changed significantly, and the response to ITE and TCDD was remarkably similar, both qualitatively and quantitatively. Induced gene sets included the expected battery of AhR-dependent xenobiotic-metabolizing enzymes, as well as several sets that reflect the inflammatory role of lung fibroblasts. Real time quantitative RT-qPCR assay of several selected genes confirmed these microarray data and further suggested that there may be kinetic differences in expression between ligands. These data suggest that ITE and TCDD elicit an analogous change in AhR conformation such that the initial transcription response is the same. Furthermore, if the difference in toxicity between TCDD and ITE is mediated by differences in gene expression, then it is likely that secondary changes enabled by the persistent TCDD, but not by the shorter lived ITE, are responsible.

TCDD and a Putative Endogenous AhR Ligand, ITE, Elicit the Same Immediate Changes in Gene Expression in Mouse Lung Fibroblasts

PubMed Central

Henry, Ellen C.; Welle, Stephen L.; Gasiewicz, Thomas A.

2010-01-01

The aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, mediates toxicity of several classes of xenobiotics and also has important physiological roles in differentiation, reproduction, and immunity, although the endogenous ligand(s) mediating these functions is/are as yet unidentified. One candidate endogenous ligand, 2-(1′H-indolo-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), is a potent AhR agonist in vitro, activates the murine AhR in vivo, but does not induce toxicity. We hypothesized that ITE and the toxic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), may modify transcription of different sets of genes to account for their different toxicity. To test this hypothesis, primary mouse lung fibroblasts were exposed to 0.5μM ITE, 0.2nM TCDD, or vehicle for 4 h, and total gene expression was evaluated using microarrays. After this short-term and low-dose treatment, several hundred genes were changed significantly, and the response to ITE and TCDD was remarkably similar, both qualitatively and quantitatively. Induced gene sets included the expected battery of AhR-dependent xenobiotic-metabolizing enzymes, as well as several sets that reflect the inflammatory role of lung fibroblasts. Real time quantitative RT-qPCR assay of several selected genes confirmed these microarray data and further suggested that there may be kinetic differences in expression between ligands. These data suggest that ITE and TCDD elicit an analogous change in AhR conformation such that the initial transcription response is the same. Furthermore, if the difference in toxicity between TCDD and ITE is mediated by differences in gene expression, then it is likely that secondary changes enabled by the persistent TCDD, but not by the shorter lived ITE, are responsible. PMID:19933214

COMPARISONS OF THE EFFECTS OF TCDD AND HYDROCORTISONE (HC) ON GROWTH FACTOR EXPRESSION PROVIDE INSIGHT INTO THE SYNERGISTIC INTERACTION OCCURRING IN EMBRYONIC PALATES

EPA Science Inventory

Cleft palate (CP) can be Induced In embryonic mice by a Wide range of compounds, including glucocorticoids and 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD). ydrocortisone (HC), a glucocorticoid, retards embryonic growth producing small palatal shelves, while TCDD exposure blocks t...

Inhibition of ATP Synthase by Chlorinated Adenosine Analogue

PubMed Central

Chen, Lisa S.; Nowak, Billie J.; Ayres, Mary L.; Krett, Nancy L.; Rosen, Steven T.; Zhang, Shuxing; Gandhi, Varsha

2009-01-01

8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; FO intermembrane base and F1 domain, containing α and β subunits. Crystal structures of both α and β subunits that bind to the substrate, ADP, are known in tight binding (αdpβdp) and loose binding (αtpβtp) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the αtpβtp state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (αdpβdp) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF3 −. Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase. PMID:19477165

Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gruenhagen, Jason Alan

The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activatedmore » a G{sub q}-type protein to initiate ATP release from HUVECs. Ca 2+ imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca 2+ signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K + and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol functionalized Cd

Adsorption of TCDD with 1-butyl-3-methylimidazolium dicyanamide ionic liquid: a combined molecular dynamics simulation and quantum chemistry study.

PubMed

Pan, Wenxiao; Qi, Yuanyuan; Wang, Ruoxi; Han, Zhe; Zhang, Dongju; Zhan, Jinhua

2013-04-01

The effective abatement of flue gas emissions, especially polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), is one of the challenging issues in the field of environmental science currently. Imidazolium-based dicyanamide ionic liquids (ILs) were proposed to have potential in controlling the emissions of PCDD/Fs. However, the relevant mechanism at the molecular level still remains unclear. To address this subject, we here present a combined molecular dynamics (MD) simulation and quantum chemical (QM) study on the adsorption of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener among PCDD/F family, by 1-butyl-3-methylimidazolium dicyanamide IL, a representative imidazolium dicyanoamide ILs, which were demonstrated to possess high capture capability for PCDD/Fs. The MD simulation results show that TCDD molecules can be effectively adsorbed on the IL surface to form a dense layer, but cannot enter the interior of the IL. The results of QM calculations show that the adsorption of TCDDs on the IL surface occurs via intra-molecular hydrogen bond interactions. The calculated interaction energy of the anion with TCDD molecule is two times more than that of the cation, implying that the IL anion dominates the interaction with TCDD molecules, while the cation plays a secondary role. Based on the calculated results, we propose that imidazolium dicyanamide IL films/membranes may be better materials than the corresponding bulk for capturing TCDD. The present theoretical results may be helpful to designing the functional ILs which effectively capture and concentrate PCDD/F compounds. Copyright © 2013 Elsevier Ltd. All rights reserved.

Oral Adenosine-5'-triphosphate (ATP) Administration Increases Postexercise ATP Levels, Muscle Excitability, and Athletic Performance Following a Repeated Sprint Bout.

PubMed

Purpura, Martin; Rathmacher, John A; Sharp, Matthew H; Lowery, Ryan P; Shields, Kevin A; Partl, Jeremy M; Wilson, Jacob M; Jäger, Ralf

2017-01-01

Oral adenosine-5'-triphosphate (ATP) administration has failed to increase plasma ATP levels; however, chronic supplementation with ATP has shown to increase power, strength, lean body mass, and blood flow in trained athletes. The purpose of this study was to investigate the effects of ATP supplementation on postexercise ATP levels and on muscle activation and excitability and power following a repeated sprint bout. In a double-blind, placebo-controlled, randomized design, 42 healthy male individuals were given either 400 mg of ATP as disodium salt or placebo for 2 weeks prior to an exercise bout. During the exercise bout, muscle activation and excitability (ME, ratio of power output to muscle activation) and Wingate test peak power were measured during all sprints. ATP and metabolites were measured at baseline, after supplementation, and immediately following exercise. Oral ATP supplementation prevented a drop in ATP, adenosine-5'-diphosphate (ADP), and adenosine-5'-monophosphate (AMP) levels postexercise (p < 0.05). No group by time interaction was observed for muscle activation. Following the supplementation period, muscle excitability significantly decreased in later bouts 8, 9, and 10 in the placebo group (-30.5, -28.3, and -27.9%, respectively; p < 0.02), whereas ATP supplementation prevented the decline in later bouts. ATP significantly increased Wingate peak power in later bouts compared to baseline (bout 8: +18.3%, bout 10: +16.3%). Oral ATP administration prevents exercise-induced declines in ATP and its metabolite and enhances peak power and muscular excitability, which may be beneficial for sports requiring repeated high-intensity sprinting bouts.

Increases in levels of epoxyeicosatrienoic and dihydroxyeicosatrienoic acids (EETs and DHETs) in liver and heart in vivo by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and in hepatic EET:DHET ratios by cotreatment with TCDD and the soluble epoxide hydrolase inhibitor AUDA.

PubMed

Diani-Moore, Silvia; Ma, Yuliang; Gross, Steven S; Rifkind, Arleen B

2014-02-01

The environmental toxin and carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) binds and activates the transcription factor aryl hydrocarbon receptor (AHR), inducing CYP1 family cytochrome P450 enzymes. CYP1A2 and its avian ortholog CYP1A5 are highly active arachidonic acid epoxygenases. Epoxygenases metabolize arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs) and selected monohydroxyeicosatetraenoic acids (HETEs). EETs can be further metabolized by epoxide hydrolases to dihydroxyeicosatrienoic acids (DHETs). As P450-arachidonic acid metabolites affect vasoregulation, responses to ischemia, inflammation, and metabolic disorders, identification of their production in vivo is needed to understand their contribution to biologic effects of TCDD and other AHR activators. Here we report use of an acetonitrile-based extraction procedure that markedly increased the yield of arachidonic acid products by lipidomic analysis over a standard solid-phase extraction protocol. We show that TCDD increased all four EETs (5,6-, 8,9-, 11,12-, and 14,15-), their corresponding DHETs, and 18- and 20-HETE in liver in vivo and increased 5,6-EET, the four DHETs, and 18-HETE in heart, in a chick embryo model. As the chick embryo heart lacks arachidonic acid-metabolizing activity, the latter findings suggest that arachidonic acid metabolites may travel from their site of production to a distal organ, i.e., heart. To determine if the TCDD-arachidonic acid-metabolite profile could be altered pharmacologically, chick embryos were treated with TCDD and the soluble epoxide hydrolase inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA). Cotreatment with AUDA increased hepatic EET-to-DHET ratios, indicating that the in vivo profile of P450-arachidonic acid metabolites can be modified for potential therapeutic intervention.

USE OF A PHYSIOLOGICALLY BASED PHARMACOKINETIC MODEL (PBPK) FOR RATS TO STUDY THE INFLUENCE OF BODY FAT MASS AND INDUCTION OF CYP1A2 ON THE PHARMACOKINETICS OF TCDD

EPA Science Inventory

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly lipophilic chemical which distributes into adipose tissue, especially at low doses. However, at high doses TCDD sequesters in liver because it induces CYP1A2 that binds TCDD. A physiologically based pharmacokinetic (PBPK) mod...

Comparative Analysis of AhR-Mediated TCDD-Elicited Gene Expression in Human Liver Adult Stem Cells

PubMed Central

Kim, Suntae; Dere, Edward; Burgoon, Lyle D.; Chang, Chia-Cheng; Zacharewski, Timothy R.

2009-01-01

Time course and dose-response studies were conducted in HL1-1 cells, a human liver cell line with stem cell–like characteristics, to assess the differential gene expression elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) compared with other established models. Cells were treated with 0.001, 0.01, 0.1, 1, 10, or 100nM TCDD or dimethyl sulfoxide vehicle control for 12 h for the dose-response study, or with 10nM TCDD or vehicle for 1, 2, 4, 8, 12, 24, or 48 h for the time course study. Elicited changes were monitored using a human cDNA microarray with 6995 represented genes. Empirical Bayes analysis identified 144 genes differentially expressed at one or more time points following treatment. Most genes exhibited dose-dependent responses including CYP1A1, CYP1B1, ALDH1A3, and SLC7A5 genes. Comparative analysis of HL1-1 differential gene expression to human HepG2 data identified 74 genes with comparable temporal expression profiles including 12 putative primary responses. HL1-1–specific changes were related to lipid metabolism and immune responses, consistent with effects elicited in vivo. Furthermore, comparative analysis of HL1-1 cells with mouse Hepa1c1c7 hepatoma cell lines and C57BL/6 hepatic tissue identified 18 and 32 commonly regulated orthologous genes, respectively, with functions associated with signal transduction, transcriptional regulation, metabolism and transport. Although some common pathways are affected, the results suggest that TCDD elicits species- and model-specific gene expression profiles. PMID:19684285

Variable effects of the mitoK(ATP) channel modulators diazoxide and 5-HD in ATP-depleted renal epithelial cells.

PubMed

Nilakantan, Vani; Liang, Huanling; Mortensen, Jordan; Taylor, Erin; Johnson, Christopher P

2010-02-01

The role of mitochondrial K(ATP) (mitoK(ATP)) channels in renal ischemia-reperfusion injury is controversial with studies showing both protective and deleterious effects. In this study, we compared the effects of the putative mitoK(ATP) opener, diazoxide, and the mitoK(ATP) blocker, 5-hydroxydecanoate (5-HD) on cytotoxicity and apoptosis in tubular epithelial cells derived from rat (NRK-52E) and pig (LLC-PK1) following in vitro ischemic injury. Following ATP depletion-recovery, there was a significant increase in cytotoxicity in both NRK cells and LLC-PK1 cells although NRK cells were more sensitive to the injury. Diazoxide treatment attenuated cytotoxicity in both cell types and 5-HD treatment-increased cytotoxicity in the sensitive NRK cells in a superoxide-dependant manner. The protective effect of diazoxide was also reversed in the presence of 5-HD in ATP-depleted NRK cells. The ATP depletion-mediated increase in superoxide was enhanced by both diazoxide and 5-HD with the effect being more pronounced in the cells undergoing 5-HD treatment. Further, ATP depletion-induced activation of caspase-3 was decreased by diazoxide in NRK cells. In order to determine the signaling pathways involved in apoptosis, we examined the activation of Erk and JNK in ATP-depleted NRK cells. Diazoxide-activated Erk in ATP-depleted cells, but did not have any effect on JNK activation. In contrast, 5-HD did not impact Erk levels but increased JNK activation even under controlled conditions. Further, the use of a JNK inhibitor with 5-HD reversed the deleterious effects of 5-HD. This study demonstrates that in cells that are sensitive to ATP depletion-recovery, mitoK(ATP) channels protect against ATP depletion-mediated cytotoxicity and apoptosis through Erk- and JNK-dependant mechanisms.

Eicosapentaenoic acid prevents TCDD-induced oxidative stress and inflammatory response by modulating MAP kinases and redox-sensitive transcription factors

PubMed Central

Palanisamy, Kalaiselvi; Krishnaswamy, Rajashree; Paramasivan, Poornima; Chih-Yang, Huang; Vishwanadha, Vijaya Padma

2015-01-01

Background and Purpose Oxidative stress and subsequent activation of inflammatory responses is a widely accepted consequence of exposure to environmental toxins. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a well-known environmental toxin, exerts its toxicity through many signalling mechanisms, with liver being the principal organ affected. However, an effective antidote to TCDD-induced toxicity is unknown. The present study evaluated the effect of eicosapentaenoic acid (EPA), an n3 fatty acid, on TCDD-induced toxicity. Experimental Approach In cultures of HepG2 cells, the EPA/AA ratio was determined using gas chromatography, oxidative stress and inflammatory responses through reactive oxygen species (ROS) levels, antioxidant status, [Ca2+]i, nuclear migration of two redox-sensitive transcription factors, NF-κB p65 and Nrf-2, expression of MAP kinase (p-Erk, p-p38), NF-κB p65, COX-2 and Nrf-2. Cellular changes in ΔΨm, acidic vesicular organelle formation, cell cycle analysis and scanning electron microscopy analysis were performed. Key Results EPA offered significant cytoprotection by increasing EPA/AA ratios in cell membranes, inhibiting ROS generation, enhancing antioxidant status and modulating nuclear translocation of redox-sensitive transcription factors (NF-κB p65 and Nrf-2) and expression of NF-κB p65, COX-2 and Nrf-2. Furthermore, TCDD-induced upstream events of MAPK phosphorylation, the increase in [Ca2+]i levels and cell surface changes in microvilli were significantly inhibited by EPA. EPA treatment maintained ΔΨm and prevented formation of acidic vesicular organelles. Conclusion and Implications The present study demonstrates for the first time some underlying molecular mechanisms of cytoprotection exerted by EPA against TCDD-induced oxidative stress and inflammatory responses. PMID:26177858

ATP Dependence of Na+/H+ Exchange

PubMed Central

Demaurex, Nicolas; Romanek, Robert R.; Orlowski, John; Grinstein, Sergio

1997-01-01

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pHi) by microfluorimetry. Na+/H+ exchange activity was measured as the Na+-driven pHi recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na+/H+ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-d-glucose and oligomycin. In cells dialyzed in the presence of ATP, no “run-down” was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at ∼5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na+/H+ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca2+ exchanger, this requirement has been attributed to an aminophospholipid translocase, or “flippase.” The involvement of this enzyme in Na+/H+ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na+/H+ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATPγS were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In

Handler, bystander and reentry exposure to TCDD from application of Agent Orange by C-123 aircraft during the Vietnam War.

PubMed

Ross, John H; Hewitt, Andrew; Armitage, James; Solomon, Keith; Watkins, Deborah K; Ginevan, Michael E

2015-02-01

Using validated models and methods routinely employed by pesticide regulatory agencies, the absorbed dosages of Agent Orange (AO) herbicide contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were estimated for mixer/loaders, applicators, and individuals in the vicinity of applications of AO by C-123 aircraft during the Vietnam War. Resulting dosages of TCDD were then transformed to estimates of adipose residues, and compared to population biomonitoring of known mixer/loaders and applicators as well as ground troops in Vietnam and civilians in the U.S. Results demonstrate that mixer/loaders and applicators had the greatest exposures and their measured residues of TCDD in adipose were consistent with the estimated exposures. Further, the potentially exposed ground troops, including those who could have been directly sprayed during aerial defoliation, had measured adipose residues that were consistent with those in civilian U.S. populations with no defined source of exposure exposures and both of those cohorts had orders of magnitude less exposure than the mixer/loaders or applicators. Despite the availability of validated exposure modeling methods for decades, the quantitative TCDD dose estimates presented here are the first of their kind for the Vietnam conflict. Copyright © 2014 Elsevier B.V. All rights reserved.

Biallelic Mutations in ATP5F1D , which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oláhová, Monika; Yoon, Wan Hee; Thompson, Kyle

ATP synthase, H + transporting, mitochondrial F1 complex, δ subunit (ATP5F1D; formerly ATP5D) is a subunit of mitochondrial ATP synthase and plays an important role in coupling proton translocation and ATP production. Here, we describe two individuals, each with homozygous missense variants in ATP5F1D, who presented with episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia. Subject 1, homozygous for c.245C>T (p.Pro82Leu), presented with recurrent metabolic decompensation starting in the neonatal period, and subject 2, homozygous for c.317T>G (p.Val106Gly), presented with acute encephalopathy in childhood. Cultured skin fibroblasts from these individuals exhibited impaired assembly of F 1F O ATP synthase andmore » subsequent reduced complex V activity. Cells from subject 1 also exhibited a significant decrease in mitochondrial cristae. Knockdown of Drosophila ATPsynδ, the ATP5F1D homolog, in developing eyes and brains caused a near complete loss of the fly head, a phenotype that was fully rescued by wild-type human ATP5F1D. In contrast, expression of the ATP5F1D c.245C>T and c.317T>G variants rescued the head-size phenotype but recapitulated the eye and antennae defects seen in other genetic models of mitochondrial oxidative phosphorylation deficiency. Our data establish c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) in ATP5F1D as pathogenic variants leading to a Mendelian mitochondrial disease featuring episodic metabolic decompensation.« less

Biallelic Mutations in ATP5F1D, which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder.

PubMed

Oláhová, Monika; Yoon, Wan Hee; Thompson, Kyle; Jangam, Sharayu; Fernandez, Liliana; Davidson, Jean M; Kyle, Jennifer E; Grove, Megan E; Fisk, Dianna G; Kohler, Jennefer N; Holmes, Matthew; Dries, Annika M; Huang, Yong; Zhao, Chunli; Contrepois, Kévin; Zappala, Zachary; Frésard, Laure; Waggott, Daryl; Zink, Erika M; Kim, Young-Mo; Heyman, Heino M; Stratton, Kelly G; Webb-Robertson, Bobbie-Jo M; Snyder, Michael; Merker, Jason D; Montgomery, Stephen B; Fisher, Paul G; Feichtinger, René G; Mayr, Johannes A; Hall, Julie; Barbosa, Ines A; Simpson, Michael A; Deshpande, Charu; Waters, Katrina M; Koeller, David M; Metz, Thomas O; Morris, Andrew A; Schelley, Susan; Cowan, Tina; Friederich, Marisa W; McFarland, Robert; Van Hove, Johan L K; Enns, Gregory M; Yamamoto, Shinya; Ashley, Euan A; Wangler, Michael F; Taylor, Robert W; Bellen, Hugo J; Bernstein, Jonathan A; Wheeler, Matthew T

2018-03-01

ATP synthase, H + transporting, mitochondrial F1 complex, δ subunit (ATP5F1D; formerly ATP5D) is a subunit of mitochondrial ATP synthase and plays an important role in coupling proton translocation and ATP production. Here, we describe two individuals, each with homozygous missense variants in ATP5F1D, who presented with episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia. Subject 1, homozygous for c.245C>T (p.Pro82Leu), presented with recurrent metabolic decompensation starting in the neonatal period, and subject 2, homozygous for c.317T>G (p.Val106Gly), presented with acute encephalopathy in childhood. Cultured skin fibroblasts from these individuals exhibited impaired assembly of F 1 F O ATP synthase and subsequent reduced complex V activity. Cells from subject 1 also exhibited a significant decrease in mitochondrial cristae. Knockdown of Drosophila ATPsynδ, the ATP5F1D homolog, in developing eyes and brains caused a near complete loss of the fly head, a phenotype that was fully rescued by wild-type human ATP5F1D. In contrast, expression of the ATP5F1D c.245C>T and c.317T>G variants rescued the head-size phenotype but recapitulated the eye and antennae defects seen in other genetic models of mitochondrial oxidative phosphorylation deficiency. Our data establish c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) in ATP5F1D as pathogenic variants leading to a Mendelian mitochondrial disease featuring episodic metabolic decompensation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Biallelic Mutations in ATP5F1D , which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder

DOE PAGES

Oláhová, Monika; Yoon, Wan Hee; Thompson, Kyle; ...

2018-02-22

ATP synthase, H + transporting, mitochondrial F1 complex, δ subunit (ATP5F1D; formerly ATP5D) is a subunit of mitochondrial ATP synthase and plays an important role in coupling proton translocation and ATP production. Here, we describe two individuals, each with homozygous missense variants in ATP5F1D, who presented with episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia. Subject 1, homozygous for c.245C>T (p.Pro82Leu), presented with recurrent metabolic decompensation starting in the neonatal period, and subject 2, homozygous for c.317T>G (p.Val106Gly), presented with acute encephalopathy in childhood. Cultured skin fibroblasts from these individuals exhibited impaired assembly of F 1F O ATP synthase andmore » subsequent reduced complex V activity. Cells from subject 1 also exhibited a significant decrease in mitochondrial cristae. Knockdown of Drosophila ATPsynδ, the ATP5F1D homolog, in developing eyes and brains caused a near complete loss of the fly head, a phenotype that was fully rescued by wild-type human ATP5F1D. In contrast, expression of the ATP5F1D c.245C>T and c.317T>G variants rescued the head-size phenotype but recapitulated the eye and antennae defects seen in other genetic models of mitochondrial oxidative phosphorylation deficiency. Our data establish c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) in ATP5F1D as pathogenic variants leading to a Mendelian mitochondrial disease featuring episodic metabolic decompensation.« less

COMPARING MIXTURES OF DIOXIN-LIKE AND NON DIOXIN-LIKE PCBS TO TCDD

EPA Science Inventory

COMPARING MIXTURES OF DIOXIN-LIKE AND NON DIOXIN-LIKE PCBS TO TCDD. D E Burgin1, J J Diliberto2 and L S Birnbaum3.1University of North Carolina/Toxicology, Chapel Hill, NC, USA; 2USEPA/ORD/NHEERL, ETD, Research Triangle Park, NC, USA; 3USEPA/ORD/NHEERL, HSD, Chapel Hill, NC, USA....

2,3,7,8-tetrachlorodibenzo-p-dioxin decrease expression of aryl hydrocarbon receptor in peripheral lymphocyte of β-thalassemia major patients.

PubMed

Ghatrehsamani, Mahdi; Soleimani, Masoud; Esfahani, Behjat Al-Sadat Moayedi; Hakemi, Mazdak Ganjalikhani; Shirzad, Hedayatollah; Eskandari, Nahid; Adib, Minoo

2015-01-01

β-thalassemia major is a hereditary disease with inefficient erythropoiesis. Level of inflammatory cytokine is elevated in these patients. In this study, we investigate the effect of aryl hydrocarbon receptor (AhR) ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the expression of inflammatory mediators in β-thalassemia major patient's lymphocytes. Peripheral blood mononuclear cells of patients and healthy participants was isolated and cultured in favor of lymphocytes increment. Based on the treatment, we divided the cell into four groups. The orders of group's treatments were no treatment, tumor necrosis factor-α (TNF-α) treatment, TNF-α and TCDD treatment, TCDD treatment in Group 1-4, respectively. After cell culture, we extracted the cells RNA and converted them to cDNA. Real-time polymerase chain reaction was performed to assessment relative expression of caspase-1, NLRP3, and AhR. We compared all patient groups with equal healthy (control) groups. Results showed that expression of caspase-1 in patients (Groups 1 and 2) was significantly lower than healthy individuals (P < 0.05). Although, no significant difference was found (Groups 1, 2, and control) in AhR gene expression (P > 0.05). Expression of AhR in other groups of patients (3 and 4) was significantly lower than control groups (P < 0.05). Expression of caspase-1 in Group 4 was significantly larger than the control group (P < 0.001). We show here that chronic inflammation decrease caspase-1 expression and exposure of human lymphocytes to TCDD promote caspase-1 expression. Furthermore, activation of AhR with TCDD decreases AhR expression in lymphocytes of β-thalassemia major disease.

NMR-based metabonomic analysis of the hepatotoxicity induced by combined exposure to PCBs and TCDD in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu Chunfeng; Department of Pharmacology, Basic Medical College, Jiamusi University, Jiamusi 154007; Wang Yimei

2010-11-01

A metabonomic approach using {sup 1}H NMR spectroscopy was adopted to investigate the metabonomic pattern of rat urine after oral administration of environmental endocrine disruptors (EDs) polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) alone or in combination and to explore the possible hepatotoxic mechanisms of combined exposure to PCBs and TCDD. {sup 1}H NMR spectra of urines collected 24 h before and after exposure were analyzed via pattern recognition by using principal component analysis (PCA). Serum biochemistry and liver histopathology indicated significant hepatotoxicity in the rats of the combined group. The PCA scores plots of urinary {sup 1}H NMR datamore » showed that all the treatment groups could be easily distinguished from the control group, so could the PCBs or TCDD group and the combined group. The loadings plots of the PCA revealed remarkable increases in the levels of lactate, glucose, taurine, creatine, and 2-hydroxy-isovaleric acid and reductions in the levels of 2-oxoglutarate, citrate, succinate, hippurate, and trimethylamine-N-oxide in rat urine after exposure. These changes were more striking in the combined group. The changed metabolites may be considered possible biomarker for the hepatotoxicity. The present study demonstrates that combined exposure to PCBs and TCDD induced significant hepatotoxicity in rats, and mitochondrial dysfunction and fatty acid metabolism perturbations might contribute to the hepatotoxicity. There was good conformity between changes in the urine metabonomic pattern and those in serum biochemistry and liver histopathology. These results showed that the NMR-based metabonomic approach may provide a promising technique for the evaluation of the combined toxicity of EDs.« less

Exposure to TCDD from base perimeter application of Agent Orange in Vietnam.

PubMed

Ross, John H; Hewitt, Andrew; Armitage, James; Solomon, Keith; Watkins, Deborah K; Ginevan, Michael E

2015-04-01

Using recognized methods routinely employed by pesticide regulatory agencies, the exposures of military personnel that were mixer/loader/applicators (M/L/A) of Agent Orange (AO) for perimeter foliage at bases during the Vietnam War were estimated. From the fraction of TCDD in AO, absorbed dosage of the manufacturing contaminant was estimated. Dermal exposure estimated from spray drift to residents of the bases was calculated using internationally recognized software that accounted for proximity, foliar density of application site, droplet size and wind speed among other factors, and produced estimates of deposition. Those that directly handled AO generally had much higher exposures than those further from the areas of use. The differences in exposure potential varied by M/L/A activity, but were typically orders of magnitude greater than bystanders. However, even the most-exposed M/L/A involved in perimeter application had lifetime exposures comparable to persons living in the U.S. at the time, i.e., ~1.3 to 5 pg TCDD/kg bodyweight. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparison of hepatic NRF2 and AHR binding in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated mice demonstrates NRF2-independent PKM2 induction.

PubMed

Nault, Rance; Doskey, Claire M; Fader, Kelly A; Rockwell, Cheryl E; Zacharewski, Timothy R

2018-05-11

2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD) induces hepatic oxidative stress following activation of the aryl hydrocarbon receptor (AhR). Our recent studies showed TCDD induced pyruvate kinase muscle isoform 2 ( Pkm2 ) as a novel antioxidant response in normal differentiated hepatocytes. To investigate cooperative regulation between nuclear factor, erythroid derived 2, like 2 ( Nrf2 ) and the AhR in the induction of Pkm2 , hepatic ChIP-seq analyses were integrated with RNA-seq time course data from mice treated with TCDD for 2 - 168h. ChIP-seq analysis 2h after TCDD treatment identified genome-wide NRF2 enrichment. Approximately 842 NRF2 enriched regions were located in the regulatory region of differentially expressed genes (DEGs) while 579 DEGs showed both NRF2 and AhR enrichment. Sequence analysis of regions with overlapping NRF2 and AhR enrichment showed over-representation of either antioxidant or dioxin response elements (ARE and DRE, respectively), although 18 possessed both motifs. NRF2 exhibited negligible enrichment within a closed Pkm chromatin region while the AhR was enriched 29-fold. Furthermore, TCDD induced Pkm2 in primary hepatocytes from wild-type and Nrf2 null mice, indicating NRF2 is not required. Although NRF2 and AhR cooperate to regulate numerous antioxidant gene expression responses, the induction of Pkm2 by TCDD is independent of ROS-mediated NRF2 activation. The American Society for Pharmacology and Experimental Therapeutics.

2,3,7, 8-TETRACHLORODIBENZO-P-DIOXIN (TCDD)-MEDIATED OXIDATIVE STRESS IN FEMALE CYP1A-2 KNOCKOUT (CYP1A2-/-) MICE

EPA Science Inventory

2,3,7,8-Tetrachlordibenzo-p-dioxin (TCDD)-Mediated Oxidative Stress in Female CYP1A2 Knockout (CYP1A2-/-) Mice

Deborah Burgin1, Janet Diliberto2, Linda Birnbaum2
1UNC Toxicology; 2USEPA/ORD/NHEERL, RTP, NC

Most of the effects due to TCDD exposure are mediated via...

Hydronephrosis in mice exposed to TCDD-contaminated breast milk: Identification of the peak period of sensitivity and assessment of potential recovery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Couture-Haws, L.; Harris, M.W.; McDonald, M.M.

1991-01-01

2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent inducer of hydronephrosis in mice both pre- and post-natally. To identify the critical period of susceptibility for development of TCDD-induced hydronephrosis in neonatal mice, as well as to characterize the potential for recovery from this renal lesion, dose-response and time-course studies were conducted using lactational exposure. Pregnant C57BL/6N mice were allowed natural delivery. In the dose-response phase of the investigation, mothers were administered 0, 3, 6, or 12 microgram TCDD/kg once by gavage on post natal day (pnd) 1, 4, 8, or 14, and dams and pups were sacrificed on pnd 26.more » In the time-course studies, dams were given a single oral dose of 0 or 9 microgram TCDD/kg on pnd 1, and mothers and litters were subsequently sacrificed on pnd 7, 13, 19, or 26. Neonatal kidneys were examined, and hydronephrotic severity was scored. The incidence and severity of hydronephrosis were significantly elevated above controls at all dose levels on pnd 26 following treatment on pnds 1 and 4, while treatment on pnd 8 or 14 was ineffective at inducing hydronephrosis.« less

Adenosine triphosphate (ATP) reduces amyloid-β protein misfolding in vitro.

PubMed

Coskuner, Orkid; Murray, Ian V J

2014-01-01

Alzheimer's disease (AD) is a devastating disease of aging that initiates decades prior to clinical manifestation and represents an impending epidemic. Two early features of AD are metabolic dysfunction and changes in amyloid-β protein (Aβ) levels. Since levels of ATP decrease over the course of the disease and Aβ is an early biomarker of AD, we sought to uncover novel linkages between the two. First and remarkably, a GxxxG motif is common between both Aβ (oligomerization motif) and nucleotide binding proteins (Rossmann fold). Second, ATP was demonstrated to protect against Aβ mediated cytotoxicity. Last, there is structural similarity between ATP and amyloid binding/inhibitory compounds such as ThioT, melatonin, and indoles. Thus, we investigated whether ATP alters misfolding of the pathologically relevant Aβ42. To test this hypothesis, we performed computational and biochemical studies. Our computational studies demonstrate that ATP interacts strongly with Tyr10 and Ser26 of Aβ fibrils in solution. Experimentally, both ATP and ADP reduced Aβ misfolding at physiological intracellular concentrations, with thresholds at ~500 μM and 1 mM respectively. This inhibition of Aβ misfolding is specific; requiring Tyr10 of Aβ and is enhanced by magnesium. Last, cerebrospinal fluid ATP levels are in the nanomolar range and decreased with AD pathology. This initial and novel finding regarding the ATP interaction with Aβ and reduction of Aβ misfolding has potential significance to the AD field. It provides an underlying mechanism for published links between metabolic dysfunction and AD. It also suggests a potential role of ATP in AD pathology, as the occurrence of misfolded extracellular Aβ mirrors lowered extracellular ATP levels. Last, the findings suggest that Aβ conformation change may be a sensor of metabolic dysfunction.

Long overlooked historical information on Agent Orange and TCDD following massive applications of 2,4,5-T-containing herbicides, Eglin Air Force Base, Florida.

PubMed

Young, Alvin L; Newton, Michael

2004-01-01

From 1961-1971, The Air Development Test Center, Eglin Air Force Base (AFB), Florida, developed, tested, and calibrated the aerial spray systems used in support of Operation RANCH HAND and the US Army Chemical Corps in Vietnam. Twenty major test and evaluation projects of aerial spray equipment were conducted on four fully instrumented test grids, each uniquely arrayed to match the needs of fixed-wing, helicopter, or jet aircraft. Each of the grids was established within the boundary of Test Area 52A of the Eglin Reservation. The tests, conducted under climatic and environmental conditions similar to those in Vietnam, included the use of the military herbicides (Agents) Orange, Purple, White, and Blue. Approximately 75,000 kg of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 76,000 kg of 2,4-dichlorophenoxyacetic acid (2,4-D) were aerially disseminated on an area of less than 3 km2 during the period 1962-1970. Data from the analysis of archived samples suggested that an estimated 3.1 kg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), present as a contaminant, were aerially released in the test area. Because most of the vegetation had been removed before establishing the test site in 1961, there was an opportunity to follow ground-based residues independent of canopy interception, and the resulting high solar exposure of initial residues. Studies of the soils, fauna, flora, and aquatic ecosystems of the test grids and associated perimeters of Test Area C-52A (an area totally more than 8 km2) were initiated in 1969 and concluded in 1984. Data from soil samples collected from 1974 through 1984 suggested that less than one percent of the TCDD that was present in soil when sampling began persisted through the ten-year period of sampling. More than 340 species of organisms were observed and identified within the test area. More than 300 biological samples were analyzed for TCDD and detectable residues were found in 16 of 45 species examined. Examination of the ecological

Binding of ATP by pertussis toxin and isolated toxin subunits

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hausman, S.Z.; Manclark, C.R.; Burns, D.L.

1990-07-03

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner;more » however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.« less

Fragrance chemicals lyral and lilial decrease viability of HaCat cells' by increasing free radical production and lowering intracellular ATP level: protection by antioxidants.

PubMed

Usta, Julnar; Hachem, Yassmine; El-Rifai, Omar; Bou-Moughlabey, Yolla; Echtay, Karim; Griffiths, David; Nakkash-Chmaisse, Hania; Makki, Rajaa Fakhoury

2013-02-01

We investigate in this study the biochemical effects on cells in culture of two commonly used fragrance chemicals: lyral and lilial. Whereas both chemicals exerted a significant effect on primary keratinocyte(s), HaCat cells, no effect was obtained with any of HepG2, Hek293, Caco2, NIH3T3, and MCF7 cells. Lyral and lilial: (a) decreased the viability of HaCat cells with a 50% cell death at 100 and 60 nM respectively; (b) decreased significantly in a dose dependant manner the intracellular ATP level following 12-h of treatment; (c) inhibited complexes I and II of electron transport chain in liver sub-mitochondrial particles; and (d) increased reactive oxygen species generation that was reversed by N-acetyl cysteine and trolox and the natural antioxidant lipoic acid, without influencing the level of free and/or oxidized glutathione. Lipoic acid protected HaCat cells against the decrease in viability induced by either compound. Dehydrogenation of lyral and lilial produce α,β-unsaturated aldehydes, that reacts with lipoic acid requiring proteins resulting in their inhibition. We propose lyral and lilial as toxic to mitochondria that have a direct effect on electron transport chain, increase ROS production, derange mitochondrial membrane potential, and decrease cellular ATP level, leading thus to cell death. Copyright © 2012 Elsevier Ltd. All rights reserved.

Induction of a chloracne phenotype in an epidermal equivalent model by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on aryl hydrocarbon receptor activation and is not reproduced by aryl hydrocarbon receptor knock down

PubMed Central

Forrester, Alison R.; Elias, Martina S.; Woodward, Emma L.; Graham, Mark; Williams, Faith M.; Reynolds, Nick J.

2014-01-01

Background 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent activator of the aryl hydrocarbon receptor (AhR) and causes chloracne in humans. The pathogenesis and role of AhR in chloracne remains incompletely understood. Objective To elucidate the mechanisms contributing to the development of the chloracne-like phenotype in a human epidermal equivalent model and identify potential biomarkers. Methods Using primary normal human epidermal keratinocytes (NHEK), we studied AhR activation by XRE-luciferase, AhR degradation and CYP1A1 induction. We treated epidermal equivalents with high affinity TCDD or two non-chloracnegens: β-naphthoflavone (β-NF) and 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Using Western blotting and immunochemistry for filaggrin (FLG), involucrin (INV) and transglutaminase-1 (TGM-1), we compared the effects of the ligands on keratinocyte differentiation and development of the chloracne-like phenotype by H&E. Results In NHEKs, activation of an XRE-luciferase and CYP1A1 protein induction correlated with ligand binding affinity: TCDD > β-NF > ITE. AhR degradation was induced by all ligands. In epidermal equivalents, TCDD induced a chloracne-like phenotype, whereas β-NF or ITE did not. All three ligands induced involucrin and TGM-1 protein expression in epidermal equivalents whereas FLG protein expression decreased following treatment with TCDD and β-NF. Inhibition of AhR by α-NF blocked TCDD-induced AhR activation in NHEKs and blocked phenotypic changes in epidermal equivalents; however, AhR knock down did not reproduce the phenotype. Conclusion Ligand-induced CYP1A1 and AhR degradation did not correlate with their chloracnegenic potential, indicating that neither CYP1A1 nor AhR are suitable biomarkers. Mechanistic studies showed that the TCDD-induced chloracne-like phenotype depends on AhR activation whereas AhR knock down did not appear sufficient to induce the phenotype. PMID:24161567

Structure of ATP-Bound Human ATP:Cobalamin Adenosyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schubert,H.; Hill, C.

Mutations in the gene encoding human ATP:cobalamin adenosyltransferase (hATR) can result in the metabolic disorder known as methylmalonic aciduria (MMA). This enzyme catalyzes the final step in the conversion of cyanocobalamin (vitamin B{sub 12}) to the essential human cofactor adenosylcobalamin. Here we present the 2.5 {angstrom} crystal structure of ATP bound to hATR refined to an R{sub free} value of 25.2%. The enzyme forms a tightly associated trimer, where the monomer comprises a five-helix bundle and the active sites lie on the subunit interfaces. Only two of the three active sites within the trimer contain the bound ATP substrate, therebymore » providing examples of apo- and substrate-bound-active sites within the same crystal structure. Comparison of the empty and occupied sites indicates that twenty residues at the enzyme's N-terminus become ordered upon binding of ATP to form a novel ATP-binding site and an extended cleft that likely binds cobalamin. The structure explains the role of 20 invariant residues; six are involved in ATP binding, including Arg190, which hydrogen bonds to ATP atoms on both sides of the scissile bond. Ten of the hydrogen bonds are required for structural stability, and four are in positions to interact with cobalamin. The structure also reveals how the point mutations that cause MMA are deficient in these functions.« less

Capture and quality control mechanisms for ATP binding

PubMed Central

Li, Li; Martinis, Susan A.

2013-01-01

The catalytic events in members of the nucleotidylyl transferase superfamily are initiated by a millisecond binding of ATP in the active site. Through metadynamics simulations on a class I aminoacyl-tRNA synthetase (aaRSs), the largest group in the superfamily, we calculate the free energy landscape of ATP selection and binding. Mutagenesis studies and fluorescence spectroscopy validated the identification of the most populated intermediate states. The rapid first binding step involves formation of encounter complexes captured through a fly-casting mechanism that acts up on the triphosphate moiety of ATP. In the slower nucleoside binding step, a conserved histidine in the HxxH motif orients the incoming ATP through base-stacking interactions resulting in a deep minimum in the free energy surface. Mutation of this histidine significantly decreases the binding affinity measured experimentally and computationally. The metadynamics simulations further reveal an intermediate quality control state that the synthetases and most likely other members of the superfamily use to select ATP over other nucleoside triphosphates. PMID:23276298

Hydrostatic pressure activates ATP-sensitive K+ channels in lung epithelium by ATP release through pannexin and connexin hemichannels.

PubMed

Richter, Katrin; Kiefer, Kevin P; Grzesik, Benno A; Clauss, Wolfgang G; Fronius, Martin

2014-01-01

Lungs of air-breathing vertebrates are constantly exposed to mechanical forces and therefore are suitable for investigation of mechanotransduction processes in nonexcitable cells and tissues. Freshly dissected Xenopus laevis lungs were used for transepithelial short-circuit current (ISC) recordings and were exposed to increased hydrostatic pressure (HP; 5 cm fluid column, modified Ussing chamber). I(SC) values obtained under HP (I(5cm)) were normalized to values before HP (I(0cm)) application (I(5cm)/I(0cm)). Under control conditions, HP decreased I(SC) (I(5cm)/I(0cm)=0.84; n=68; P<0.0001). This effect was reversible and repeatable ≥30 times. Preincubation with ATP-sensitive K(+) channel (K(ATP)) inhibitors (HMR1098 and glibenclamide) prevented the decrease in I(SC) (I(5cm)/I(0cm): HMR1098=1.19, P<0.0001; glibenclamide=1.11, P<0.0001). Similar effects were observed with hemichannel inhibitors (I(5cm)/I(0cm): meclofenamic acid=1.09, P<0.0001; probenecid=1.0, P<0.0001). The HP effect was accompanied by release of ATP (P<0.05), determined by luciferin-luciferase luminescence in perfusion solution from the luminal side of an Ussing chamber. ATP release was abrogated by both meclofenamic acid and probenecid. RT-PCR experiments revealed the expression of pannexin and connexin hemichannels and KATP subunit transcripts in X. laevis lung. These data show an activation of KATP in pulmonary epithelial cells in response to HP that is induced by ATP release through mechanosensitive pannexin and connexin hemichannels. These findings represent a novel mechanism of mechanotransduction in nonexcitable cells.

ATP13A2 variability in Parkinson disease

PubMed Central

Vilariño-Güell, Carles; Soto, Alexandra I.; Lincoln, Sarah J.; Yahmed, Samia Ben; Kefi, Mounir; Heckman, Michael G.; Hulihan, Mary M.; Chai, Hua; Diehl, Nancy N.; Amouri, Rim; Rajput, Alex; Mash, Deborah C.; Dickson, Dennis W.; Middleton, Lefkos T.; Gibson, Rachel A.; Hentati, Faycal; Farrer, Matthew J.

2008-01-01

Recessively inherited mutations in ATP13A2 result in Kufor-Rakeb syndrome, whereas genetic variability and elevated ATP13A2 expression have been implicated in Parkinson disease (PD). Given this background, ATP13A2 was comprehensively assessed to support or refute its contribution to PD. Sequencing of ATP13A2 exons and intron-exon boundaries was performed in 89 probands with familial parkinsonism from Tunisia. The segregation of mutations with parkinsonism was subsequently assessed within pedigrees. The frequency of genetic variants and evidence for association was also examined in 240 patients with non-familial PD and 372 healthy controls. ATP13A2 mRNA expression was also quantified in brain tissues from 38 patients with non-familial PD and 38 healthy subjects from the US. Sequencing analysis revealed 37 new variants; seven missense, six silent and 24 that were noncoding. However, no single ATP13A2 mutation segregated with familial parkinsonism in either a dominant or recessive manner. Four markers showed marginal association with non-familial PD, prior to correction for multiple testing. ATP13A2 mRNA expression was marginally decreased in PD brains compared with tissue from control subjects. In conclusion, neither ATP13A2 genetic variability nor quantitative gene expression in brain appears to contribute to familial parkinsonism or non-familial PD. PMID:19085912

Effects and mechanism of acid rain on plant chloroplast ATP synthase.

PubMed

Sun, Jingwen; Hu, Huiqing; Li, Yueli; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

2016-09-01

Acid rain can directly or indirectly affect plant physiological functions, especially photosynthesis. The enzyme ATP synthase is the key in photosynthetic energy conversion, and thus, it affects plant photosynthesis. To clarify the mechanism by which acid rain affects photosynthesis, we studied the effects of acid rain on plant growth, photosynthesis, chloroplast ATP synthase activity and gene expression, chloroplast ultrastructure, intracellular H(+) level, and water content of rice seedlings. Acid rain at pH 4.5 remained the chloroplast structure unchanged but increased the expression of six chloroplast ATP synthase subunits, promoted chloroplast ATP synthase activity, and increased photosynthesis and plant growth. Acid rain at pH 4.0 or less decreased leaf water content, destroyed chloroplast structure, inhibited the expression of six chloroplast ATP synthase subunits, decreased chloroplast ATP synthase activity, and reduced photosynthesis and plant growth. In conclusion, acid rain affected the chloroplast ultrastructure, chloroplast ATPase transcription and activity, and P n by changing the acidity in the cells, and thus influencing the plant growth and development. Finally, the effects of simulated acid rain on the test indices were found to be dose-dependent.

Proteomic analysis of human bladder epithelial cells by 2D blue native SDS-PAGE reveals TCDD-induced alterations of calcium and iron homeostasis possibly mediated by nitric oxide.

PubMed

Verma, Nisha; Pink, Mario; Petrat, Frank; Rettenmeier, Albert W; Schmitz-Spanke, Simone

2015-01-02

A proteomic analysis of the interaction among multiprotein complexes involved in 2,3,7,8-dibenzo-p-dioxin (TCDD)-mediated toxicity in urinary bladder epithelial RT4 cells was performed using two-dimensional blue native SDS-PAGE (2D BN/SDS-PAGE). To enrich the protein complexes, unexposed and TCDD-exposed cells were fractionated. BN/SDS-PAGE of the resulting fractions led to an effective separation of proteins and protein complexes of various origins, including cell membrane, mitochondria, and other intracellular compartments. Major differences between the proteome of control and exposed cells involved the alteration of many calcium-regulated proteins (calmodulin, protein S100-A2, annexin A5, annexin A10, gelsolin isoform b) and iron-regulated proteins (ferritin, heme-binding protein 2, transferrin). On the basis of these findings, the intracellular calcium concentration was determined, revealing a significant increase after 24 h of exposure to TCDD. Moreover, the concentration of the labile iron pool (LIP) was also significantly elevated in TCDD-exposed cells. This increase was strongly inhibited by the calmodulin (CaM) antagonist W-7, which pointed toward a possible interaction between iron and calcium signaling. Because nitric oxide (NO) production was significantly enhanced in TCDD-exposed cells and was also inhibited by W-7, we hypothesize that alterations in calcium and iron homeostasis upon exposure to TCDD may be linked through NO generated by CaM-activated nitric oxide synthase. In our model, we propose that NO produced upon TCDD exposure interacts with the iron centers of iron-regulatory proteins (IRPs) that modulate the alteration of ferritin and transferrin, resulting in an augmented cellular LIP and, hence, increased toxicity.

Clusterin (Apolipoprotein J), a Molecular Chaperone That Facilitates Degradation of the Copper-ATPases ATP7A and ATP7B*

PubMed Central

Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

2011-01-01

The copper-transporting P1B-type ATPases (Cu-ATPases) ATP7A and ATP7B are key regulators of physiological copper levels. They function to maintain intracellular copper homeostasis by delivering copper to secretory compartments and by trafficking toward the cell periphery to export excess copper. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and toxicity disorders, Menkes and Wilson diseases, respectively. This report describes the interaction between the Cu-ATPases and clusterin and demonstrates a chaperone-like role for clusterin in facilitating their degradation. Clusterin interacted with both ATP7A and ATP7B in mammalian cells. This interaction increased under conditions of oxidative stress and with mutations in ATP7B that led to its misfolding and mislocalization. A Wilson disease patient mutation (G85V) led to enhanced ATP7B turnover, which was further exacerbated when cells overexpressed clusterin. We demonstrated that clusterin-facilitated degradation of mutant ATP7B is likely to involve the lysosomal pathway. The knockdown and overexpression of clusterin increased and decreased, respectively, the Cu-ATPase-mediated copper export capacity of cells. These results highlight a new role for intracellular clusterin in mediating Cu-ATPase quality control and hence in the normal maintenance of copper homeostasis, and in promoting cell survival in the context of disease. Based on our findings, it is possible that variations in clusterin expression and function could contribute to the variable clinical expression of Menkes and Wilson diseases. PMID:21242307

Modulation of ovarian follicle maturation and effects on apoptotic cell death in Holtzman rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in utero and lactationally.

PubMed

Heimler, I; Trewin, A L; Chaffin, C L; Rawlins, R G; Hutz, R J

1998-01-01

Recent reports have described the reproduction-modulating and endocrine-disrupting effects following exposure to toxic substances such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Herein, we set out (1) to determine whether TCDD exposure exerts detrimental effects on follicle maturation in the Holtzman rat ovary and (2) to determine whether the effects of TCDD are mediated in part via apoptotic cell death. In certain species, dioxin exposure is correlated with reduced fecundity, reduced ovulatory rate, an increased incidence of endometriosis, and various reproductive cancers. Although some of the effects of TCDD are mediated via the hypothalamic-pituitary axis, direct effects on the ovary have also been observed. In the present study, an oral dose of 1 microgram TCDD/kg maternal body weight was administered on Day 15 of gestation. Female pups were sacrificed on Postnatal Day 21/22, and the ovaries were excised, fixed for histologic analysis, and analyzed in a double-blind paradigm. The analysis included a count and measurement and classification of preantral and antral follicles throughout the entire ovary. The contralateral ovary from each animal was analyzed for DNA fragmentation indicative of apoptotic cell death. The results indicate that TCDD treatment significantly reduced the number of antral follicles in the size classes 50,000 to 74,999 microns2 and > 100,000 microns2. We also observed a reduction in the number of preantral follicles less than 50,000 microns2. No difference was observed in the degree of apoptotic cell death in antral (50,000 to > 100,000 microns2) and preantral follicles (50,000 microns2 to > 75,000 microns2) between TCDD-treated and control-treated tissues. These data support the hypothesis that TCDD results in a diminution in the number of antral and preantral follicles of certain size classes in animals exposed during critical periods of development, but that apoptosis does not appear to be the underlying mechanism in these particular

[ATP content in cryopreserved sperm of Siberian white cranes Grus leucogeranus (Aves: Gruiformes)].

PubMed

Maksudov, G Iu; Erokhin, A S; Nesterenko, O N; Panchenko, V G

2002-01-01

ATP contents were studied in the native and cryoconserved sperm of Siberian white cranes Grus leucogeranus using bioluminescence analysis. The ATP content in freshly obtained spermatozoa was 12.7 nmol/10(8) cells. No ATP was found in the seminal plasma. In the process of freezing-thawing, the ATP concentration in the spermatozoa decreased by 30%. The differences in the dynamics of ATP content during cryoconservation of sperm of white cranes and other birds and mammals are discussed.

Role of the cyclooxygenase 2-thromboxane pathway in 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced decrease in mesencephalic vein blood flow in the zebrafish embryo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Teraoka, Hiroki; Kubota, Akira; Dong, Wu

2009-01-01

Previously, we reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) evoked developmental toxicity required activation of aryl hydrocarbon receptor type 2 (AHR2), using zebrafish embryos. However, the downstream molecular targets of AHR2 activation are largely unknown and are the focus of the present investigation. TCDD induces cyclooxygenase 2 (COX2), a rate-limiting enzyme for prostaglandin synthesis in certain cells. In the present study, we investigated the role of the COX2-thromboxane pathway in causing a specific endpoint of TCDD developmental toxicity in the zebrafish embryo, namely, a decrease in regional blood flow in the dorsal midbrain. It was found that the TCDD-induced reduction in mesencephalic veinmore » blood flow was markedly inhibited by selective COX2 inhibitors, NS-398 and SC-236, and by a general COX inhibitor, indomethacin, but not by a selective COX1 inhibitor, SC-560. Gene knock-down of COX2 by two different types of morpholino antisense oligonucleotides, but not by their negative homologs, also protected the zebrafish embryos from mesencephalic vein circulation failure caused by TCDD. This inhibitory effect of TCDD on regional blood flow in the dorsal midbrain was also blocked by selective antagonists of the thromboxane receptor (TP). Treatment of control zebrafish embryos with a TP agonist also caused a reduction in mesencephalic vein blood flow and it too was blocked by a TP antagonist, without any effect on trunk circulation. Finally, gene knock-down of thromboxane A synthase 1 (TBXS) with morpholinos but not by the morpholinos' negative homologs provided significant protection against TCDD-induced mesencephalic circulation failure. Taken together, these results point to a role of the prostanoid synthesis pathway via COX2-TBXS-TP in the local circulation failure induced by TCDD in the dorsal midbrain of the zebrafish embryo.« less

Nisin depletes ATP and proton motive force in mycobacteria.

PubMed

Chung, H J; Montville, T J; Chikindas, M L

2000-12-01

This study examined the inhibitory effect of nisin and its mode of action against Mycobacterium smegmatis, a non-pathogenic species of mycobacteria, and M. bovis-Bacill Carmette Guerin (BCG), a vaccine strain of pathogenic M. bovis. In agar diffusion assays, 2.5 mg ml(-1) nisin was required to inhibit M. bovis-BCG. Nisin caused a slow, gradual, time- and concentration-dependent decrease in internal ATP levels in M. bovis-BCG, but no ATP efflux was detected. In mycobacteria, nisin decreased both components of proton motive force (membrane potential, Delta Psi and Delta pH) in a time- and concentration-dependent manner. However, mycobacteria maintained their intracellular ATP levels during the initial time period of Delta Psi and Delta pH dissipation. These data suggest that the mechanism of nisin in mycobacteria is similar to that in food-borne pathogens.

Environmental fate and dietary exposures of humans to TCDD as a result of the spraying of Agent Orange in upland forests of Vietnam.

PubMed

Armitage, James M; Ginevan, Michael E; Hewitt, Andrew; Ross, John H; Watkins, Deborah K; Solomon, Keith R

2015-02-15

The fate and transport of 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD) released into the environment of South Vietnam (SVN) as a consequence of the aerial application of the herbicidal defoliant Agent Orange (AO) were simulated for a generic upland forest scenario and followed over a 50-year period (1965, 1968 and 1970 onwards). Modeled concentrations of TCDD in the environment were then used as inputs to a human exposure model, which focused on long-term exposures via the food chain. Intake rates and body burdens of TCDD were estimated for adult males over the course of the simulation period and compared to available biomonitoring data. One of the most important factors determining the magnitude of the simulated human exposure to TCDD was the fraction of the chemical deposited directly to soil (where it was assumed to have a degradation half-life of 10 or 15years) relative to the fraction assumed to remain on/in the forest canopy following the spray application (where it was assumed to have a degradation half-life of ≤48h). The simulated body burdens under the various scenarios considered were broadly consistent with the biomonitoring data from SVN collected in the mid-1980s to late 1990s. Taken together, the modeling results and empirical data suggest that highly elevated exposures to TCDD (i.e., body burdens in the several 100s of pg/g lipid range and greater) were not common among people inhabiting upland forest locations in SVN sprayed with AO and that peak and average body burdens were broadly similar to those of the general population of the U.S. in the 1970s and early 1980s. The model-based assessment is consistent with the 'hot spot' hypothesis i.e., potential exposures to TCDD linked to activities conducted on or near former bases where AO was stored are greater than potential exposures in areas subjected to aerial spraying. Copyright © 2014 Elsevier B.V. All rights reserved.

Bilirubin Decreases Macrophage Cholesterol Efflux and ATP-Binding Cassette Transporter A1 Protein Expression.

PubMed

Wang, Dongdong; Tosevska, Anela; Heiß, Elke H; Ladurner, Angela; Mölzer, Christine; Wallner, Marlies; Bulmer, Andrew; Wagner, Karl-Heinz; Dirsch, Verena M; Atanasov, Atanas G

2017-04-28

Mild but chronically elevated circulating unconjugated bilirubin is associated with reduced total and low-density lipoprotein cholesterol concentration, which is associated with reduced cardiovascular disease risk. We aimed to investigate whether unconjugated bilirubin influences macrophage cholesterol efflux, as a potential mechanism for the altered circulating lipoprotein concentrations observed in hyperbilirubinemic individuals. Cholesterol efflux from THP-1 macrophages was assessed using plasma obtained from normo- and hyperbilirubinemic (Gilbert syndrome) humans (n=60 per group) or (heterozygote/homozygote Gunn) rats (n=20 per group) as an acceptor. Hyperbilirubinemic plasma from patients with Gilbert syndrome and Gunn rats induced significantly reduced cholesterol efflux compared with normobilirubinemic plasma. Unconjugated bilirubin (3-17.1 μmol/L) exogenously added to plasma- or apolipoprotein A1-supplemented media also decreased macrophage cholesterol efflux in a concentration- and time-dependent manner. We also showed reduced protein expression of the ATP-binding cassette transporter A1 (ABCA1), a transmembrane cholesterol transporter involved in apolipoprotein A1-mediated cholesterol efflux, in THP-1 macrophages treated with unconjugated bilirubin and in peripheral blood mononuclear cells obtained from hyperbilirubinemic individuals. Furthermore, we demonstrated that bilirubin accelerates the degradation rate of the ABCA1 protein in THP-1 macrophages. Cholesterol efflux from THP-1 macrophages is decreased in the presence of plasma obtained from humans and rats with mild hyperbilirubinemia. A direct effect of unconjugated bilirubin on cholesterol efflux was demonstrated and is associated with decreased ABCA1 protein expression. These data improve our knowledge concerning bilirubin's impact on cholesterol transport and represent an important advancement in our understanding of bilirubin's role in cardiovascular disease. © 2017 The Authors. Published on

Aldehyde dehydrogenase inhibition combined with phenformin treatment reversed NSCLC through ATP depletion.

PubMed

Kang, Joon Hee; Lee, Seon-Hyeong; Lee, Jae-Seon; Nam, Boas; Seong, Tae Wha; Son, Jaekyoung; Jang, Hyonchol; Hong, Kyeong Man; Lee, Cheolju; Kim, Soo-Youl

2016-08-02

Among ALDH isoforms, ALDH1L1 in the folate pathway showed highly increased expression in non-small-cell lung cancer cells (NSCLC). Based on the basic mechanism of ALDH converting aldehyde to carboxylic acid with by-product NADH, we suggested that ALDH1L1 may contribute to ATP production using NADH through oxidative phosphorylation. ALDH1L1 knockdown reduced ATP production by up to 60% concomitantly with decrease of NADH in NSCLC. ALDH inhibitor, gossypol, also reduced ATP production in a dose dependent manner together with decrease of NADH level in NSCLC. A combination treatment of gossypol with phenformin, mitochondrial complex I inhibitor, synergized ATP depletion, which efficiently induced cell death. Pre-clinical xenograft model using human NSCLC demonstrated a remarkable therapeutic response to the combined treatment of gossypol and phenformin.

Differential susceptibilities of Holtzman and Sprague-Dawley rats to fetal death and placental dysfunction induced by 2,3,7,8-teterachlorodibenzo-p-dioxin (TCDD) despite the identical primary structure of the aryl hydrocarbon receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawakami, Takashige; Department of Hygiene-Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510; Ishimura, Ryuta

2006-05-01

A single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioin (TCDD) administered to pregnant Holtzman (HLZ) rats on gestational days 15 (GD15) caused placental dysfunction, resulting in fetal death (Ishimura, R., Ohsako, S., Miyabara, Y., Sakaue, M., Kawakami, T., Aoki, Y., Yonemoto, J., Tohyama, C., 2002a. Increased glycogen content and glucose transporter 3 mRNA level in the placenta of Holtzman rats after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 178, 161-171; Ishimura, R., Ohsako, S., Kawakami, T., Sakaue, M., Aoki, Y., Tohyama, C., 2002b. Altered protein profile and possible hypoxia in the placenta of 2,3,7,8-tetrachlorodibenzo-p-dioxin-exposed rats. Toxicol. Appl. Pharmacol. 185, 197-206). In order to investigatemore » the mechanism underlying the TCDD-induced fetal death, we compared two outbred strains of rats, namely, the HLZ and the Sprague-Dawley International Genetic Standard rats (SD-IGS), a strain with characteristics resembling those of the HLZ rats. Pregnant HLZ and SD-IGS rats were administered TCDD as a single dose by gavage on GD15, as described within the parentheses (HLZ, 0, 1.6 {mu}g TCDD/kg; SD-IGS, 0, 2, 5, 10 {mu}g TCDD/kg). Whereas a high incidence (14%) of fetal death was observed on GD20 in the HLZ rats, no fetal deaths occurred in the SD-IGS rats, even at the highest dose of TCDD. A histological marker of cellular abnormality at the placental junctional zone, i.e., delay in the disappearance of the glycogen cells and cysts filled with an eosinophilic material (GC-EM), which normally disappear by GD20, was observed in the HLZ rats after exposure to the lowest dose of TCDD (1.6 {mu}g TCDD/kg), but not in the SD-IGS rats even after exposure to the highest dose of TCDD. Furthermore, maternal blood sinusoids in the labyrinth zone were constricted following exposure to TCDD in the HLZ, but not SD-IGS rats. These observations indicate that HLZ rats are more susceptible to the adverse effects of TCDD on fetal

EXPRESSION OF AHR AND ARNT MRNA IN CULTURED HUMAN ENDOMETRIAL EXPLANTS EXPOSED TO TCDD

EPA Science Inventory

Expression of AhR and ARNT mRNA in cultured human endometrial explants exposed to TCDD.

Pitt JA, Feng L, Abbott BD, Schmid J, Batt RE, Costich TG, Koury ST, Bofinger DP.

Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC 27599, USA.

Endom...

Microwave irradiation decreases ATP, increases free [Mg2+], and alters in vivo intracellular reactions in rat brain

PubMed Central

Srivastava, Shireesh; Kashiwaya, Yoshihiro; Chen, Xuesong; Geiger, Jonathan D.; Pawlosky, Robert; Veech, Richard L.

2012-01-01

Rapid inactivation of metabolism is essential for accurately determining the concentrations of metabolic intermediates in the in vivo state. We compared a broad spectrum of energetic intermediate metabolites and neurotransmitters in brains obtained by microwave irradiation to those obtained by freeze blowing, the most rapid method of extracting and freezing rat brain. The concentrations of many intermediates, cytosolic free NAD(P)+/NAD(P)H ratios, as well as neurotransmitters were not affected by the microwave procedure. However, the brain concentrations of ATP were about 30% lower, whereas those of ADP, AMP, and GDP were higher in the microwave-irradiated compared with the freeze-blown brains. In addition, the hydrolysis of approximately 1 μmol/g of ATP, a major in vivo Mg2+-binding site, was related to approximately five-fold increase in free [Mg2+] (0.53 ± 0.07 mM in freeze blown vs. 2.91 mM ± 0.48 mM in microwaved brains), as determined from the ratio [citrate]/[isocitrate]. Consequently, many intracellular properties, such as the phosphorylation potential and the ΔG’ of ATP hydrolysis were significantly altered in microwaved tissue. The determinations of some glycolytic and TCA cycle metabolites, the phosphorylation potential, and the ΔG’ of ATP hydrolysis do not represent the in vivo state when using microwave-fixed brain tissue. PMID:23013291

Diadenosine tetraphosphate-gating of recombinant pancreatic ATP-sensitive K(+) channels.

PubMed

Jovanovic, S; Jovanovic, A

2001-02-01

Diadenosine tetraphosphate (Ap4A) has been recently discovered in the pancreatic beta cells where targets ATP-sensitive K(+) (K(ATP)) channels, depolarizes the cell membrane and induces insulin secretion. However, whether Ap4A inhibit pancreatic K(ATP) channels by targeting protein channel complex itself was unknown. Therefore, we coexpressed pancreatic K(ATP) channel subunits, Kir6.2 and SUR1, in COS-7 cells and examined the effect of Ap4A on the single channel behavior using the inside-out configuration of the patch-clamp technique. Ap4A inhibited channel opening in a concentration-dependent manner. Analysis of single channels demonstrated that Ap4A did not change intraburst kinetic behavior of K(ATP) channels, but rather decreased burst duration and increased between-burst duration. It is concluded that Ap4A antagonizes K(ATP) channel opening by targeting channel subunits themselves and by keeping channels longer in closed interburst states.

ATP11B mediates platinum resistance in ovarian cancer

PubMed Central

Moreno-Smith, Myrthala; Halder, J.B.; Meltzer, Paul S.; Gonda, Tamas A.; Mangala, Lingegowda S.; Rupaimoole, Rajesha; Lu, Chunhua; Nagaraja, Archana S.; Gharpure, Kshipra M.; Kang, Yu; Rodriguez-Aguayo, Cristian; Vivas-Mejia, Pablo E.; Zand, Behrouz; Schmandt, Rosemarie; Wang, Hua; Langley, Robert R.; Jennings, Nicholas B.; Ivan, Cristina; Coffin, Jeremy E.; Armaiz, Guillermo N.; Bottsford-Miller, Justin; Kim, Sang Bae; Halleck, Margaret S.; Hendrix, Mary J.C.; Bornman, William; Bar-Eli, Menashe; Lee, Ju-Seog; Siddik, Zahid H.; Lopez-Berestein, Gabriel; Sood, Anil K.

2013-01-01

Platinum compounds display clinical activity against a wide variety of solid tumors; however, resistance to these agents is a major limitation in cancer therapy. Reduced platinum uptake and increased platinum export are examples of resistance mechanisms that limit the extent of DNA damage. Here, we report the discovery and characterization of the role of ATP11B, a P-type ATPase membrane protein, in cisplatin resistance. We found that ATP11B expression was correlated with higher tumor grade in human ovarian cancer samples and with cisplatin resistance in human ovarian cancer cell lines. ATP11B gene silencing restored the sensitivity of ovarian cancer cell lines to cisplatin in vitro. Combined therapy of cisplatin and ATP11B-targeted siRNA significantly decreased cancer growth in mice bearing ovarian tumors derived from cisplatin-sensitive and -resistant cells. In vitro mechanistic studies on cellular platinum content and cisplatin efflux kinetics indicated that ATP11B enhances the export of cisplatin from cells. The colocalization of ATP11B with fluorescent cisplatin and with vesicular trafficking proteins, such as syntaxin-6 (STX6) and vesicular-associated membrane protein 4 (VAMP4), strongly suggests that ATP11B contributes to secretory vesicular transport of cisplatin from Golgi to plasma membrane. In conclusion, inhibition of ATP11B expression could serve as a therapeutic strategy to overcome cisplatin resistance. PMID:23585472

Aldehyde dehydrogenase inhibition combined with phenformin treatment reversed NSCLC through ATP depletion

PubMed Central

Lee, Jae-Seon; Nam, Boas; Seong, Tae Wha; Son, Jaekyoung; Jang, Hyonchol; Hong, Kyeong Man; Lee, Cheolju; Kim, Soo-Youl

2016-01-01

Among ALDH isoforms, ALDH1L1 in the folate pathway showed highly increased expression in non-small-cell lung cancer cells (NSCLC). Based on the basic mechanism of ALDH converting aldehyde to carboxylic acid with by-product NADH, we suggested that ALDH1L1 may contribute to ATP production using NADH through oxidative phosphorylation. ALDH1L1 knockdown reduced ATP production by up to 60% concomitantly with decrease of NADH in NSCLC. ALDH inhibitor, gossypol, also reduced ATP production in a dose dependent manner together with decrease of NADH level in NSCLC. A combination treatment of gossypol with phenformin, mitochondrial complex I inhibitor, synergized ATP depletion, which efficiently induced cell death. Pre-clinical xenograft model using human NSCLC demonstrated a remarkable therapeutic response to the combined treatment of gossypol and phenformin. PMID:27384481

ATP depletion inhibits glucocorticoid-induced thymocyte apoptosis.

PubMed Central

Stefanelli, C; Bonavita, F; Stanic', I; Farruggia, G; Falcieri, E; Robuffo, I; Pignatti, C; Muscari, C; Rossoni, C; Guarnieri, C; Caldarera, C M

1997-01-01

In quiescent thymocytes, mitochondrial de-energization was not correlated to apoptotic death. In fact, thymocytes treated with oligomycin, a highly specific inhibitor of ATP synthase, alone or with atractyloside to block ATP translocation from the cytoplasm, were alive, even if their mitochondria were depolarized, as revealed by flow cytometry after Rhodamine 123 staining. Furthermore, oligomycin was a powerful inhibitor of apoptosis induced in rat thymocytes by dexamethasone and, to a lesser extent, by the calcium ionophore A23187 and etoposide, but was without effect when apoptosis was induced by staurosporine, and increased cell death in mitogen-treated thymocytes. The inhibition of apoptosis was confirmed by morphological criteria, inhibition of inter-nucleosomal DNA fragmentation and inhibition of the loss of membrane integrity. The anti-apoptotic effect of oligomycin in cells treated with A23187 or etoposide was correlated to the inhibition of protein synthesis, while inhibition of apoptosis induced by dexamethasone, already evident at an oligomycin concentration of 10 ng/ml, was instead strictly correlated to the effect exerted on the cellular ATP level. Thymocyte apoptosis triggered by dexamethasone was blocked or delayed by inhibitors of respiratory-chain uncouplers, inhibitors of ATP synthase and antioxidants: a lasting protection from dexamethasone-induced apoptosis was always correlated to a drastic and rapid reduction in ATP level (31-35% of control), while a delay in the death process was characterized by a moderate decrease in ATP (73-82% of control). Oligomycin inhibited the specific binding of radioactive corticosteroid to thymocyte nuclei, confirming the inhibitory effect of ATP depletion on glucocorticoid binding and suggesting that ATP depletion is a common mediator of the anti-apoptotic action of different effectors in glucocorticoid-induced apoptosis. In conclusion, the reported data indicate that ATP may act as a cellular modulator of some

Hypophosphatemia promotes lower rates of muscle ATP synthesis.

PubMed

Pesta, Dominik H; Tsirigotis, Dimitrios N; Befroy, Douglas E; Caballero, Daniel; Jurczak, Michael J; Rahimi, Yasmeen; Cline, Gary W; Dufour, Sylvie; Birkenfeld, Andreas L; Rothman, Douglas L; Carpenter, Thomas O; Insogna, Karl; Petersen, Kitt Falk; Bergwitz, Clemens; Shulman, Gerald I

2016-10-01

Hypophosphatemia can lead to muscle weakness and respiratory and heart failure, but the mechanism is unknown. To address this question, we noninvasively assessed rates of muscle ATP synthesis in hypophosphatemic mice by using in vivo saturation transfer [ 31 P]-magnetic resonance spectroscopy. By using this approach, we found that basal and insulin-stimulated rates of muscle ATP synthetic flux (V ATP ) and plasma inorganic phosphate (P i ) were reduced by 50% in mice with diet-induced hypophosphatemia as well as in sodium-dependent P i transporter solute carrier family 34, member 1 (NaPi2a)-knockout (NaPi2a -/- ) mice compared with their wild-type littermate controls. Rates of V ATP normalized in both hypophosphatemic groups after restoring plasma P i concentrations. Furthermore, V ATP was directly related to cellular and mitochondrial P i uptake in L6 and RC13 rodent myocytes and isolated muscle mitochondria. Similar findings were observed in a patient with chronic hypophosphatemia as a result of a mutation in SLC34A3 who had a 50% reduction in both serum P i content and muscle V ATP After oral P i repletion and normalization of serum P i levels, muscle V ATP completely normalized in the patient. Taken together, these data support the hypothesis that decreased muscle ATP synthesis, in part, may be caused by low blood P i concentrations, which may explain some aspects of muscle weakness observed in patients with hypophosphatemia.-Pesta, D. H., Tsirigotis, D. N., Befroy, D. E., Caballero, D., Jurczak, M. J., Rahimi, Y., Cline, G. W., Dufour, S., Birkenfeld, A. L., Rothman, D. L., Carpenter, T. O., Insogna, K., Petersen, K. F., Bergwitz, C., Shulman, G. I. Hypophosphatemia promotes lower rates of muscle ATP synthesis. © The Author(s).

MECHANISMS OF TCDD-INDUCTION OF CLEFT PALATE: INSIGHTS FROM IN VIVO AND IN VITRO APPROACHES

EPA Science Inventory

TCDD induced cleft palate (CP) in C57BL6N embryos by altering proliferation and differentiation of palatal medial epithelial cells. hese effects correlated with altered expression of the growth factors, TGF-a,EGF. TGF-B1 and TGF-B2, and the EGF receptor. ynergistic interactions b...

COMPARING ENVIRONMENTALLY RELEVANT PCBS TO TCDD IN CYP1A2 NULL AND WILDTYPE MICE

EPA Science Inventory

The role of CYP1A2 on the interactions of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, dioxin), dioxin-like (DL) and non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) was compared in multiple responses of different laboratory-defined mixtures, based on mass ratios found in...

Renal epithelial cells can release ATP by vesicular fusion

PubMed Central

Bjaelde, Randi G.; Arnadottir, Sigrid S.; Overgaard, Morten T.; Leipziger, Jens; Praetorius, Helle A.

2013-01-01

Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30), which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1) cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin) reduced both the spontaneous and hypotonically (80%)-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1) and vesicular transport (nocodazole). These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ~90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP) or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50%) or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8 and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells. PMID:24065923

S-Sulfhydration of ATP synthase by hydrogen sulfide stimulates mitochondrial bioenergetics.

PubMed

Módis, Katalin; Ju, YoungJun; Ahmad, Akbar; Untereiner, Ashley A; Altaany, Zaid; Wu, Lingyun; Szabo, Csaba; Wang, Rui

2016-11-01

(when systemic H 2 S levels decrease) S-sulfhydration of ATP5A1 decreased as well. In conclusion, H 2 S induces S-sulfhydration of ATP5A1 at C244 and C294. This post-translational modification may be a physiological mechanism to maintain ATP synthase in a physiologically activated state, thereby supporting mitochondrial bioenergetics. The sulfhydration of ATP synthase may be a dynamic process, which may be regulated by endogenous H 2 S levels under various pathophysiological conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparison of PFDA (Perfluoro-n-Decanoic Acid) and TCDD on Heart Membranes.

DTIC Science & Technology

1986-06-18

AD-A171 960 COMPARISON OF PFDA ( PERFLUORO -N-DECANOIC ACID) AND TCDD 1/1 ON HEART NEMBRANES(U) WRIGHT STATE UNIY DAYTON OH SCHOOL OF MEDICINE A E...1986) Toxicol. Appl. Pharmacol. Perfluoro -n-decanoic acid ( PFDA ) is a synthetic chemical resembling a 10 carbon fatty acid. Several studies have...3 INTRODUCTION Perfluoro -n-decanoic acid ( PFDA ; nonadecafluorodecanoic acid, C10 F19 0 2H) is a straight-chain 10 carbon carboxylic acid with fluorine

Substrate recognition by the hetero-octameric ATP phosphoribosyltransferase from Lactococcus lactis†

PubMed Central

Champagne, Karen S.; Piscitelli, Elise; Francklyn, Christopher S.

2008-01-01

Two families of ATP phosphoribosyl transferases (ATP-PRT) join ATP and 5-phosphoribosyl-1 pyrophosphate (PRPP) in the first reaction of histidine biosynthesis. These consist of a homohexameric form found in all three kingdoms, and a hetero-octameric form largely restricted to bacteria. Hetero-octameric ATP-PRTs consist of four HisGS catalytic subunits related to periplasmic binding proteins, and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. To clarify the relationship between the two families of ATP-PRTs, and among phosphoribosyltransferases in general, we determined the steady state kinetics for the hetero-octameric form, and characterized the active site by mutagenesis. The Km PRPP (18.4 ± 3.5 μM) and kcat (2.7 ± 0.3 sec−1) values for the PRPP substrate are similar to those of hexameric ATP-PRTs, but the Km for ATP (2.7 ± 0.3 mM) is 4-fold higher, suggestive of tighter regulation by energy charge. Histidine and AMP were determined to be non-competitive (Ki = 81.1 μM) and competitive (Ki= 1.44 mM) inhibitors, respectively, with values that approximate their intracellular concentrations. Mutagenesis experiments investigating the side chains recognizing PRPP showed that 5′ phosphate contacts (T159A and T162A) had the largest (25- and 155-fold) decreases in kcat/Km, while smaller decreases were seen with mutants making cross subunit contacts (K50A and K8A) to the pyrophosphate moiety, or contacts to the 2′ OH. Despite their markedly different quaternary structures, hexameric and hetero-octameric ATRP-PRTs exhibit similar functional parameters, and employ mechanistic strategies reminiscent of the broader PRT superfamily. PMID:17154531

Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport

NASA Technical Reports Server (NTRS)

Tang, Wenqiang; Brady, Shari R.; Sun, Yu; Muday, Gloria K.; Roux, Stanley J.

2003-01-01

Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

Mechanisms of charge transfer in human copper ATPases ATP7A and ATP7B.

PubMed

Tadini-Buoninsegni, Francesco; Smeazzetto, Serena

2017-04-01

ATP7A and ATP7B are Cu + -transporting ATPases of subclass IB and play a fundamental role in intracellular copper homeostasis. ATP7A/B transfer Cu + ions across the membrane from delivery to acceptor proteins without establishing a free Cu + gradient. Transfer of copper across the membrane is coupled to ATP hydrolysis. Current measurements on solid supported membranes (SSM) were performed to investigate the mechanism of copper-related charge transfer across ATP7A and ATP7B. SSM measurements demonstrated that electrogenic copper displacement occurs within ATP7A/B following addition of ATP and formation of the phosphorylated intermediate. Comparison of the time constants for cation displacement in ATP7A/B and sarcoplasmic reticulum Ca 2+ -ATPase is consistent with the slower phosphoenzyme formation in copper ATPases. Moreover, ATP-dependent copper transfer in ATP7A/B is not affected by varying the pH, suggesting that net proton counter-transport may not occur in copper ATPases. Platinum anticancer drugs activate ATP7A/B and are subjected to ATP-dependent vectorial displacement with a mechanism analogous to that of copper. © 2016 IUBMB Life, 69(4):218-225, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

ADVERSE EFFECTS OF TCDD ON MAMMARY GLAND DEVELOPMENT IN LONG EVANS RATS: A TWO GENERATIONAL STUDY

EPA Science Inventory

Recent studies have demonstrated variable effects on mammary gland development in rat offspring exposed to TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, 1 ug/kg, gavage) on day 15 of gestation. We have characterized these effects in Long Evans rats, in both one and two-generational...

ATP-induced noncooperative thermal unfolding of hen lysozyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Honglin; Yin, Peidong; He, Shengnan

To understand the role of ATP underlying the enhanced amyloidosis of hen egg white lysozyme (HEWL), the synchrotron radiation circular dichroism, combined with tryptophan fluorescence, dynamic light-scattering, and differential scanning calorimetry, is used to examine the alterations of the conformation and thermal unfolding pathway of the HEWL in the presence of ATP, Mg{sup 2+}-ATP, ADP, AMP, etc. It is revealed that the binding of ATP to HEWL through strong electrostatic interaction changes the secondary structures of HEWL and makes the exposed residue W62 move into hydrophobic environments. This alteration of W62 decreases the {beta}-domain stability of HEWL, induces a noncooperativemore » unfolding of the secondary structures, and produces a partially unfolded intermediate. This intermediate containing relatively rich {alpha}-helix and less {beta}-sheet structures has a great tendency to aggregate. The results imply that the ease of aggregating of HEWL is related to the extent of denaturation of the amyloidogenic region, rather than the electrostatic neutralizing effect or monomeric {beta}-sheet enriched intermediate.« less

Lipidomic and metabolic changes in the P4-type ATPase ATP10D deficient C57BL/6J wild type mice upon rescue of ATP10D function.

PubMed

Sigruener, Alexander; Wolfrum, Christian; Boettcher, Alfred; Kopf, Thomas; Liebisch, Gerhard; Orsó, Evelyn; Schmitz, Gerd

2017-01-01

Sequence variants near the human gene for P4-type ATPase, class V, type 10D (ATP10D) were shown to significantly associate with circulating hexosylceramide d18:1/16:0 and d18:1/24:1 levels, obesity, insulin resistance, plasma high density lipoprotein (HDL), coronary stenotic index and intracranial atherosclerotic index. In mice Atp10d is associated with HDL modulation and C57BL/6 mice expressing a truncated, non-functional form of ATP10D easily develop obesity and insulin resistance on high-fat diet. We analyzed metabolic differences of ATP10D deficient C57BL/6J wild type and ATP10D transgenic C57BL/6J BAC129 mice. ATP10D transgenic mice gain 25% less weight on high-fat diet concomitant with a reduced increase in fat cell mass but independent of adipocyte size change. ATP10D transgenic mice also had 26% lower triacylglycerol levels with approximately 76% bound to very low density lipoprotein while in ATP10D deficient wild type mice 57% are bound to low density lipoprotein. Furthermore increased oxygen consumption and CO2 production, 38% lower glucose and 69% lower insulin levels and better insulin sensitivity were observed in ATP10D transgenic mice. Besides decreased hexosylceramide species levels were detected. Part of these effects may be due to reduced hepatic stearoyl-CoA desaturase 1 (SCD1) expression in ATP10D transgenic mice, which was reflected by altered fatty acid and lipid species patterns. There was a significant decrease in the hepatic 18:1 to 18:0 free fatty acid ratio in transgenic mice. The ratio of 16:1 to 16:0 was not significantly different. Interestingly both ratios were significantly reduced in plasma total fatty acids. In summary we found that ATP10D reduces high-fat diet induced obesity and improves insulin sensitivity. ATP10D transgenic mice showed altered hepatic expression of lipid-metabolism associated genes, including Scd1, along with changes in hepatic and plasma lipid species and plasma lipoprotein pattern.

ATP synthase.

PubMed

Junge, Wolfgang; Nelson, Nathan

2015-01-01

Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms.

Piezo1 regulates mechanotransductive release of ATP from human RBCs.

PubMed

Cinar, Eyup; Zhou, Sitong; DeCourcey, James; Wang, Yixuan; Waugh, Richard E; Wan, Jiandi

2015-09-22

Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca(2+)) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca(2+) influx decrease significantly. Remarkably, a critical extracellular Ca(2+) concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease.

Extracellular ATP Acts on Jasmonate Signaling to Reinforce Plant Defense.

PubMed

Tripathi, Diwaker; Zhang, Tong; Koo, Abraham J; Stacey, Gary; Tanaka, Kiwamu

2018-01-01

Damaged cells send various signals to stimulate defense responses. Recent identification and genetic studies of the plant purinoceptor, P2K1 (also known as DORN1), have demonstrated that extracellular ATP is a signal involved in plant stress responses, including wounding, perhaps to evoke plant defense. However, it remains largely unknown how extracellular ATP induces plant defense responses. Here, we demonstrate that extracellular ATP induces plant defense mediated through activation of the intracellular signaling of jasmonate (JA), a well-characterized defense hormone. In Arabidopsis ( Arabidopsis thaliana ) leaves, ATP pretreatment induced resistance against the necrotrophic fungus, Botrytis cinerea The induced resistance was enhanced in the P2K1 receptor overexpression line, but reduced in the receptor mutant, dorn1 - 3 Mining the transcriptome data revealed that ATP induces a set of JA-induced genes. In addition, the P2K1-associated coexpression network contains defense-related genes, including those encoding jasmonate ZIM-domain (JAZ) proteins, which play key roles as repressors of JA signaling. We examined whether extracellular ATP impacts the stability of JAZ1 in Arabidopsis. The results showed that the JAZ1 stability decreased in response to ATP addition in a proteasome-dependent manner. This reduction required intracellular signaling via second messengers-cytosolic calcium, reactive oxygen species, and nitric oxide. Interestingly, the ATP-induced JAZ1 degradation was attenuated in the JA receptor mutant, coi1 , but not in the JA biosynthesis mutant, aos , or upon addition of JA biosynthesis inhibitors. Immunoprecipitation analysis demonstrated that ATP increases the interaction between COI1 and JAZ1, suggesting direct cross talk between extracellular ATP and JA in intracellular signaling events. Taken together, these results suggest that extracellular ATP signaling directly impacts the JA signaling pathway to maximize plant defense responses. © 2018

INFLUENCE OF TYPE II DIABETES AND OBESITY ON THE DISPOSITION AND ELIMINATION OF TCDD IN MICE

EPA Science Inventory

INFLUENCE OF TYPE II DIABETES AND OBESITY ON THE DISPOSTION AND ELIMINATION OF TCDD IN MICE. MJ DeVito', JJ Diliberto', DG Ross', C Emond2, VM Richardson', and LS Birnbaum', 'ETD, NHEERL, ORD, US EPA, RTP, NC, 27711, USA, 2National Research Council.
One possible explanation fo...

Contractile activity of ATP and diadenosine tetraphosphate on urinary bladder in the rats: role of superoxide anion and urothelium.

PubMed

Khattab, M M; Al-Hrasen, M N

2006-04-01

Both ATP and diadenosine tetraphosphate (AP(4)A) produced a dose-dependent contraction of rat isolated urinary bladder rings. The AP(4)A dose-response curve was to the left of that of ATP, and the maximum response was greater than that produced by ATP. Mechanical removal of the urothelium increased the contractile response to ATP by between 53% and 71%, and that to AP(4)A by 42% (at highest AP(4)A concentration) to 68% at lower concentration. Inhibition of Cu/Zn superoxide dismutase with diethylthiocarbamate (DETCA, 5 mm) significantly reduced the ATP-evoked contraction by 31% (at high ATP concentration) to 40% at low ATP concentration. Similarly, the AP(4)A-induced contractions were significantly decreased by 27% at low AP(4)A level to 38% at higher concentrations. Induction of exogenous superoxide anion stress by the use of the superoxide anion generator, pyrogallol (0.5 mm), significantly decreased both ATP- and AP(4)A-induced contractions of the rat urinary bladder over the whole dose range. Contractile responses to ATP decreased by 36-40%, and those to AP(4)A by 44-49%. In conclusion, the urinary bladder urothelium exerts an inhibitory control over the purinergic contractility produced by adenine mononucleotides and dinucleotides. Superoxide anion stress, whether endogenous or exogenous, attenuates the ATP-induced as well as AP(4)A-induced contractility.

The effect of dose on 2,3,7,8-TCDD tissue distribution, metabolism and elimination in CYP1A2(-/_) knockout and C57BL/6N parental strains of mice

EPA Science Inventory

Numerous metabolism studies have demonstrated that the toxic contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is poorly metabolized. A hallmark feature of TCDD exposure is induction of hepatic CYP1A2 and subsequent sequestration leading to high liver-to-fat concentration ra...

Contraction of small mesenteric arteries induced by micromolar concentrations of ATP released from caged ATP.

PubMed

Sjöblom-Widfeldt, N; Arner, A; Nilsson, H

1993-01-01

The concentration dependence of ATP-induced contractions in isolated resistance arteries was estimated using photolysis of caged ATP. Rat mesenteric vessels were isolated and mounted for force registration in a small chamber allowing illumination from a xenon-flash lamp. Photolysis of 100 microM caged ATP, which released about 20 microM ATP within a few milliseconds in the vessel, induced a transient contraction with an amplitude approximately 40-50% of the response induced by 10 microM noradrenaline. The responses could neither be induced by the light flash as such nor by caged ATP alone nor by photolysis of caged phosphate. The amplitude of the contractions was dependent on the concentration of caged ATP, and the effective concentration for ATP was estimated to be in the range of 1-10 microM. In contrast, when ATP was introduced by diffusion, about a 100-fold higher concentration was required. Thus photolytic release of ATP minimizes metabolism before its action on receptors and reveals action of ATP in a concentration range consistent with a role of ATP as a transmitter in nervous regulation of the tone of resistance vessels.

Percutaneous Absorption of 2,3,7,8-Tetrachlorodibenzo-P-Dioxin (TCDD) From Soil (Journal Article)

EPA Science Inventory

Eight dermal absorption (two in vivo; six in vitro) and one intravenous experiment were conducted using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) either neat (high dose at ~250 µg/cm2 and low dose at 10 ng/cm2) or sorbed on a low organic (LOS) or high organic (HOS) soil at 1 ppm...

LASER CAPTURE MICRODISSECTION AND GENE ARRAY ANALYSIS OF PALATAL EPITHELIAL AND MESENCHYMAL CELLS EXPOSED TO TCDD

EPA Science Inventory

Palatal shelves from embryos exposed on gestation day (GD) 12 to either retinoic acid (RA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) contact but fail to fuse. It is of interest to know if diverse agents that induce clefting via the same etiology also activate the same biochem...

Failure of ATP supply to match ATP demand: the mechanism of toxicity of the lampricide, 3-trifluoromethyl-4-nitrophenol (TFM), used to control sea lamprey (Petromyzon marinus) populations in the Great Lakes.

PubMed

Birceanu, Oana; McClelland, Grant B; Wang, Yuxiang S; Wilkie, Michael P

2009-10-04

Although the pesticide, 3-trifluoromethyl-4-nitrophenol (TFM), has been extensively used to control invasive sea lamprey (Petromyzon marinus) populations in the Great Lakes, it is surprising that its mechanism(s) of toxicity is unresolved. A better knowledge of the mode of toxicity of this pesticide is needed for predicting and improving the effectiveness of TFM treatments on lamprey, and for risk assessments regarding potential adverse effects on invertebrate and vertebrate non-target organisms. We investigated two hypotheses of TFM toxicity in larval sea lamprey. The first was that TFM interferes with oxidative ATP production by mitochondria, causing rapid depletion of energy stores in vital, metabolically active tissues such as the liver and brain. The second was that TFM toxicity resulted from disruption of gill-ion uptake, adversely affecting ion homeostasis. Exposure of larval sea lamprey to 4.6 m gl(-1) TFM (12-h LC50) caused glycogen concentrations in the brain to decrease by 80% after 12h, suggesting that the animals increased their reliance on glycolysis to generate ATP due to a shortfall in ATP supply. This conclusion was reinforced by a 9-fold increase in brain lactate concentration, a 30% decrease in brain ATP concentration, and an 80% decrease in phosphocreatine (PCr) concentration after 9 and 12h. A more pronounced trend was noted in the liver, where glycogen decreased by 85% and ATP was no longer detected after 9 and 12h. TFM led to marginal changes in whole body Na(+), Cl(-), Ca(2+) and K(+), as well as in plasma Na(+) and Cl(-), which were unlikely to have contributed to toxicity. TFM had no adverse effect on Na(+) uptake rates or gill Na(+)/K(+)-ATPase activity. We conclude that TFM toxicity in the sea lamprey is due to a mismatch between ATP consumption and ATP production rates, leading to a depletion of glycogen in the liver and brain, which ultimately leads to neural arrest and death.

Dynamics of shear-induced ATP release from red blood cells.

PubMed

Wan, Jiandi; Ristenpart, William D; Stone, Howard A

2008-10-28

Adenosine triphosphate (ATP) is a regulatory molecule for many cell functions, both for intracellular and, perhaps less well known, extracellular functions. An important example of the latter involves red blood cells (RBCs), which help regulate blood pressure by releasing ATP as a vasodilatory signaling molecule in response to the increased shear stress inside arterial constrictions. Although shear-induced ATP release has been observed widely and is believed to be triggered by deformation of the cell membrane, the underlying mechanosensing mechanism inside RBCs is still controversial. Here, we use an in vitro microfluidic approach to investigate the dynamics of shear-induced ATP release from human RBCs with millisecond resolution. We demonstrate that there is a sizable delay time between the onset of increased shear stress and the release of ATP. This response time decreases with shear stress, but surprisingly does not depend significantly on membrane rigidity. Furthermore, we show that even though the RBCs deform significantly in short constrictions (duration of increased stress <3 ms), no measurable ATP is released. This critical timescale is commensurate with a characteristic membrane relaxation time determined from observations of the cell deformation by using high-speed video. Taken together our results suggest a model wherein the retraction of the spectrin-actin cytoskeleton network triggers the mechanosensitive ATP release and a shear-dependent membrane viscosity controls the rate of release.

Deformities, PCBs, and TCDD-equivalents in double-crested cormorants (Phalacrocorax auritus) and Caspian terns (Hydroprogne caspia) of the Upper Great Lakes 1986–1991: Testing a cause-effect hypothesis

USGS Publications Warehouse

Ludwig, James P.; Kurita-Matsuba, Hiroko; Auman, Heidi J.; Ludwig, Matthew E.; Summer, Cheryl L.; Giesy, John P.; Tillitt, Donald E.; Jones, Paul D.

1996-01-01

Deformities have been reported in many species of colonial waterbirds from several localities on the Laurentian Great Lakes. The hypothesis that deformities were caused by either polychlorinated biphenyls (PCBs) or contaminants measured as 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQs) is tested in this review of available data on concentrations of contaminants in eggs and observed deformities in embryos and chicks of double-crested cormorants (Phalacrocorax auritus) and Caspian terns (Hydroprogne caspia) between 1986 and 1991. Hatched chicks, live and dead eggs retrieved from 37 colonies in the upper Great Lakes were assessed for gross anatomical deformities. Rates of embryo death from seven regions of the upper Great Lakes were measured annually between 1986–1991. Half the embryos found dead in eggs were deformed. Nineteen types of abnormalities or deformities were observed. Subcutaneous edema in cormorants and gastroschisis in terns were the most common abnormalities in live or dead eggs. One of ten crossed-billed cormorant embryos survived to hatch. No bill-deformed terns hatched, although tern embryos had a greater rate of crossed-bills than cormorants. The suite of deformities and abnormalities found was similar to that produced in chickens by exposure to planar polychlorinated biphenyl (pPCB) and dioxin congeners. Hatching and deformity rates were correlated with concentrations ofpPCBs and TCDD-EQs. Planar PCB congeners that contributed most of the TCDD-EQs were present at concentrations sufficient to cause the observed effects. TCDD-EQs measured by H4IIE rat hepatoma cell 7-ethoxyresorufin O-deethylase (EROD) bioassay were highly correlated with deformity rates observed in cormorant chicks, live and dead eggs, and egg death rates. Similar correlations of TCDD-EQs with deformity rates were found in hatched tern chicks, dead eggs, and egg death rates, but not in live eggs. TCDD-EQs were more highly correlated to deformity and embryo death rates

Sensitivity of small myosin II ensembles from different isoforms to mechanical load and ATP concentration.

PubMed

Erdmann, Thorsten; Bartelheimer, Kathrin; Schwarz, Ulrich S

2016-11-01

Based on a detailed crossbridge model for individual myosin II motors, we systematically study the influence of mechanical load and adenosine triphosphate (ATP) concentration on small myosin II ensembles made from different isoforms. For skeletal and smooth muscle myosin II, which are often used in actomyosin gels that reconstitute cell contractility, fast forward movement is restricted to a small region of phase space with low mechanical load and high ATP concentration, which is also characterized by frequent ensemble detachment. At high load, these ensembles are stalled or move backwards, but forward motion can be restored by decreasing ATP concentration. In contrast, small ensembles of nonmuscle myosin II isoforms, which are found in the cytoskeleton of nonmuscle cells, are hardly affected by ATP concentration due to the slow kinetics of the bound states. For all isoforms, the thermodynamic efficiency of ensemble movement increases with decreasing ATP concentration, but this effect is weaker for the nonmuscle myosin II isoforms.

Inhibition of plasma membrane Ca(2+)-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca(2+)-occluded state.

PubMed

Moreira, Otacilio C; Rios, Priscila F; Barrabin, Hector

2005-07-15

The bidentate complex of ATP with Cr(3+), CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca(2+)-ATPase and the Na(+),K(+)-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca(2+) and Na(+), respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca(2+)-ATPase. The complex inhibited with similar efficiency the Ca(2+)-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly (T(1/2)=30 min at 37 degrees C) with a K(i)=28+/-9 microM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg(2+) but unaltered when Ca(2+) was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca(2+) occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La(3+) with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca(2+) at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca(2+) promoted by the plasma membrane Ca(2+)-ATPase goes through an enzymatic phospho-intermediate that maintains Ca(2+) ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.

The Effect of Dose on 2,3,7,8-TCDD Tissue Distribution, Metabolism and Elimination in CYP1A2 (-/-) Knockout and C57BL/6N Parental Strains of Mice

USDA-ARS?s Scientific Manuscript database

Numerous metabolism studies have demonstrated that the highly toxic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is poorly metabolized. A hallmark feature of TCDD exposure is induction of hepatic CYP1A2 and subsequent sequestration leading to high liver to fat concentration ratios. This study was in...

ATPase activity and light scattering of acto-heavy meromyosin: dependence on ATP concentration and on ionic strength.

PubMed

Dancker, P

1975-01-01

1. The dependence on ATP concentration of ATPase activity and light scattering decrease of acto-HMM could be described at very low ionic strength by one hyperbolic adsorption isotherm with a dissociation constant of 3 X 10(-6)M. Hence the increase of ATP ase activity was paralleled by a decrease in light scattering. At higher values of ionic strength ATPase activity stopped rising before HMM was completely saturated with ATP. Higher ionic strength prevented ATPase activity from further increasing when the rigor links (links between actin and nucleotide-free myosin), which have formerly protected the ATPase against the suppressing action of higher ionic strength have fallen below a certain amount. This protecting influence of rigor links did not require tropomyosin-troponin. 2. For complete activation of ATPase activity by actin less actin was needed when HMM was incompletely saturated with ATP than when it was completely saturated with ATP. 3. The apparent affinity of ATP to regulated acto-HMM (which contained tropomyosin-troponin) was lower than to unregulated acto-HMM (which was devoid of tropomyosin-troponin). In the presence of rigor complexes (indicated by an incomplete decrease of light scattering) the ATPase activity of regulated acto-HMM was higher than that of unregulated acto-HMM. At increasing ATP concentrations the ATPase activity of regulated acto-HMM stopped rising at a similar degree of saturation with ATP as the ATPase activity of unregulated acto-HMM at the same ionic strength.

Chi-square analysis of the reduction of ATP levels in L-02 hepatocytes by hexavalent chromium.

PubMed

Yuan, Yang; Peng, Li; Gong-Hua, Hu; Lu, Dai; Xia-Li, Zhong; Yu, Zhou; Cai-Gao, Zhong

2012-06-01

This study explored the reduction of adenosine triphosphate (ATP) levels in L-02 hepatocytes by hexavalent chromium (Cr(VI)) using chi-square analysis. Cells were treated with 2, 4, 8, 16, or 32 μM Cr(VI) for 12, 24, or 36 h. Methyl thiazolyl tetrazolium (MTT) experiments and measurements of intracellular ATP levels were performed by spectrophotometry or bioluminescence assays following Cr(VI) treatment. The chi-square test was used to determine the difference between cell survival rate and ATP levels. For the chi-square analysis, the results of the MTT or ATP experiments were transformed into a relative ratio with respect to the control (%). The relative ATP levels increased at 12 h, decreased at 24 h, and increased slightly again at 36 h following 4, 8, 16, 32 μM Cr(VI) treatment, corresponding to a "V-shaped" curve. Furthermore, the results of the chi-square analysis demonstrated a significant difference of the ATP level in the 32-μM Cr(VI) group (P < 0.05). The results suggest that the chi-square test can be applied to analyze the interference effects of Cr(VI) on ATP levels in L-02 hepatocytes. The decreased ATP levels at 24 h indicated disruption of mitochondrial energy metabolism and the slight increase of ATP levels at 36 h indicated partial recovery of mitochondrial function or activated glycolysis in L-02 hepatocytes.

A futile cycle, formed between two ATP-dependant gamma-glutamyl cycle enzymes, gamma-glutamyl cysteine synthetase and 5-oxoprolinase: the cause of cellular ATP depletion in nephrotic cystinosis?

PubMed

Kumar, Akhilesh; Bachhawat, Anand Kumar

2010-03-01

Cystinosis, an inherited disease caused by a defect in the lysosomal cystine transporter (CTNS), is characterized by renal proximal tubular dysfunction. Adenosine triphosphate (ATP) depletion appears to be a key event in the pathophysiology of the disease, even though the manner in which ATP depletion occurs is still a puzzle. We present a model that explains how a futile cycle that is generated between two ATP-utilizing enzymes of the gamma-glutamyl cycle leads to ATP depletion. The enzyme gamma-glutamyl cysteine synthetase (gamma-GCS), in the absence of cysteine, forms 5-oxoproline (instead of the normal substrate, gamma-glutamyl cysteine) and the 5-oxoproline is converted into glutamate by the ATP-dependant enzyme, 5-oxoprolinase. Thus, in cysteine-limiting conditions, glutamate is cycled back into glutamate via 5-oxoproline at the cost of two ATP molecules without production of glutathione and is the cause of the decreased levels of glutathione synthesis, as well as the ATP depletion observed in these cells. The model is also compatible with the differences seen in the human patients and the mouse model of cystinosis, where renal failure is not observed.

SIRT3 Deacetylates ATP Synthase F1 Complex Proteins in Response to Nutrient- and Exercise-Induced Stress

PubMed Central

Vassilopoulos, Athanassios; Pennington, J. Daniel; Andresson, Thorkell; Rees, David M.; Bosley, Allen D.; Fearnley, Ian M.; Ham, Amy; Flynn, Charles Robb; Hill, Salisha; Rose, Kristie Lindsey; Kim, Hyun-Seok; Walker, John E.

2014-01-01

Abstract Aims: Adenosine triphosphate (ATP) synthase uses chemiosmotic energy across the inner mitochondrial membrane to convert adenosine diphosphate and orthophosphate into ATP, whereas genetic deletion of Sirt3 decreases mitochondrial ATP levels. Here, we investigate the mechanistic connection between SIRT3 and energy homeostasis. Results: By using both in vitro and in vivo experiments, we demonstrate that ATP synthase F1 proteins alpha, beta, gamma, and Oligomycin sensitivity-conferring protein (OSCP) contain SIRT3-specific reversible acetyl-lysines that are evolutionarily conserved and bind to SIRT3. OSCP was further investigated and lysine 139 is a nutrient-sensitive SIRT3-dependent deacetylation target. Site directed mutants demonstrate that OSCPK139 directs, at least in part, mitochondrial ATP production and mice lacking Sirt3 exhibit decreased ATP muscle levels, increased ATP synthase protein acetylation, and an exercise-induced stress-deficient phenotype. Innovation: This work connects the aging and nutrient response, via SIRT3 direction of the mitochondrial acetylome, to the regulation of mitochondrial energy homeostasis under nutrient-stress conditions by deacetylating ATP synthase proteins. Conclusion: Our data suggest that acetylome signaling contributes to mitochondrial energy homeostasis by SIRT3-mediated deacetylation of ATP synthase proteins. Antioxid. Redox Signal. 21, 551–564. PMID:24252090

Imaging Adenosine Triphosphate (ATP)

PubMed Central

Rajendran, Megha; Dane, Eric; Conley, Jason; Tantama, Mathew

2016-01-01

Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provides valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific for ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies that are available to visualize ATP in living cells and identify areas where new tools and approaches are needed to expand our capabilities. PMID:27638696

Imaging Adenosine Triphosphate (ATP).

PubMed

Rajendran, Megha; Dane, Eric; Conley, Jason; Tantama, Mathew

2016-08-01

Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provide valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to the organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific to ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies available for visualizing ATP in living cells, and identify areas where new tools and approaches are needed to expand our capabilities. © 2016 Marine Biological Laboratory.

Conformational dynamics of ATP/Mg:ATP in motor proteins via data mining and molecular simulation.

PubMed

Bojovschi, A; Liu, Ming S; Sadus, Richard J

2012-08-21

The conformational diversity of ATP/Mg:ATP in motor proteins was investigated using molecular dynamics and data mining. Adenosine triphosphate (ATP) conformations were found to be constrained mostly by inter cavity motifs in the motor proteins. It is demonstrated that ATP favors extended conformations in the tight pockets of motor proteins such as F(1)-ATPase and actin whereas compact structures are favored in motor proteins such as RNA polymerase and DNA helicase. The incorporation of Mg(2+) leads to increased flexibility of ATP molecules. The differences in the conformational dynamics of ATP/Mg:ATP in various motor proteins was quantified by the radius of gyration. The relationship between the simulation results and those obtained by data mining of motor proteins available in the protein data bank is analyzed. The data mining analysis of motor proteins supports the conformational diversity of the phosphate group of ATP obtained computationally.

Comparative hemodynamic effects of hypotension induced by diadenosine tetraphosphate (AP4A) and ATP in dogs.

PubMed

Takeda, Shohei; Inada, Yutaka; Fukui, Noriyuki; Tomaru, Teruaki

1997-03-01

ATP and diadenosine tetraphosphate (AP 4 A) have been shown to produce vasodilation mediated by P 1 - and P 2 -purinoceptor, respectively. The differing mechanisms involved in this vasodilating activity may induce different systemic hemodynamic changes. We compared the hemodynamic effects of AP 4 A-induced hypotension with those induced by ATP. Fourteen mongrel dogs were anesthetized with 0.87% halothane in oxygen (1 MAC). After the baseline period, mean arterial pressure was reduced to 60 mmHg for 60 min by the infusion of AP 4 A or ATP. The ATP- and AP 4 A-induced hypotension resulted in a maximum reduction in systemic vascular resistance of 43% and 46%, respectively (P<0.01), associated with a significant increase in stroke volume index. With ATP, a 20% of maximum increase (P<0.05) in cardiac index (CI) was observed during the induced hypotension. In contrast, AP 4 A-induced hypotension did not result in any changes in CI throughout the observation period. The varying results concerning CI during the ATP- and AP 4 A-induced hypotension were probably due to differences in ventricular filling pressure, since AP 4 A-induced hypotension was associated with decreases (P<0.01) in both right atrial and pulmonary capillary wedge pressures, whereas neither of these variables significantly changed with ATP. The hypotension induced by either ATP or AP 4 A was associated with a significant decrease in heart rate (HR). However, both the magnitude and duration of decreases in HR due to ATP-induced hypotension were more pronounced than those seen with AP 4 A. In conclusion, while both drugs were equally capable of inducing hypotension, our results suggest that AP 4 A was more suitable for induced hypotension because of its potent vasodilatory action with venodilation and less negative chronotropic action.

Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

PubMed Central

Kucharczyk, Roza; Ezkurdia, Nahia; Couplan, Elodie; Procaccio, Vincent; Ackerman, Sharon H.; Blondel, Marc; di Rago, Jean-Paul

2010-01-01

Summary Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). BN-PAGE analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of subcomplexes (F1, Atp9p-ring, unassembled α-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the

Integration of Genome-Wide Computation DRE Search, AhR ChIP-chip and Gene Expression Analyses of TCDD-Elicited Responses in the Mouse Liver

PubMed Central

2011-01-01

Background The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor (TF) that mediates responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Integration of TCDD-induced genome-wide AhR enrichment, differential gene expression and computational dioxin response element (DRE) analyses further elucidate the hepatic AhR regulatory network. Results Global ChIP-chip and gene expression analyses were performed on hepatic tissue from immature ovariectomized mice orally gavaged with 30 μg/kg TCDD. ChIP-chip analysis identified 14,446 and 974 AhR enriched regions (1% false discovery rate) at 2 and 24 hrs, respectively. Enrichment density was greatest in the proximal promoter, and more specifically, within ± 1.5 kb of a transcriptional start site (TSS). AhR enrichment also occurred distal to a TSS (e.g. intergenic DNA and 3' UTR), extending the potential gene expression regulatory roles of the AhR. Although TF binding site analyses identified over-represented DRE sequences within enriched regions, approximately 50% of all AhR enriched regions lacked a DRE core (5'-GCGTG-3'). Microarray analysis identified 1,896 number of TCDD-responsive genes (|fold change| ≥ 1.5, P1(t) > 0.999). Integrating this gene expression data with our ChIP-chip and DRE analyses only identified 625 differentially expressed genes that involved an AhR interaction at a DRE. Functional annotation analysis of differentially regulated genes associated with AhR enrichment identified overrepresented processes related to fatty acid and lipid metabolism and transport, and xenobiotic metabolism, which are consistent with TCDD-elicited steatosis in the mouse liver. Conclusions Details of the AhR regulatory network have been expanded to include AhR-DNA interactions within intragenic and intergenic genomic regions. Moreover, the AhR can interact with DNA independent of a DRE core suggesting there are alternative mechanisms of AhR-mediated gene regulation. PMID:21762485

Interaction between ATP, metal ions, glycine, and several minerals

NASA Technical Reports Server (NTRS)

Rishpon, J.; Ohara, P. J.; Lawless, J. G.; Lahav, N.

1982-01-01

Interactions between ATP, glycine and montmorillonite and kaolinite clay minerals in the presence of various metal cations are investigated. The adsorption of adenine nucleotides on clays and Al(OH)3 was measured as a function of pH, and glycine condensation was followed in the presence of ATP, ZnCl2, MgCl2 and either kaolinite or montmorillonite. The amounts of ATP and ADP adsorbed are found to decrease with increasing Ph, and to be considerably enhanced in experiments with Mg(2+)- and Zn(2+)-montmorillonite with respect to Na(+)-montmorillonite. The effects of divalent cations are less marked in kaolinite. Results for Al(OH)3 show the importance of adsorption at clay platelet edges at high pH. The decomposition of ATP during drying at high temperature is observed to be inhibited by small amounts of clay, vacuum, or Mg(2+) or Zn(2+) ions, and to be accompanied by peptide formation in the presence of glycine. Results suggest the importance of Zn(2+) and Mg(2+) in chemical evolution.

Effect of K+ATP channel and adenosine receptor blockade during rest and exercise in congestive heart failure.

PubMed

Traverse, Jay H; Chen, YingJie; Hou, MingXiao; Li, Yunfang; Bache, Robert J

2007-06-08

K(+)(ATP) channels are important metabolic regulators of coronary blood flow (CBF) that are activated in the setting of reduced levels of ATP or perfusion pressure. In the normal heart, blockade of K(+)(ATP) channels results in a approximately 20% reduction in resting CBF but does not impair the increase in CBF that occurs during exercise. In contrast, adenosine receptor blockade fails to alter CBF or myocardial oxygen consumption (MVO(2)) in the normal heart but contributes to the increase in CBF during exercise when vascular K(+)(ATP) channels are blocked. Congestive heart failure (CHF) is associated with a decrease in CBF that is matched to a decrease in MVO(2) suggesting downregulation of myocardial energy utilization. Because myocardial ATP levels and coronary perfusion pressure are reduced in CHF, this study was undertaken to examine the role of K(+)(ATP) channels and adenosine in dogs with pacing-induced CHF. Myocardial blood flow (MBF) and MVO(2) were measured during rest and treadmill exercise before and after K(+)(ATP) channel blockade with glibenclamide (50 microg/kg/min ic) or adenosine receptor blockade with 8-phenyltheophylline (8-PT; 5 mg/kg iv). Inhibition of K(+)(ATP) channels resulted in a decrease in CBF and MVO(2) at rest and during exercise without a change in the relationship between CBF and MVO(2). In contrast, adenosine receptor blockade caused a significant increase in CBF that occurred secondary to an increase of MVO(2). These findings demonstrate that coronary K(+)(ATP) channel activity contribute to the regulation of resting MBF in CHF, and that endogenous adenosine may act to inhibit MVO(2) in the failing heart.

Study on ATP concentration changes in cytosol of individual cultured neurons during glutamate-induced deregulation of calcium homeostasis.

PubMed

Surin, A M; Gorbacheva, L R; Savinkova, I G; Sharipov, R R; Khodorov, B I; Pinelis, V G

2014-02-01

For the first time, simultaneous monitoring of changes in the concentration of cytosolic ATP ([ATP]c), pH (pHc), and intracellular free Ca2+ concentration ([Ca2+]i) of the individual neurons challenged with toxic glutamate (Glu) concentrations was performed. To this end, the ATP-sensor AT1.03, which binds to ATP and therefore enhances the efficiency of resonance energy transfer between blue fluorescent protein (energy donor) and yellow-green fluorescent protein (energy acceptor), was expressed in cultured hippocampal neurons isolated from 1-2-day-old rat pups. Excitation of fluorescence in the acceptor protein allowed monitoring changes in pHc. Cells were loaded with fluorescent low-affinity Ca2+ indicators Fura-FF or X-rhod-FF to register [Ca2+]i. It was shown that Glu (20 µM, glycine 10 µM, Mg2+-free) produced a rapid acidification of the cytosol and decrease in [ATP]c. An approximately linear relationship (r(2) = 0.56) between the rate of [ATP]c decline and latency of glutamate-induced delayed calcium deregulation (DCD) was observed: higher rate of [ATP]c decrease corresponded to shorter DCD latency period. DCD began with a decrease in [ATP]c of as much as 15.9%. In the phase of high [Ca2+]i, the plateau of [ATP]c dropped to 10.4% compared to [ATP]c in resting neurons (100%). In the presence of the Na+/K+-ATPase inhibitor ouabain (0.5 mM), glutamate-induced reduction in [ATP]c in the phase of the high [Ca2+]i plateau was only 36.6%. Changes in [ATP]c, [Ca2+]i, mitochondrial potential, and pHc in calcium-free or sodium-free buffers, as well as in the presence of the inhibitor of Na+/K+-ATPase ouabain (0.5 mM), led us to suggest that in addition to increase in proton conductivity and decline in [ATP]c, one of the triggering factors of DCD might be a reversion of the neuronal plasma membrane Na+/Ca2+ exchange.

Controlled rotation of the F1-ATPase reveals differential and continuous binding changes for ATP synthesis

PubMed Central

Adachi, Kengo; Oiwa, Kazuhiro; Yoshida, Masasuke; Nishizaka, Takayuki; Kinosita, Kazuhiko

2012-01-01

F1-ATPase is an ATP-driven rotary molecular motor that synthesizes ATP when rotated in reverse. To elucidate the mechanism of ATP synthesis, we imaged binding and release of fluorescently labelled ADP and ATP while rotating the motor in either direction by magnets. Here we report the binding and release rates for each of the three catalytic sites for 360° of the rotary angle. We show that the rates do not significantly depend on the rotary direction, indicating ATP synthesis by direct reversal of the hydrolysis-driven rotation. ADP and ATP are discriminated in angle-dependent binding, but not in release. Phosphate blocks ATP binding at angles where ADP binding is essential for ATP synthesis. In synthesis rotation, the affinity for ADP increases by >104, followed by a shift to high ATP affinity, and finally the affinity for ATP decreases by >104. All these angular changes are gradual, implicating tight coupling between the rotor angle and site affinities. PMID:22929779

Decreased ATP synthesis and lower pH may lead to abnormal muscle contraction and skin sensitivity in human skin.

PubMed

Kim, Eun Ju; Lee, Dong Hun; Kim, Yeon Kyung; Kim, Min-Kyoung; Kim, Jung Yun; Lee, Min Jung; Choi, Won Woo; Eun, Hee Chul; Chung, Jin Ho

2014-12-01

Sensitive skin represents hyperactive sensory symptoms showing exaggerated reactions in response to internal stimulants or external irritants. Although sensitive skin is a very common condition affecting an estimated 50% of the population, its pathophysiology remains largely elusive, particularly with regard to its metabolic aspects. The objective of our study was to investigate the pathogenesis of sensitive skin. We recruited healthy participants with 'sensitive' or 'non-sensitive' skin based on standardized questionnaires and 10% lactic acid stinging test, and obtained skin samples for microarray analysis and subsequent experiments. Microarray transcriptome profiling revealed that genes involved in muscle contraction, carbohydrate and lipid metabolism, and ion transport and balance were significantly decreased in sensitive skin. These altered genes could account for the abnormal muscle contraction, decreased ATP amount in sensitive skin. In addition, pain-related transcripts such as TRPV1, ASIC3 and CGRP were significantly up-regulated in sensitive skin, compared with non-sensitive skin. Our findings suggest that sensitive skin is closely associated with the dysfunction of muscle contraction and metabolic homeostasis. Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Increased Glycolytic ATP Synthesis Is Associated with Tafenoquine Resistance in Leishmania major▿

PubMed Central

Manzano, José Ignacio; Carvalho, Luis; Pérez-Victoria, José M.; Castanys, Santiago; Gamarro, Francisco

2011-01-01

Tafenoquine (TFQ), an 8-aminoquinoline used to treat and prevent Plasmodium infections, could represent an alternative therapy for leishmaniasis. Indeed, TFQ has shown significant leishmanicidal activity both in vitro and in vivo, where it targets Leishmania mitochondria and activates a final apoptosis-like process. In order not to jeopardize the life span of this potential antileishmania drug, it is important to determine the likelihood that Leishmania will develop resistance to TFQ and the mechanisms of resistance induced. To address this issue, a TFQ-resistant Leishmania major promastigote line (R4) was selected. This resistance, which is unstable in a drug-free medium (revertant line), was maintained in intramacrophage amastigote forms, and R4 promastigotes were found to be cross-resistant to other 8-aminoquinolines. A decreased TFQ uptake, which is probably associated with an alkalinization of the intracellular pH rather than drug efflux, was observed for both the R4 and revertant lines. TFQ induces a decrease in ATP synthesis in all Leishmania lines, although total ATP levels were maintained at higher values in R4 parasites. In contrast, ATP synthesis by glycolysis was significantly increased in R4 parasites, whereas mitochondrial ATP synthesis was similar to that in wild-type parasites. We therefore conclude that increased glycolytic ATP synthesis is the main mechanism underlying TFQ resistance in Leishmania. PMID:21199921

Increased glycolytic ATP synthesis is associated with tafenoquine resistance in Leishmania major.

PubMed

Manzano, José Ignacio; Carvalho, Luis; Pérez-Victoria, José M; Castanys, Santiago; Gamarro, Francisco

2011-03-01

Tafenoquine (TFQ), an 8-aminoquinoline used to treat and prevent Plasmodium infections, could represent an alternative therapy for leishmaniasis. Indeed, TFQ has shown significant leishmanicidal activity both in vitro and in vivo, where it targets Leishmania mitochondria and activates a final apoptosis-like process. In order not to jeopardize the life span of this potential antileishmania drug, it is important to determine the likelihood that Leishmania will develop resistance to TFQ and the mechanisms of resistance induced. To address this issue, a TFQ-resistant Leishmania major promastigote line (R4) was selected. This resistance, which is unstable in a drug-free medium (revertant line), was maintained in intramacrophage amastigote forms, and R4 promastigotes were found to be cross-resistant to other 8-aminoquinolines. A decreased TFQ uptake, which is probably associated with an alkalinization of the intracellular pH rather than drug efflux, was observed for both the R4 and revertant lines. TFQ induces a decrease in ATP synthesis in all Leishmania lines, although total ATP levels were maintained at higher values in R4 parasites. In contrast, ATP synthesis by glycolysis was significantly increased in R4 parasites, whereas mitochondrial ATP synthesis was similar to that in wild-type parasites. We therefore conclude that increased glycolytic ATP synthesis is the main mechanism underlying TFQ resistance in Leishmania.

Mitochondrial Genome Integrity Mutations Uncouple the Yeast Saccharomyces cerevisiae ATP Synthase*║

PubMed Central

Wang, Yamin; Singh, Usha; Mueller, David M.

2013-01-01

The mitochondrial ATP synthase is a molecular motor, which couples the flow of rotons with phosphorylation of ADP. Rotation of the central stalk within the core of ATP synthase effects conformational changes in the active sites driving the synthesis of ATP. Mitochondrial genome integrity (mgi) mutations have been previously identified in the α-, β-, and γ-subunits of ATP synthase in yeast Kluyveromyces lactis and trypanosome Trypanosoma brucei. These mutations reverse the lethality of the loss of mitochondrial DNA in petite negative strains. Introduction of the homologous mutations in Saccharomyces cerevisiae results in yeast strains that lose mitochondrial DNA at a high rate and accompanied decreases in the coupling of the ATP synthase. The structure of yeast F1-ATPase reveals that the mgi residues cluster around the γ-subunit and selectively around the collar region of F1. These results indicate that residues within the mgi complementation group are necessary for efficient coupling of ATP synthase, possibly acting as a support to fix the axis of rotation of the central stalk. PMID:17244612

31P-NMR measurements of ATP, ADP, 2,3-diphosphoglycerate and Mg2+ in human erythrocytes.

PubMed

Petersen, A; Kristensen, S R; Jacobsen, J P; Hørder, M

1990-08-17

Absolute 31P-NMR measurements of ATP, ADP and 2,3-diphosphoglycerate (2,3-DPG) in oxygenated and partly deoxygenated human erythrocytes, compared to measurements by standard assays after acid extraction, show that ATP is only 65% NMR visible, ADP measured by NMR is unexpectedly 400% higher than the enzymatic measurement and 2,3-DPG is fully NMR visible, regardless of the degree of oxygenation. These results show that binding to hemoglobin is unlikely to cause the decreased visibility of ATP in human erythrocytes as deoxyhemoglobin binds the phosphorylated metabolites more tightly than oxyhemoglobin. The high ADP visibility is unexplained. The levels of free Mg2+ [( Mg2+]free) in human erythrocytes are 225 mumol/l at an oxygen saturation of 98.6% and instead of the expected increase, the level decreased to 196 mumol/l at an oxygen saturation of 38.1% based on the separation between the alpha- and beta-ATP peaks. [Mg2+]free in the erythrocytes decreased to 104 mumol/l at a high 2,3-DPG concentration of 25.4 mmol/l red blood cells (RBC) and a normal ATP concentration of 2.05 mmol/l RBC. By increasing the ATP concentration to 3.57 mmol/l RBC, and with a high 2,3-DPG concentration of 24.7 mmol/l RBC, the 31P-NMR measured [Mg2+]free decreased to 61 mumol/l. These results indicate, that the 31P-NMR determined [Mg2+]free in human erythrocytes, based solely on the separation of the alpha- and beta-ATP peaks, does not give a true measure of intracellular free Mg2+ changes with different oxygen saturation levels. Furthermore the measurement is influenced by the concentration of the Mg2+ binding metabolites ATP and 2,3-DPG. Failure to take these factors into account when interpreting 31P-NMR data from human erythrocytes may explain some discrepancies in the literature regarding [Mg2+]free.

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons

PubMed Central

Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Houlden, Henry

2017-01-01

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP. Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. PMID:28360103

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons.

PubMed

Ludtmann, Marthe H R; Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Preza, Elisavet; Carro, Eva; Houlden, Henry; Gandhi, Sonia; Wray, Selina; Abramov, Andrey Y

2017-05-26

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Respiratory ATP cost and benefit of arbuscular mycorrhizal symbiosis with Nicotiana tabacum at different growth stages and under salinity.

PubMed

Del-Saz, Néstor Fernández; Romero-Munar, Antonia; Alonso, David; Aroca, Ricardo; Baraza, Elena; Flexas, Jaume; Ribas-Carbo, Miquel

2017-11-01

Growth and maintenance partly depend on both respiration and ATP production during oxidative phosphorylation in leaves. Under stress, ATP is needed to maintain the accumulated biomass. ATP production mostly proceeds from the cytochrome oxidase pathway (COP), while respiration via the alternative oxidase pathway (AOP) may decrease the production of ATP per oxygen consumed, especially under phosphorus (P) limitation and salinity conditions. Symbiosis with arbuscular mycorrhizal (AM) fungi is reputed by their positive effect on plant growth under stress at mature stages of colonization; however, fungal colonization may decrease plant growth at early stages. Thus, the present research is based on the hypothesis that AM fungus colonization will increase both foliar respiration and ATP production at mature stages of plant growth while decreasing them both at early stages. We used the oxygen-isotope-fractionation technique to study the in vivo respiratory activities and ATP production of the COP and AOP in AM and non-AM (NM) tobacco plants grown under P-limiting and saline conditions in sand at different growth stages (14, 28 and 49days). Our results suggest that AM symbiosis represents an ATP cost detrimental for shoot growth at early stages, whilst it represents a benefit on ATP allowing for faster rates of growth at mature stages, even under salinity conditions. Copyright © 2017 Elsevier GmbH. All rights reserved.

Caged ATP - an internal calibration method for ATP bioluminescence assays.

PubMed

Calvert, R M; Hopkins, H C; Reilly, M J; Forsythe, S J

2000-03-01

ATP bioluminescence, based on the firefly luciferase system, is used for the rapid determination of hygienic practices in the food industry. This study has demonstrated the use of caged ATP as an internal ATP standard and quantified the effects of industrial cleansing solutions, alcoholic beverages and pH on firefly luciferase activity. The light signal was quenched 6-47% by five cleansing solutions at standard working concentrations. Ethanol at 1% (v/v) inhibited bioluminescence by 15% (w/v) whereas concentrations above 4% enhanced the light output. The light signal was quenched by 20-25% at pH values below pH 4 and above pH 10.

Enhancement of succinate yield by manipulating NADH/NAD+ ratio and ATP generation.

PubMed

Li, Jiaojiao; Li, Yikui; Cui, Zhiyong; Liang, Quanfeng; Qi, Qingsheng

2017-04-01

We previously engineered Escherichia coli YL104 to efficiently produce succinate from glucose. In this study, we investigated the relationships between the NADH/NAD + ratio, ATP level, and overall yield of succinate production by using glucose as the carbon source in YL104. First, the use of sole NADH dehydrogenases increased the overall yield of succinate by 7% and substantially decreased the NADH/NAD + ratio. Second, the soluble fumarate reductase from Saccharomyces cerevisiae was overexpressed to manipulate the anaerobic NADH/NAD + ratio and ATP level. Third, another strategy for reducing the ATP level was applied by introducing ATP futile cycling for improving succinate production. Finally, a combination of these methods exerted a synergistic effect on improving the overall yield of succinate, which was 39% higher than that of the previously engineered strain YL104. The study results indicated that regulation of the NADH/NAD + ratio and ATP level is an efficient strategy for succinate production.

Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+.

PubMed

Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D

2005-03-01

Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.

Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+

NASA Technical Reports Server (NTRS)

Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D.

2005-01-01

Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.

A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells*

PubMed Central

Dai, Feihan F.; Bhattacharjee, Alpana; Liu, Ying; Batchuluun, Battsetseg; Zhang, Ming; Wang, Xinye Serena; Huang, Xinyi; Luu, Lemieux; Zhu, Dan; Gaisano, Herbert; Wheeler, Michael B.

2015-01-01

GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H+-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca2+ influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion. PMID:26272612

ATP and microfilaments in cellular oxidant injury.

PubMed Central

Hinshaw, D. B.; Armstrong, B. C.; Burger, J. M.; Beals, T. F.; Hyslop, P. A.

1988-01-01

Oxidant injury produces dramatic changes in cytoskeletal organization and cell shape. ATP synthetic pathways are major targets of oxidant injury resulting in rapid depletion of cellular ATP following oxidant exposure. The relation of ATP depletion to the changes in microfilament organization seen following H2O2 exposure were examined in the P388D1 cell line. Three hours of glucose depletion alone resulted in a decline in cellular ATP levels to less than 10% of controls, which was comparable to ATP levels in cells 30 to 60 minutes after exposure to 5 mM H2O2 in the presence of glucose. Adherent cells stained with rhodamine phalloidin, a probe specific for polymerized (F) actin, revealed a progressive shortening of microfilaments into globular aggregates within cells depleted of glucose over 3 hours, a pattern similar to earlier observations of H2O2-injured cells after 1 hour. The changes in cellular ATP associated with glucose depletion or H2O2 exposure were then correlated with G actin content measured by the DNAse 1 inhibition assay. No real differences in G actin content as a percentage of total actin were seen in P388D1 cells following 3 hours of glucose depletion or 30 to 60 minutes after exposure to 5 mM H2O2. But 2 to 3 hours after exposure to H2O2 there was a progressive decrease in G actin as a percentage of total actin within the cells. Transmission electron microscopy of cells depleted of glucose for 3 h or 1 hour after exposure to H2O2 revealed the presence of side-to-side aggregates or bundles of microfilaments within the cells. These observations suggest that declining levels of ATP either from metabolic inhibition or H2O2 injury are correlated with the fragmentation and shortening of microfilaments into aggregates. No net change in monomeric or polymeric actin was necessary for this to occur. However, at later time points after H2O2 exposure some actin assembly did occur. Images p[484]-a p481-a p482-a Figure 2 Figure 3 PMID:3414780

Effect of exogenous ATP on the postharvest properties and pectin degradation of mung bean sprouts (Vigna radiata).

PubMed

Chen, Lin; Zhou, Yige; He, Zhenyun; Liu, Qin; Lai, Shaojuan; Yang, Hongshun

2018-06-15

The effects of exogenous ATP on the postharvest quality, browning and softening of mung bean (Vigna radiata) sprouts were evaluated. ATP treatment significantly alleviated the quality loss and browning events during the storage of 3 days. It also reduced the oxidant damage by inducing high activities of peroxidase (9.3-13.9%) and superoxide dismutase (8.8-10.3%) which scavenged the reactive oxygen species (ROS) effectively. Transcriptional results indicated that ATP treatment decreased VrPL1, VrPME and VrPG1 gene expression levels more than 2 folds at some time points. Furthermore, the atomic force microscope (AFM) images revealed that the pectin degradation was notably slowed by ATP treatment and the width and height of pectin backbone were better maintained (47.1% and 45.6% higher than control without ATP treatment). The cooperative effects of ROS scavenging and decreased expressions of pectin-related genes might contribute to the deferred pectin deterioration and firmness loss by ATP treatment. Copyright © 2018 Elsevier Ltd. All rights reserved.

The regulation of ATP release from the urothelium by adenosine and transepithelial potential.

PubMed

Dunning-Davies, Bryony M; Fry, Christopher H; Mansour, Dina; Ferguson, Douglas R

2013-03-01

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Stretch of the urothelium, as occurs during bladder filling, is associated with a release of ATP that is postulated to act as a sensory neurotransmitter. The regulation of ATP release is poorly understood and in particular if there is a feedback mechanism provided by ATP itself. Adenosine, a breakdown product of ATP, is a potent inhibitor of stretch-induced ATP release, acting through and A1 receptor; endogenous levels are about 0.6μM. Data are consistent with ATP release relying on the rise of intracellular Ca2+. Transepithelial potential also controls ATP release, also acting via an A1 receptor-dependent pathway. To test the hypothesis that distension-induced ATP release from the bladder urothelium is regulated by adenosine as well as changes to transurothelial potential (TEP). To examine the role of changes to intracellular [Ca(2+) ] in ATP release. Rabbit urothelium/suburothelium membranes were used in an Ussing chamber system. Distension was induced by fluid removal from the chamber bathing the serosal (basolateral) membrane face. The TEP and short-circuit current were measured. ATP was measured in samples aspirated from the serosal chamber by a luciferin-luciferase assay. Intracellular [Ca(2+) ] was measured in isolated urothelial cells using the fluorochrome Fura-2. All experiments were performed at 37°C. Distension-induced ATP release was decreased by adenosine (1-10 μm) and enhanced by adenosine deaminase and A1- (but not A2-) receptor antagonists. Distension-induced ATP release was reduced by 2-APB, nifedipine and capsazepine; capsaicin induced ATP release in the absence of distension. ATP and capsaicin, but not adenosine, generated intracellular Ca(2+) transients; adenosine did not affect the ATP-generated Ca(2+) transient. ATP release was dependent on a finite transepithelial potential. Changes to TEP, in the absence of distension, generated ATP release that was in turn reduced by adenosine

Global gene profiling of aging lungs in Atp8b1 mutant mice.

PubMed

Soundararajan, Ramani; Stearns, Timothy M; Czachor, Alexander; Fukumoto, Jutaro; Turn, Christina; Westermann-Clark, Emma; Breitzig, Mason; Tan, Lee; Lockey, Richard F; King, Benjamin L; Kolliputi, Narasaiah

2016-09-29

Recent studies implicate cardiolipin oxidation in several age-related diseases. Atp8b1 encoding Type 4 P-type ATPases is a cardiolipin transporter. Mutation in Atp8b1 gene or inflammation of the lungs impairs the capacity of Atp8b1 to clear cardiolipin from lung fluid. However, the link between Atp8b1 mutation and age-related gene alteration is unknown. Therefore, we investigated how Atp8b1 mutation alters age-related genes. We performed Affymetrix gene profiling of lungs isolated from young (7-9 wks, n=6) and aged (14 months, 14 M, n=6) C57BL/6 and Atp8b1 mutant mice. In addition, Ingenuity Pathway Analysis (IPA) was performed. Differentially expressed genes were validated by quantitative real-time PCR (qRT-PCR). Global transcriptome analysis revealed 532 differentially expressed genes in Atp8b1 lungs, 157 differentially expressed genes in C57BL/6 lungs, and 37 overlapping genes. IPA of age-related genes in Atp8b1 lungs showed enrichment of Xenobiotic metabolism and Nrf2-mediated signaling pathways. The increase in Adamts2 and Mmp13 transcripts in aged Atp8b1 lungs was validated by qRT-PCR. Similarly, the decrease in Col1a1 and increase in Cxcr6 transcripts was confirmed in both Atp8b1 mutant and C57BL/6 lungs. Based on transcriptome profiling, our study indicates that Atp8b1 mutant mice may be susceptible to age-related lung diseases.

K+ depolarization evokes ATP, adenosine and glutamate release from glia in rat hippocampus: a microelectrode biosensor study

PubMed Central

Heinrich, A; Andó, RD; Túri, G; Rózsa, B; Sperlágh, B

2012-01-01

BACKGROUND AND PURPOSE This study was undertaken to characterize the ATP, adenosine and glutamate outflow evoked by depolarization with high K+ concentrations, in slices of rat hippocampus. EXPERIMENTAL APPROACH We utilized the microelectrode biosensor technique and extracellular electrophysiological recording for the real-time monitoring of the efflux of ATP, adenosine and glutamate. KEY RESULTS ATP, adenosine and glutamate sensors exhibited transient and reversible current during depolarization with 25 mM K+, with distinct kinetics. The ecto-ATPase inhibitor ARL67156 enhanced the extracellular level of ATP and inhibited the prolonged adenosine efflux, suggesting that generation of adenosine may derive from the extracellular breakdown of ATP. Stimulation-evoked ATP, adenosine and glutamate efflux was inhibited by tetrodotoxin, while exposure to Ca2+-free medium abolished ATP and adenosine efflux from hippocampal slices. Extracellular elevation of ATP and adenosine were decreased in the presence of NMDA receptor antagonists, D-AP-5 and ifenprodil, whereas non-NMDA receptor blockade by CNQX inhibited glutamate but not ATP and adenosine efflux. The gliotoxin fluoroacetate and P2X7 receptor antagonists inhibited the K+-evoked ATP, adenosine and glutamate efflux, while carbenoxolone in low concentration and probenecid decreased only the adenosine efflux. CONCLUSIONS AND IMPLICATIONS Our results demonstrated activity-dependent gliotransmitter release in the hippocampus in response to ongoing neuronal activity. ATP and glutamate were released by P2X7 receptor activation into extracellular space. Although the increased extracellular levels of adenosine did derive from released ATP, adenosine might also be released directly via pannexin hemichannels. LINKED ARTICLE This article is commented on by Sershen, pp. 1000–1002 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02072.x PMID:22394324

The effects of gamma radiation, UV and visible light on ATP levels in yeast cells depend on cellular melanization.

PubMed

Bryan, Ruth; Jiang, Zewei; Friedman, Matthew; Dadachova, Ekaterina

2011-10-01

Previously we have shown that growth of melanized fungi is stimulated by low levels of gamma radiation. The goal of this study was to examine the effects of visible light, UV light, and gamma radiation on the energy level (ATP concentration) in melanized Cryptococcus neoformans cells. Melanized C. neoformans cells as well as non-melanized controls were subjected to visible, UV or gamma radiation, and ATP was quantified by measuring the amount of light emitted by the ATP-dependent reaction of luciferase with luciferin. We found that all three forms of radiation led to a reduction in the ATP levels in melanized C. neoformans cells. This points to a universal melanin-related mechanism underlying observation of ATP decrease in irradiated melanized cells. In contrast, in non-melanized cells visible light led to increase in ATP levels; gamma radiation did not cause any changes while UV exposure resulted in some ATP decrease, however, much less pronounced than in melanized cells. Copyright © 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

[Stabilization of Cadmium Contaminated Soils by Ferric Ion Modified Attapulgite (Fe/ATP)--Characterizations and Stabilization Mechanism].

PubMed

Rong, Yang; Li, Rong-bo; Zhou, Yong-li; Chen, Jing; Wang, Lin-ling; Lu, Xiao-hua

2015-08-01

Ferric ion modified attapulgite (Fe/ATP) was prepared by impregnation and its structure and morphology were characterized. The toxicity characteristic leaching procedure (TCLP) was used to evaluate the effect of Cadmium( Cd) stabilization in soil with the addition of attapulgite (ATP) and Fe/ATP. The stabilization mechanism of Cd was further elucidated by comparing the morphologies and structure of ATP and Fe/ATP before and after Cd adsorption. Fe/ATP exhibited much better adsorption capacity than ATP, suggesting different adsorption mechanisms occurred between ATP and Fe/ATP. The leaching concentrations of Cd in soil decreased by 45% and 91% respectively, with the addition of wt. 20% ATP and Fe/ATP. The former was attributed to the interaction between Cd2 and --OH groups by chemical binding to form inner-sphere complexes in ATP and the attachment between Cd2+ and the defect sites in ATP framework. Whereas Cd stabilization with Fe/ATP was resulted from the fact that the active centers (--OH bonds or O- sites) on ATP could react with Fe3+ giving Fe--O--Cd-- bridges, which helped stabilize Cd in surface soil. What'more, the ferric oxides and metal hydroxides on the surface of ATP could interact with Cd, probably by the formation of cadmium ferrite. In conclusion, Fe/ATP, which can be easily prepared, holds promise as a potential low-cost and environmental friendly stabilizing agent for remediation of soil contaminated with heavy metals.

The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

PubMed Central

Suszynski, T.M.; Wildey, G.M.; Falde, E.J.; Cline, G.W.; Maynard, K. Stewart; Ko, N.; Sotiris, J.; Naji, A.; Hering, B.J.; Papas, K.K.

2009-01-01

Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio. PMID:18374063

Contribution of extracellular ATP on the cell-surface F1F0-ATP synthase-mediated intracellular triacylglycerol accumulation.

PubMed

Kita, Toshiyuki; Arakaki, Naokatu

2015-01-01

Cell-surface F1F0-ATP synthase was involved in the cell signaling mediating various biological functions. Recently, we found that cell-surface F1F0-ATP synthase plays a role on intracellular triacylglycerol accumulation in adipocytes, and yet, the underlying mechanisms remained largely unknown. In this study, we investigated the role of extracellular ATP on the intracellular triacylglycerol accumulation. We demonstrated that significant amounts of ATP were produced extracellularly by cultured 3T3-L1 adipocytes and that the antibodies against α and β subunits of F1F0-ATP synthase inhibited the extracellular ATP production. Piceatannol, a F1F0-ATP synthase inhibitor, and apyrase, an enzyme which degrades extracellular ATP, suppressed triacylglycerol accumulation. The selective P2Y1 receptor antagonist MRS2500 significantly inhibited triacylglycerol accumulation, whereas the selective P2X receptor antagonist NF279 has less effect. The present results indicate that cell-surface F1F0-ATP synthase on adipocytes is functional in extracellular ATP production and that the extracellular ATP produced contributes, at least in part, to the cell-surface F1F0-ATP synthase-mediated intracellular triacylglycerol accumulation in adipocytes through P2Y1 receptor.

Defining suitable reference genes for RT-qPCR analysis on human sertoli cells after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure.

PubMed

Ribeiro, Mariana Antunes; dos Reis, Mariana Bisarro; de Moraes, Leonardo Nazário; Briton-Jones, Christine; Rainho, Cláudia Aparecida; Scarano, Wellerson Rodrigo

2014-11-01

Quantitative real-time RT-PCR (qPCR) has proven to be a valuable molecular technique to quantify gene expression. There are few studies in the literature that describe suitable reference genes to normalize gene expression data. Studies of transcriptionally disruptive toxins, like tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes. The present study was designed to validate potential reference genes in human Sertoli cells after exposure to TCDD. 32 candidate reference genes were analyzed to determine their applicability. geNorm and NormFinder softwares were used to obtain an estimation of the expression stability of the 32 genes and to identify the most suitable genes for qPCR data normalization.

Keratinocytes mediate innocuous and noxious touch via ATP-P2X4 signaling

PubMed Central

Moehring, Francie; Cowie, Ashley M; Menzel, Anthony D; Weyer, Andy D; Grzybowski, Michael; Arzua, Thiago; Geurts, Aron M; Palygin, Oleg

2018-01-01

The first point of our body’s contact with tactile stimuli (innocuous and noxious) is the epidermis, the outermost layer of skin that is largely composed of keratinocytes. Here, we sought to define the role that keratinocytes play in touch sensation in vivo and ex vivo. We show that optogenetic inhibition of keratinocytes decreases behavioral and cellular mechanosensitivity. These processes are inherently mediated by ATP signaling, as demonstrated by complementary cutaneous ATP release and degradation experiments. Specific deletion of P2X4 receptors in sensory neurons markedly decreases behavioral and primary afferent mechanical sensitivity, thus positioning keratinocyte-released ATP to sensory neuron P2X4 signaling as a critical component of baseline mammalian tactile sensation. These experiments lay a vital foundation for subsequent studies into the dysfunctional signaling that occurs in cutaneous pain and itch disorders, and ultimately, the development of novel topical therapeutics for these conditions. PMID:29336303

TERATOGENICITY OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN MICE LACKING THE EXPRESSION OF EGF AND/OR TGFALPHA

EPA Science Inventory

Abstract
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure produces hydronephrosis and cleft palate in mice. These responses are correlated with disruption of expression of epidermal growth factor (EGF) receptor ligands, primarily EGF and transforming growth factor-alpha ...

ATP analogues at a glance.

PubMed

Bagshaw, C

2001-02-01

ATP has long been known to play a central role in the energetics of cells both in transduction mechanisms and in metabolic pathways, and is involved in regulation of enzyme, channel and receptor activities. Numerous ATP analogues have been synthesised to probe the role of ATP in biosystems (Yount, 1975; Jameson and Eccleston, 1997; Bagshaw, 1998). In general, two contrasting strategies are employed. Modifications may be introduced deliberately to change the properties of ATP (e.g. making it non-hydrolysable) so as to perturb the chemical steps involved in its action. Typically these involve modification of the phosphate chain. Alternatively, derivatives (e.g. fluorescent probes) are designed to report on the action of ATP but have a minimal effect on its properties. ATP-utilising systems vary enormously in their specificity; so what acts as a good analogue in one case may be very poor in another. The accompanying poster shows a representative selection of derivatives that have been synthesised and summarises their key properties.

Optogenetic control of ATP release

NASA Astrophysics Data System (ADS)

Lewis, Matthew A.; Joshi, Bipin; Gu, Ling; Feranchak, Andrew; Mohanty, Samarendra K.

2013-03-01

Controlled release of ATP can be used for understanding extracellular purinergic signaling. While coarse mechanical forces and hypotonic stimulation have been utilized in the past to initiate ATP release from cells, these methods are neither spatially accurate nor temporally precise. Further, these methods cannot be utilized in a highly effective cell-specific manner. To mitigate the uncertainties regarding cellular-specificity and spatio-temporal release of ATP, we herein demonstrate use of optogenetics for ATP release. ATP release in response to optogenetic stimulation was monitored by Luciferin-Luciferase assay (North American firefly, photinus pyralis) using luminometer as well as mesoscopic bioluminescence imaging. Our result demonstrates repetitive release of ATP subsequent to optogenetic stimulation. It is thus feasible that purinergic signaling can be directly detected via imaging if the stimulus can be confined to single cell or in a spatially-defined group of cells. This study opens up new avenue to interrogate the mechanisms of purinergic signaling.

Erythrocyte haemolysate interacts with ATP-Fe to form a complex containing iron, ATP and 13 800 MW polypeptide.

PubMed

Weaver, J; Zhan, H; Pollack, S

1993-01-01

Iron first entering the reticulocyte is bound to ATP in the low MW cytosolic pool; some is also 'loosely bound' to haemoglobin, coeluting with haemoglobin from a molecular sieve column though not incorporated into haem. When haemolysate is mixed with ATP-Fe in vitro a similar high MW iron-containing complex is formed: the ATP-Fe interacts with a non-haemoglobin constituent of the haemolysate to form a high MW ATP-Fe complex in which the ratio of ATP:Fe (originally 6:1) is reversed, so that the complex contains more iron than ATP. The high MW ATP-Fe complex is formed even when ATP is in 150-fold molar excess and is formed without detectable hydrolysis of the ATP. The activity of haemolysate in forming the high MW ATP-Fe complex is not diminished by dialysis; all of the activity is recovered in the haemoglobin-containing fraction obtained from an Ultrogel AcA 44 column. The activity does not derive from haemoglobin since 85% of the activity is removed when haemoglobin is purified from haemolysate with DEAE-Sephadex. The chelatable iron pool of the cell probably includes both the high MW ATP-Fe complex and low MW ATP-Fe. Shunting of ATP-Fe to a high MW aggregate reduces the amount of iron present in the highly reactive low MW form and thus probably serves to limit the formation of cell damaging radicals.

Exposure reconstruction for the TCDD-exposed NIOSH cohort using a concentration- and age-dependent model of elimination.

PubMed

Aylward, Lesa L; Brunet, Robert C; Starr, Thomas B; Carrier, Gaétan; Delzell, Elizabeth; Cheng, Hong; Beall, Colleen

2005-08-01

Recent studies demonstrating a concentration dependence of elimination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suggest that previous estimates of exposure for occupationally exposed cohorts may have underestimated actual exposure, resulting in a potential overestimate of the carcinogenic potency of TCDD in humans based on the mortality data for these cohorts. Using a database on U.S. chemical manufacturing workers potentially exposed to TCDD compiled by the National Institute for Occupational Safety and Health (NIOSH), we evaluated the impact of using a concentration- and age-dependent elimination model (CADM) (Aylward et al., 2005) on estimates of serum lipid area under the curve (AUC) for the NIOSH cohort. These data were used previously by Steenland et al. (2001) in combination with a first-order elimination model with an 8.7-year half-life to estimate cumulative serum lipid concentration (equivalent to AUC) for these workers for use in cancer dose-response assessment. Serum lipid TCDD measurements taken in 1988 for a subset of the cohort were combined with the NIOSH job exposure matrix and work histories to estimate dose rates per unit of exposure score. We evaluated the effect of choices in regression model (regression on untransformed vs. ln-transformed data and inclusion of a nonzero regression intercept) as well as the impact of choices of elimination models and parameters on estimated AUCs for the cohort. Central estimates for dose rate parameters derived from the serum-sampled subcohort were applied with the elimination models to time-specific exposure scores for the entire cohort to generate AUC estimates for all cohort members. Use of the CADM resulted in improved model fits to the serum sampling data compared to the first-order models. Dose rates varied by a factor of 50 among different combinations of elimination model, parameter sets, and regression models. Use of a CADM results in increases of up to five-fold in AUC estimates for the more

ATP Maintenance via Two Types of ATP Regulators Mitigates Pathological Phenotypes in Mouse Models of Parkinson's Disease.

PubMed

Nakano, Masaki; Imamura, Hiromi; Sasaoka, Norio; Yamamoto, Masamichi; Uemura, Norihito; Shudo, Toshiyuki; Fuchigami, Tomohiro; Takahashi, Ryosuke; Kakizuka, Akira

2017-08-01

Parkinson's disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances), which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors) are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist) against cell death in Parkinson's disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor) were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinson's disease. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Ionotropic P2X ATP Receptor Channels Mediate Purinergic Signaling in Mouse Odontoblasts

PubMed Central

Shiozaki, Yuta; Sato, Masaki; Kimura, Maki; Sato, Toru; Tazaki, Masakazu; Shibukawa, Yoshiyuki

2017-01-01

ATP modulates various functions in the dental pulp cells, such as intercellular communication and neurotransmission between odontoblasts and neurons, proliferation of dental pulp cells, and odontoblast differentiation. However, functional expression patterns and their biophysical properties of ionotropic ATP (P2X) receptors (P2X1–P2X7) in odontoblasts were still unclear. We examined these properties of P2X receptors in mouse odontoblasts by patch-clamp recordings. K+-ATP, nonselective P2X receptor agonist, induced inward currents in odontoblasts in a concentration-dependent manner. K+-ATP-induced currents were inhibited by P2X4 and P2X7 selective inhibitors (5-BDBD and KN62, respectively), while P2X1 and P2X3 inhibitors had no effects. P2X7 selective agonist (BzATP) induced inward currents dose-dependently. We could not observe P2X1, 2/3, 3 selective agonist (αβ-MeATP) induced currents. Amplitudes of K+-ATP-induced current were increased in solution without extracellular Ca2+, but decreased in Na+-free extracellular solution. In the absence of both of extracellular Na+ and Ca2+, K+-ATP-induced currents were completely abolished. K+-ATP-induced Na+ currents were inhibited by P2X7 inhibitor, while the Ca2+ currents were sensitive to P2X4 inhibitor. These results indicated that odontoblasts functionally expressed P2X4 and P2X7 receptors, which might play an important role in detecting extracellular ATP following local dental pulp injury. PMID:28163685

Ionotropic P2X ATP Receptor Channels Mediate Purinergic Signaling in Mouse Odontoblasts.

PubMed

Shiozaki, Yuta; Sato, Masaki; Kimura, Maki; Sato, Toru; Tazaki, Masakazu; Shibukawa, Yoshiyuki

2017-01-01

ATP modulates various functions in the dental pulp cells, such as intercellular communication and neurotransmission between odontoblasts and neurons, proliferation of dental pulp cells, and odontoblast differentiation. However, functional expression patterns and their biophysical properties of ionotropic ATP (P2X) receptors (P2X 1 -P2X 7 ) in odontoblasts were still unclear. We examined these properties of P2X receptors in mouse odontoblasts by patch-clamp recordings. K + -ATP, nonselective P2X receptor agonist, induced inward currents in odontoblasts in a concentration-dependent manner. K + -ATP-induced currents were inhibited by P2X 4 and P2X 7 selective inhibitors (5-BDBD and KN62, respectively), while P2X 1 and P2X 3 inhibitors had no effects. P2X 7 selective agonist (BzATP) induced inward currents dose-dependently. We could not observe P2X 1, 2/3, 3 selective agonist (αβ-MeATP) induced currents. Amplitudes of K + -ATP-induced current were increased in solution without extracellular Ca 2+ , but decreased in Na + -free extracellular solution. In the absence of both of extracellular Na + and Ca 2+ , K + -ATP-induced currents were completely abolished. K + -ATP-induced Na + currents were inhibited by P2X 7 inhibitor, while the Ca 2+ currents were sensitive to P2X 4 inhibitor. These results indicated that odontoblasts functionally expressed P2X 4 and P2X 7 receptors, which might play an important role in detecting extracellular ATP following local dental pulp injury.

ATP depletion during mitotic arrest induces mitotic slippage and APC/CCdh1-dependent cyclin B1 degradation.

PubMed

Park, Yun Yeon; Ahn, Ju-Hyun; Cho, Min-Guk; Lee, Jae-Ho

2018-04-27

ATP depletion inhibits cell cycle progression, especially during the G1 phase and the G2 to M transition. However, the effect of ATP depletion on mitotic progression remains unclear. We observed that the reduction of ATP after prometaphase by simultaneous treatment with 2-deoxyglucose and NaN 3 did not arrest mitotic progression. Interestingly, ATP depletion during nocodazole-induced prometaphase arrest resulted in mitotic slippage, as indicated by a reduction in mitotic cells, APC/C-dependent degradation of cyclin B1, increased cell attachment, and increased nuclear membrane reassembly. Additionally, cells successfully progressed through the cell cycle after mitotic slippage, as indicated by EdU incorporation and time-lapse imaging. Although degradation of cyclin B during normal mitotic progression is primarily regulated by APC/C Cdc20 , we observed an unexpected decrease in Cdc20 prior to degradation of cyclin B during mitotic slippage. This decrease in Cdc20 was followed by a change in the binding partner preference of APC/C from Cdc20 to Cdh1; consequently, APC/C Cdh1 , but not APC/C Cdc20 , facilitated cyclin B degradation following ATP depletion. Pulse-chase analysis revealed that ATP depletion significantly abrogated global translation, including the translation of Cdc20 and Cdh1. Additionally, the half-life of Cdh1 was much longer than that of Cdc20. These data suggest that ATP depletion during mitotic arrest induces mitotic slippage facilitated by APC/C Cdh1 -dependent cyclin B degradation, which follows a decrease in Cdc20 resulting from reduced global translation and the differences in the half-lives of the Cdc20 and Cdh1 proteins.

ATP-induced current in isolated outer hair cells of guinea pig cochlea.

PubMed

Nakagawa, T; Akaike, N; Kimitsuki, T; Komune, S; Arima, T

1990-05-01

1. Electrical and pharmacologic properties of ATP-induced current in outer hair cells isolated from guinea pig cochlea were investigated in the whole-cell recording mode by the use of a conventional patch-clamp technique. 2. Under current-clamp conditions, rapid application of ATP depolarized the outer hair cells resulting in an increase in conductance. The ATP-induced response did not show any desensitization during a continuous application. 3. At a holding potential of -70 mV, the ATP-induced inward current increased in a sigmoidal fashion over the concentration range between 3 microM and 1 mM. The half-maximum concentration (EC50) was 12 microM and the Hill coefficient was 0.93. 4. The ATP-induced current had a reversal potential near 6 mV, which was close to the theoretical value (1 mV) calculated from the Goldman-Hodgkin-Katz equation for permeable intra- and extracellular cations. 5. In the current-voltage (I-V) relationship for the ATP response, a slight inward-going rectification was observed at more positive potentials than the reversal potential. 6. The substitution of extracellular Na+ by equimolar choline+ shifted the reversal potential of the ATP-induced current to more negative values. The substitution of Cs+ in the internal solution by N-methyl-D-glucamine+ (NMG+) shifted it in the positive direction. The reversal potential of ATP-induced current was also shifted to positive values with increasing extracellular Ca2+ concentration. A decrease of intracellular Cl- by gluconate- did not affect the reversal potential, thereby indicating that the ATP-induced current is carried through a large cation channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shin, Youn Ho; Lee, Seo Jin; Jung, Junyang, E-mail: jjung@khu.ac.kr

Highlights: Black-Right-Pointing-Pointer ATP-treated sciatic explants shows the decreased expression of p75NGFR. Black-Right-Pointing-Pointer Extracellular ATP inhibits the expression of phospho-ERK1/2. Black-Right-Pointing-Pointer Lysosomal exocytosis is involved in Schwann cell dedifferentiation. Black-Right-Pointing-Pointer Extracellular ATP blocks Schwann cell proliferation in sciatic explants. -- Abstract: After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibitsmore » Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.« less

FTIR Study of ATP-Induced Changes in Na+/K+-ATPase from Duck Supraorbital Glands

PubMed Central

Pratap, Promod R.; Dediu, Oana; Nienhaus, G. Ulrich

2003-01-01

The Na+/K+-ATPase uses energy from the hydrolysis of ATP to pump Na+ ions out of and K+ ions into the cell. ATP-induced conformational changes in the protein have been examined in the Na+/K+-ATPase isolated from duck supraorbital salt glands using Fourier transform infrared spectroscopy. Both standard transmission and attenuated total internal reflection sample geometries have been employed. Under transmission conditions, enzyme at 75 mg/ml was incubated with dimethoxybenzoin-caged ATP. ATP was released by flashing with a UV laser pulse at 355 nm, which resulted in a large change in the amide I band. The absorbance at 1659 cm−1 decreased with a concomitant increase in the absorbance at 1620 cm−1. These changes are consistent with a partial conversion of protein secondary structure from α-helix to β-sheet. The changes were ∼8% of the total absorbance, much larger than those seen with other P-type ATPases. Using attenuated total internal reflection Fourier transform infrared spectroscopy, the decrease in absorbance at ∼1650 cm−1 was titrated with ATP, and the titration midpoint K0.5 was determined under different ionic conditions. In the presence of metal ions (Na+, Na+ and K+, or Mg2+), K0.5 was on the order of a few μM. In the absence of these ions, K0.5 was an order of magnitude lower (0.1 μM), indicating a higher apparent affinity. This effect suggests that the equilibrium for the ATP-induced conformational changes is dependent on the presence of metal ions. PMID:14645062

Modulation of nucleotide sensitivity of ATP-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase.

PubMed

Shyng, S L; Barbieri, A; Gumusboga, A; Cukras, C; Pike, L; Davis, J N; Stahl, P D; Nichols, C G

2000-01-18

ATP-sensitive potassium channels (K(ATP) channels) regulate cell excitability in response to metabolic changes. K(ATP) channels are formed as a complex of a sulfonylurea receptor (SURx), a member of the ATP-binding cassette protein family, and an inward rectifier K(+) channel subunit (Kir6.x). Membrane phospholipids, in particular phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)), activate K(ATP) channels and antagonize ATP inhibition of K(ATP) channels when applied to inside-out membrane patches. To examine the physiological relevance of this regulatory mechanism, we manipulated membrane PIP(2) levels by expressing either the wild-type or an inactive form of PI-4-phosphate 5-kinase (PIP5K) in COSm6 cells and examined the ATP sensitivity of coexpressed K(ATP) channels. Channels from cells expressing the wild-type PIP5K have a 6-fold lower ATP sensitivity (K(1/2), the half maximal inhibitory concentration, approximately 60 microM) than the sensitivities from control cells (K(1/2) approximately 10 microM). An inactive form of the PIP5K had little effect on the K(1/2) of wild-type channels but increased the ATP-sensitivity of a mutant K(ATP) channel that has an intrinsically lower ATP sensitivity (from K(1/2) approximately 450 microM to K(1/2) approximately 100 microM), suggesting a decrease in membrane PIP(2) levels as a consequence of a dominant-negative effect of the inactive PIP5K. These results show that PIP5K activity, which regulates PIP(2) and PI-3,4,5-P(3) levels, is a significant determinant of the physiological nucleotide sensitivity of K(ATP) channels.

An ATP-gated cation channel with some P2Z-like characteristics in gastric smooth muscle cells of toad.

PubMed Central

Ugur, M; Drummond, R M; Zou, H; Sheng, P; Singer, J J; Walsh, J V

1997-01-01

1. Whole-cell and single-channel currents elicited by extracellular ATP were studied in freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus using standard patch clamp and microfluorimetric techniques. 2. This ATP-gated cation channel shares a number of pharmacological and functional properties with native rat myometrium receptors, certain native P2Z purinoceptors and the recently cloned P2X7 purinoceptor. But, unlike the last two, the ATP-gated channel does not mediate the formation of large non-specific pores. Thus, it may represent a novel member of the P2X or P2Z class. 3. Extracellular application of ATP (> or = 150 microM) elicited an inward whole-cell current at negative holding potentials that was inwardly rectifying and showed no sign of desensitization. Na+, Cs+ and, to a lesser degree, the organic cation choline served as charge carriers, but Cl- did not. Ratiometric fura-2 measurements indicated that the current is carried in part by Ca2+. The EC50 for ATP was 700 microM in solutions with a low divalent cation concentration. 4. ATP (> or = 100 microM) at the extracellular surface of cell-attached or excised patches elicited inwardly rectifying single-channel currents with a 22 pS conductance. Cl- did not serve as a charge carrier but both Na+ and Cs+ did, as did choline to a lesser extent. The mean open time of the channel was quite long, with a range in hundreds of milliseconds at a holding potential of -70 mV. 5. Mg2+ and Ca2+ decreased the magnitude of the ATP-induced whole-cell currents. Mg2+ decreased both the amplitude and the activity of ATP-activated single-channel currents. 6. ADP, UTP, P1, P5-di-adenosine pentaphosphate (AP5A), adenosine and alpha, beta-methylene ATP (alpha, beta-Me-ATP) did not induce significant whole-cell current. ATP-gamma-S and 2-methylthio ATP (2-Me-S-ATP) were significantly less effective than ATP in inducing whole-cell currents, whereas benzoylbenzoyl ATP (BzATP) was more effective. BzATP

Down-regulation of the detoxifying enzyme NAD(P)H:quinone oxidoreductase 1 by vanadium in Hepa 1c1c7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anwar-Mohamed, Anwar; El-Kadi, Ayman O.S.

2009-05-01

Recent data suggest that vanadium (V{sup 5+}) compounds exert protective effects against chemical-induced carcinogenesis, mainly through modifying various xenobiotic metabolizing enzymes. In fact, we have shown that V{sup 5+} down-regulates the expression of Cyp1a1 at the transcriptional level through an ATP-dependent mechanism. However, incongruously, there is increasing evidence that V{sup 5+} is found in higher amounts in cancer cells and tissues than in normal cells or tissues. Therefore, the current study aims to address the possible effect of this metal on the regulation of expression of an enzyme that helps maintain endogenous antioxidants used to protect tissues/cells from mutagens, carcinogens,more » and oxidative stress damage, NAD(P)H:quinone oxidoreductase 1 (Nqo1). In an attempt to examine these effects, Hepa 1c1c7 cells and its AhR-deficient version, c12, were treated with increasing concentrations of V{sup 5+} in the presence of two distinct Nqo1 inducers, the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and isothiocyanate sulforaphane (SUL). Our results showed that V{sup 5+} inhibits the TCDD- and SUL-mediated induction of Nqo1 at mRNA, protein, and catalytic activity levels. At transcriptional level, V{sup 5+} was able to decrease the TCDD- and SUL-induced nuclear accumulation of Nrf2 and the subsequent binding to antioxidant responsive element (ARE) without affecting Nrf2 protein levels. Looking at post-transcriptional level; we found that V{sup 5+} did not affect Nqo1 mRNA transcripts turn-over rates. However, at the post-translational level V{sup 5+} increased Nqo1 protein half-life. In conclusion, the present study demonstrates that V{sup 5+} down-regulates Nqo1 at the transcriptional level, possibly through inhibiting the ATP-dependent activation of Nrf2.« less

Effects of 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) injected into the yolks of chicken (Gallus domesticus) eggs prior to incubation

USGS Publications Warehouse

Powell, D.C.; Aulerich, R.J.; Meadows, J.C.; Tillitt, D.E.; Giesy, J.P.; Stromborg, K.L.; Bursian, S.J.

1996-01-01

The yolks of White Leghorn chicken (Gallus domesticus) eggs were injected prior to incubation with either 3,3′,4,4′,5- pentachlorobiphenyl (PCB 126) at doses ranging from 0.1 to 12.8 μg/kg egg or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at doses ranging from 0.04 to 0.64 μg/kg egg. Chicks were subjected to necropsy within 24 h of hatching. The brain, bursa, heart, liver, and spleen were removed and weighed. Assessment of the rate of hatching indicated an LD50±S.E. of 2.3±0.19 μg/kg egg (7.1±0.58 nmol/kg egg) for PCB 126 and 0.15±0.012 μg/kg egg (0.47±0.037 nmol/kg egg) for TCDD. No significant differences in the incidence of developmental abnormalities (structural defects and edema) were observed in TCDD-exposed embryos, while PCB 126 caused significantly more developmental abnormalities at 3.2, 6.4, and 12.8 μg/kg egg than the vehicle control. PCB 126 caused lower hatchling weights and greater relative brain, heart, and liver weights when compared to the vehicle control group at a dose of 3.2 μg/kg egg which is greater than the LD50. TCDD at 0.08 μg/kg egg caused relative bursa weights to be less than those of the vehicle control. A toxic equivalency factor (TEF) of 0.07 was determined for PCB 126 in relation to TCDD based on overt lethality.

TCDD and omeprazole prime platelets through the aryl hydrocarbon receptor (AhR) non-genomic pathway.

PubMed

Pombo, Mónica; Lamé, Michael W; Walker, Naomi J; Huynh, Danh H; Tablin, Fern

2015-05-19

The role of the aryl hydrocarbon receptor (AhR) in hemostasis has recently gained increased attention. Here, we demonstrate, by qRT-PCR and western blot, that human platelets express both AhR mRNA and AhR protein. AhR protein levels increase in a dose dependent manner when incubated with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or omeprazole. Treatment of platelets with puromycin blocks increased AhR protein synthesis in the presence of AhR activators. Additionally, treatment of platelets with either activator results in phosphorylation of p38MAPK and cPLA2, two key signaling molecules in platelet activation pathways. Using the AhR competitive inhibitors alpha naphthoflavone and CH-223191, we show that phosphorylation of p38MAPK is AhR dependent. Further, inhibition of p38MAPK blocks downstream cPLA2 phosphorylation induced by TCDD or omeprazole. Treatment with AhR activators results in platelet priming, as demonstrated by increased platelet aggregation, which is inhibited by AhR antagonists. Our data support a model of the platelet AhR non-genomic pathway in which treatment with AhR activators results in increased expression of the AhR, phosphorylation of p38MAPK and cPLA2, leading to platelet priming in response to agonist. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Developmental Exposure of Mice to TCDD Elicits a Similar Uterine Phenotype in Adult Animals as Observed in Women with Endometriosis

PubMed Central

Nayyar, Tultul; Bruner-Tran, Kaylon L.; Piestrzeniewicz-Ulanska, Dagmara; Osteen, Kevin G.

2007-01-01

Whether environmental toxicants impact an individual woman’s risk for developing endometriosis remains uncertain. Although the growth of endometrial glands and stroma at extra-uterine sites is associated with retrograde menstruation, our studies suggest that reduced responsiveness to progesterone may increase the invasive capacity of endometrial tissue in women with endometriosis. Interestingly, our recent studies using isolated human endometrial cells in short-term culture suggest that experimental exposure to the environmental contaminant 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) can alter the expression of progesterone receptor isotypes. Compared to adult exposure, toxicant exposure during development can exert a significantly greater biological impact, potentially affecting the incidence of endometriosis in adults. To address this possibility, we exposed mice to TCDD at critical developmental time points and subsequently examined uterine progesterone receptor expression and steroid responsive transforming growth factor-β2 expression in adult animals. We find that the uterine phenotype of toxicant-exposed mice is markedly similarly to the endometrial phenotype of women with endometriosis. PMID:17056225

CD73 and AMPD3 deficiency enhance metabolic performance via erythrocyte ATP that decreases hemoglobin oxygen affinity.

PubMed

O'Brien, William G; Berka, Vladimir; Tsai, Ah-Lim; Zhao, Zhaoyang; Lee, Cheng Chi

2015-08-07

Erythrocytes are the key target in 5'-AMP induced hypometabolism. To understand how regulation of endogenous erythrocyte AMP levels modulates systemic metabolism, we generated mice deficient in both CD73 and AMPD3, the key catabolic enzymes for extracellular and intra-erythrocyte AMP, respectively. Under physiological conditions, these mice displayed enhanced capacity for physical activity accompanied by significantly higher food and oxygen consumption, compared to wild type mice. Erythrocytes from Ampd3(-/-) mice exhibited higher half-saturation pressure of oxygen (p50) and about 3-fold higher levels of ATP and ADP, while they maintained normal 2,3-bisphosphoglycerate (2,3-BPG), methemoglobin levels and intracellular pH. The affinity of mammalian hemoglobin for oxygen is thought to be regulated primarily by 2,3-BPG levels and pH (the Bohr effect). However, our results show that increased endogenous levels of ATP and ADP, but not AMP, directly increase the p50 value of hemoglobin. Additionally, the rise in erythrocyte p50 directly correlates with an enhanced capability of systemic metabolism.

PERSISTENT ABNORMALITIES IN THE RAT MAMMARY GLAND FOLLOWING GESTATIONAL AND LACTATIONAL EXPOSURE TO 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD)

EPA Science Inventory

SUMMARY

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure during gestation has revealed reproductive anomalies in rat offspring, including inconclusive reports of stunted mammary development in females (Brown et al., 1998, Lewis et al., 2001). The current studies wer...

Dioxin exposure reduces the steroidogenic capacity of mouse antral follicles mainly at the level of HSD17B1 without altering atresia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karman, Bethany N., E-mail: bklement@illinois.edu; Basavarajappa, Mallikarjuna S., E-mail: mbshivapur@gmail.com; Hannon, Patrick, E-mail: phannon2@illinois.edu

2012-10-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent ovarian toxicant. Previously, we demonstrated that in vitro TCDD (1 nM) exposure decreases production/secretion of the sex steroid hormones progesterone (P4), androstenedione (A4), testosterone (T), and 17β-estradiol (E2) in mouse antral follicles. The purpose of this study was to determine the mechanism by which TCDD inhibits steroidogenesis. Specifically, we examined the effects of TCDD on the steroidogenic enzymes, atresia, and the aryl hydrocarbon receptor (AHR) protein. TCDD exposure for 48 h increased levels of A4, without changing HSD3B1 protein, HSD17B1 protein, estrone (E1), T or E2 levels. Further, TCDD did not alter atresia ratings comparedmore » to vehicle at 48 h. TCDD, however, did down regulate the AHR protein at 48 h. TCDD exposure for 96 h decreased transcript levels for Cyp11a1, Cyp17a1, Hsd17b1, and Cyp19a1, but increased Hsd3b1 transcript. TCDD exposure particularly lowered both Hsd17b1 transcript and HSD17B1 protein. However, TCDD exposure did not affect levels of E1 in the media nor atresia ratings at 96 h. TCDD, however, decreased levels of the proapoptotic factor Bax. Collectively, these data suggest that TCDD exposure causes a major block in the steroidogenic enzyme conversion of A4 to T and E1 to E2 and that it regulates apoptotic pathways, favoring survival over death in antral follicles. Finally, the down‐regulation of the AHR protein in TCDD exposed follicles persisted at 96 h, indicating that the activation and proteasomal degradation of this receptor likely plays a central role in the impaired steroidogenic capacity and altered apoptotic pathway of exposed antral follicles. -- Highlights: ► TCDD disrupts steroidogenic enzymes in mouse antral follicles. ► TCDD particularly affects the HSD17B1 enzyme in mouse antral follicles. ► TCDD does not affect atresia ratings in mouse antral follicles. ► TCDD decreases levels of the proapoptitic factor Bax in mouse antral

ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells

PubMed Central

Song, Shanshan; Jacobson, Krista N.; McDermott, Kimberly M.; Reddy, Sekhar P.; Cress, Anne E.; Tang, Haiyang; Dudek, Steven M.; Black, Stephen M.; Garcia, Joe G. N.; Makino, Ayako

2015-01-01

Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca2+ signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca2+] ([Ca2+]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca2+ eliminated the plateau phase increase of [Ca2+]cyt in lung cancer cells, indicating that the plateau phase of [Ca2+]cyt increase is due to Ca2+ influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca2+ or chelating intracellular Ca2+ with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca2+]cyt through Ca2+ influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression. PMID:26491047

[P4-ATP-ase Atp8b1/FIC1: structural properties and (patho)physiological functions].

PubMed

Korneenko, T V; Pestov, N B; Okkelman, I A; Modyanov, N N; Shakhparonov, M I

2015-01-01

P4-ATP-ases comprise an interesting family among P-type ATP-ases, since they are thought to play a major role in the transfer of phospholipids such as phosphatydylserine from the outer leaflet to the inner leaflet. Isoforms of P4-ATP-ases are partially interchangeable but peculiarities of tissue-specific expression of their genes, intracellular localization of proteins, as well as regulatory pathways lead to the fact that, on the organismal level, serious pathologies may develop in the presence of structural abnormalities in certain isoforms. Among P4-ATP-ases a special place is occupied by ATP8B1, for which several mutations are known that lead to serious hereditary diseases: two forms of congenital cholestasis (PFIC1 or Byler disease and benign recurrent intrahepatic cholestasis) with extraliver symptoms such as sensorineural hearing loss. The physiological function of the Atp8b1/FIC1 protein is known in general outline: it is responsible for transport of certain phospholipids (phosphatydylserine, cardiolipin) for the outer monolayer of the plasma membrane to the inner one. It is well known that perturbation of membrane asymmetry, caused by the lack of Atp8B1 activity, leads to death of hairy cells of the inner ear, dysfunction of bile acid transport in liver-cells that causes cirrhosis. It is also probable that insufficient activity of Atp8b1/FIC1 increases susceptibility to bacterial pneumonia.Regulatory pathways of Atp8b1/FIC1 activity in vivo remain to be insufficiently studied and this opens novel perspectives for research in this field that may allow better understanding of molecular processes behind the development of certain pathologies and to reveal novel therapeutical targets.

Ethanol Dose- and Time-dependently Increases α and β Subunits of Mitochondrial ATP Synthase of Cultured Neonatal Rat Cardiomyocytes.

PubMed

Mashimo, Keiko; Arthur, Peter G; Ohno, Youkichi

2015-01-01

Mitochondria are target subcellular organelles of ethanol. In this study, the effects of ethanol on protein composition was examined with 2-dimensional electrophoresis of protein extracts from cultured neonatal rat cardiomyocytes exposed to 100 mM ethanol for 24 hours. A putative β subunit of mitochondrial ATP synthase was increased, which was confirmed by Western blot. The cellular protein abundances in the α and β subunits of ATP synthase increased in dose (0, 10, 50, and 100 mM) - and time (0.5 hour and 24 hours) -dependent manners. The DNA microarray analysis of total RNA extract demonstrated that gene expression of the corresponding messenger RNAs of these subunit proteins did not significantly alter due to 24-hour ethanol exposure. Therefore, protein expression of these nuclear-encoded mitochondrial proteins may be regulated at the translational, rather than the transcriptional, level. Alternatively, degradation of these subunit proteins might be decreased. Additionally, cellular ATP content of cardiomyocytes scarcely decreased following 24-hour exposure to any examined concentrations of ethanol. Previous studies, together with this study, have demonstrated that protein abundance of the α subunit or β subunit or both subunits of ATP synthase after ethanol exposure or dysfunctional conditions might differ according to tissue: significant increases in heart but decreases in liver and brain. Thus, it is suggested that the abundance of subunit proteins of mitochondrial ATP synthase in the ethanol-exposed heart, being different from that in the liver and brain, should increase dose-dependently through either translational upregulation or decreased degradation or both to maintain ATP production, as the heart requires much more energy than other tissues for continuing sustained contractions.

2,3,7,8-Tetrachlorodibenzo-P-Dioxin (TCDD) Dose-Response Studies: Preliminary Literature Search Results and Request for Additional Studies

EPA Science Inventory

EPA invited the public to comment on the preliminary list of in vivo mammalian dose-response citations for 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). This list was compiled as a first step in the development of EPA’s response to the National Academy of Sciences comments (NAS, 2...

Influence of glucagon or 5-(tetradecyloxy)-2-furoic acid on binding to mitochondria and phosphorylation of ATP-citrate lyase.

PubMed

Janski, A M; Cornell, N W

1982-02-01

To study the binding to mitochondria and the phosphorylation of ATP-citrate lyase (EC 4.1.3.8), isolated rat hepatocytes were fractionated by exposure to digitonin. After incubation of hepatocytes with the hypolipidemic agent 5-(tetradecyloxy)-2-furoic acid, which decreases the cellular CoA, the amount of bound ATP-citrate lyase was increased, but the content of acid-stable phosphate in the enzyme was diminished. Glucagon, in contrast, decreased the amount of bound enzyme but increased phosphorylation. This inverse relationship might indicate either that the bound ATP-citrate lyase is less readily phosphorylated or that the phosphorylated enzyme binds less readily to mitochondria.

Activation of an ATP-dependent K(+) conductance in Xenopus oocytes by expression of adenylate kinase cloned from renal proximal tubules.

PubMed

Brochiero, E; Coady, M J; Klein, H; Laprade, R; Lapointe, J Y

2001-02-09

In rabbit proximal convoluted tubules, an ATP-sensitive K(+) (K(ATP)) channel has been shown to be involved in membrane cross-talk, i.e. the coupling (most likely mediated through intracellular ATP) between transepithelial Na(+) transport and basolateral K(+) conductance. This K(+) conductance is inhibited by taurine. We sought to isolate this K(+) channel by expression cloning in Xenopus oocytes. Injection of renal cortex mRNA into oocytes induced a K(+) conductance, largely inhibited by extracellular Ba(2+) and intracellular taurine. Using this functional test, we isolated from our proximal tubule cDNA library a unique clone, which induced a large K(+) current which was Ba(2+)-, taurine- and glibenclamide-sensitive. Surprisingly, this clone is not a K(+) channel but an adenylate kinase protein (AK3), known to convert NTP+AMP into NDP+ADP (N could be G, I or A). AK3 expression resulted in a large ATP decrease and activation of the whole-cell currents including a previously unknown, endogenous K(+) current. To verify whether ATP decrease was responsible for the current activation, we demonstrated that inhibition of glycolysis greatly reduces oocyte ATP levels and increases an inwardly rectifying K(+) current. The possible involvement of AK in the K(ATP) channel's regulation provides a means of explaining their observed activity in cytosolic environments characterized by high ATP concentrations.

ATP-induced changes in rat skeletal muscle contractility.

PubMed

Gabdrakhmanov, A I; Khayrullin, A E; Grishin, C H; Ziganshin, A U

2015-01-01

considered as typical effects of ATP and other purines on skeletal muscles and could not be extrapolated to all warm-blooded animals. Furthermore the role of ATP and its derivatives in the accumulation of vertebrate muscular effort has not been investigated.It is known that in physiological conditions vertebrates may mobilize only up to a third of the maximum muscle force. Why the two-thirds of muscular strength are not used normally but may be used at stress, remains unknown.It is known that the body's adaptive response to stress is a change in the activity of the endocrine system. The leading role in this is given to catechol amines and glucocorticoids, mobilized in significant quantities in blood under stress.We have found previously that incubation of frog sartorius muscle with hydrocortisone resulted in a decrease of contraction amplitude. However, when hydrocortisone was used in combination with ATP, its inhibitory effect on contractile responses disappeared. It is interesting that hydrocortisone had no effect on the inhibitory effect of adenosine. In the following experiments, assessing the effect of hydrocortisone on rat soleus muscle, it was established that hydrocortisone and purines had similar inhibitory effect. When ATP and hydrocortisone were given together the same oppression occurred. To study the effects of ATP and adenosine on contraction parameters of rat skeletal muscle and assess the impact of the catechol amines on these processes. Contractions of rat soleus muscles were recorded isometrically by mechanical sensor Linton FSG-01 (UK) according to standard procedures. The average of muscle parameters received within 30 seconds (30 responses) was treated as one result. Amplitude and time characteristics of the curve reductions were estimated. During all experiments standard Krebs solution flowed through the bath continuously to which agents were added at necessary concentrations. All experimental animals were maintained and prepared for dissection under

Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae

PubMed Central

Peters, Theodore W.; Miller, Aaron W.; Tourette, Cendrine; Agren, Hannah; Hubbard, Alan; Hughes, Robert E.

2015-01-01

Adenosine triphosphate (ATP) plays an important role as a primary molecule for the transfer of chemical energy to drive biological processes. ATP also functions as an extracellular signaling molecule in a diverse array of eukaryotic taxa in a conserved process known as purinergic signaling. Given the important roles of extracellular ATP in cell signaling, we sought to comprehensively elucidate the pathways and mechanisms governing ATP efflux from eukaryotic cells. Here, we present results of a genomic analysis of ATP efflux from Saccharomyces cerevisiae by measuring extracellular ATP levels in cultures of 4609 deletion mutants. This screen revealed key cellular processes that regulate extracellular ATP levels, including mitochondrial translation and vesicle sorting in the late endosome, indicating that ATP production and transport through vesicles are required for efflux. We also observed evidence for altered ATP efflux in strains deleted for genes involved in amino acid signaling, and mitochondrial retrograde signaling. Based on these results, we propose a model in which the retrograde signaling pathway potentiates amino acid signaling to promote mitochondrial respiration. This study advances our understanding of the mechanism of ATP secretion in eukaryotes and implicates TOR complex 1 (TORC1) and nutrient signaling pathways in the regulation of ATP efflux. These results will facilitate analysis of ATP efflux mechanisms in higher eukaryotes. PMID:26585826

ATP mediates flow-induced NO production in thick ascending limbs

PubMed Central

Hong, Nancy J.; Garvin, Jeffrey L.

2012-01-01

Mechanical stimulation caused by increasing flow induces nucleotide release from many cells. Luminal flow and extracellular ATP stimulate production of nitric oxide (NO) in thick ascending limbs. However, the factors that mediate flow-induced NO production are unknown. We hypothesized that luminal flow stimulates thick ascending limb NO production via ATP. We measured NO in isolated, perfused rat thick ascending limbs using the fluorescent dye DAF FM. The rate of increase in dye fluorescence reflects NO accumulation. Increasing luminal flow from 0 to 20 nl/min stimulated NO production from 17 ± 16 to 130 ± 37 arbitrary units (AU)/min (P < 0.02). Increasing flow from 0 to 20 nl/min raised ATP release from 4 ± 1 to 21 ± 6 AU/min (P < 0.04). Hexokinase (10 U/ml) plus glucose, which consumes ATP, completely prevented the measured increase in ATP. Luminal flow did not increase NO production in the presence of luminal and basolateral hexokinase (10 U/ml). When flow was increased with the ATPase apyrase in both luminal and basolateral solutions (5 U/ml), NO levels did not change significantly. The P2 receptor antagonist suramin (300 μmol/l) reduced flow-induced NO production by 83 ± 25% (P < 0.03) when added to both and basolateral sides. Luminal hexokinase decreased flow-induced NO production from 205.6 ± 85.6 to 36.6 ± 118.6 AU/min (P < 0.02). Basolateral hexokinase also reduced flow-induced NO production. The P2X receptor-selective antagonist NF023 (200 μmol/l) prevented flow-induced NO production when added to the basolateral side but not the luminal side. We conclude that ATP mediates flow-induced NO production in the thick ascending limb likely via activation of P2Y receptors in the luminal and P2X receptors in the basolateral membrane. PMID:22496412

Regulation of Cl^- Channels in Normal and Cystic Fibrosis Airway Epithelial Cells by Extracellular ATP

NASA Astrophysics Data System (ADS)

Stutts, M. J.; Chinet, T. C.; Mason, S. J.; Fullton, J. M.; Clarke, L. L.; Boucher, R. C.

1992-03-01

The rate of Cl^- secretion by human airway epithelium is determined, in part, by apical cell membrane Cl^- conductance. In cystic fibrosis airway epithelia, defective regulation of Cl^- conductance decreases the capability to secrete Cl^-. Here we report that extracytosolic ATP in the luminal bath of cultured human airway epithelia increased transepithelial Cl^- secretion and apical membrane Cl^- permeability. Single-channel studies in excised membrane patches revealed that ATP increased the open probability of outward rectifying Cl^- channels. The latter effect occurs through a receptor mechanism that requires no identified soluble second messengers and is insensitive to probes of G protein function. These results demonstrate a mode of regulation of anion channels by binding ATP at the extracellular surface. Regulation of Cl^- conductance by external ATP is preserved in cystic fibrosis airway epithelia.

Mitochondrial flashes regulate ATP homeostasis in the heart

PubMed Central

Wang, Xianhua; Zhang, Xing; Wu, Di; Huang, Zhanglong; Hou, Tingting; Jian, Chongshu; Yu, Peng; Lu, Fujian; Zhang, Rufeng; Sun, Tao; Li, Jinghang; Qi, Wenfeng; Wang, Yanru; Gao, Feng; Cheng, Heping

2017-01-01

The maintenance of a constant ATP level (‘set-point’) is a vital homeostatic function shared by eukaryotic cells. In particular, mammalian myocardium exquisitely safeguards its ATP set-point despite 10-fold fluctuations in cardiac workload. However, the exact mechanisms underlying this regulation of ATP homeostasis remain elusive. Here we show mitochondrial flashes (mitoflashes), recently discovered dynamic activity of mitochondria, play an essential role for the auto-regulation of ATP set-point in the heart. Specifically, mitoflashes negatively regulate ATP production in isolated respiring mitochondria and, their activity waxes and wanes to counteract the ATP supply-demand imbalance caused by superfluous substrate and altered workload in cardiomyocytes. Moreover, manipulating mitoflash activity is sufficient to inversely shift the otherwise stable ATP set-point. Mechanistically, the Bcl-xL-regulated proton leakage through F1Fo-ATP synthase appears to mediate the coupling between mitoflash production and ATP set-point regulation. These findings indicate mitoflashes appear to constitute a digital auto-regulator for ATP homeostasis in the heart. DOI: http://dx.doi.org/10.7554/eLife.23908.001 PMID:28692422

Adenosine monophosphate as a mediator of ATP effects at P1 purinoceptors

PubMed Central

Ross, Fiona M; Brodie, Martin J; Stone, Trevor W

1998-01-01

When perfused with a medium containing no added magnesium and 4-aminopyridine (4AP) (50 μM) hippocampal slices generated epileptiform bursts of an interictal nature. We have shown in a previous study that adenosine 5′-triphosphate (ATP) depressed epileptiform activity and that this effect was blocked by the adenosine A1 receptor antagonist cyclopentyltheophylline but was not affected by adenosine deaminase. This implied that ATP might act indirectly at P1 receptors or at a xanthine-sensitive P2 receptor. The aim of the present study was to investigate further the action of ATP on epileptiform activity.ATP can be metabolized by ecto-nucleotidases to adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP) and adenosine, respectively. Each of these metabolites can activate receptors in its own right: P2 receptors for ADP and P1 receptors for AMP and adenosine.We now show that both AMP and ATP (50 μM) significantly decrease epileptiform discharge rate in a rapid and reversible manner. 5′Adenylic acid deaminase (AMP deaminase, AMPase) (0.2 u ml−1), when perfused alone did not significantly alter the discharge rate over the 10 min superfusion period used for drug application. When perfused concurrently with AMP (50 μM), AMP deaminase prevented the depressant effect of AMP on discharge rate.AMP deaminase, at a concentration of 0.2 u ml−1 which annulled the effect of AMP (50 μM), prevented the inhibitory activity of ATP (50 μM). A higher concentration of ATP (200 μM) depressed the frequency of spontaneous bursts to approximately 30% control and this response was also prevented by AMP deaminase.Superfusion of the slices with 5′-nucleotidase also prevented the inhibitory activity of ATP on epileptiform discharges.The results suggest that AMP mediates the inhibitory effects of ATP on epileptiform activity, a conclusion which can explain the earlier finding that cyclopentyltheophylline but not adenosine deaminase inhibited the

Ca2+-regulated-cAMP/PKA signaling in cardiac pacemaker cells links ATP supply to demand.

PubMed

Yaniv, Yael; Juhaszova, Magdalena; Lyashkov, Alexey E; Spurgeon, Harold A; Sollott, Steven J; Lakatta, Edward G

2011-11-01

In sinoatrial node cells (SANC), Ca(2+) activates adenylate cyclase (AC) to generate a high basal level of cAMP-mediated/protein kinase A (PKA)-dependent phosphorylation of Ca(2+) cycling proteins. These result in spontaneous sarcoplasmic-reticulum (SR) generated rhythmic Ca(2+) oscillations during diastolic depolarization, that not only trigger the surface membrane to generate rhythmic action potentials (APs), but, in a feed-forward manner, also activate AC/PKA signaling. ATP is consumed to pump Ca(2+) to the SR, to produce cAMP, to support contraction and to maintain cell ionic homeostasis. Since feedback mechanisms link ATP-demand to ATP production, we hypothesized that (1) both basal ATP supply and demand in SANC would be Ca(2+)-cAMP/PKA dependent; and (2) due to its feed-forward nature, a decrease in flux through the Ca(2+)-cAMP/PKA signaling axis will reduce the basal ATP production rate. O(2) consumption in spontaneous beating SANC was comparable to ventricular myocytes (VM) stimulated at 3 Hz. Graded reduction of basal Ca(2+)-cAMP/PKA signaling to reduce ATP demand in rabbit SANC produced graded ATP depletion (r(2)=0.96), and reduced O(2) consumption and flavoprotein fluorescence. Neither inhibition of glycolysis, selectively blocking contraction nor specific inhibition of mitochondrial Ca(2+) flux reduced the ATP level. Feed-forward basal Ca(2+)-cAMP/PKA signaling both consumes ATP to drive spontaneous APs in SANC and is tightly linked to mitochondrial ATP production. Interfering with Ca(2+)-cAMP/PKA signaling not only slows the firing rate and reduces ATP consumption, but also appears to reduce ATP production so that ATP levels fall. This distinctly differs from VM, which lack this feed-forward basal cAMP/PKA signaling, and in which ATP level remains constant when the demand changes. Published by Elsevier Ltd.

Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells

PubMed Central

Maddocks, Oliver D.K.; Labuschagne, Christiaan F.; Adams, Peter D.; Vousden, Karen H.

2016-01-01

Summary Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids. PMID:26774282

Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells.

PubMed

Maddocks, Oliver D K; Labuschagne, Christiaan F; Adams, Peter D; Vousden, Karen H

2016-01-21

Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Evidence for the Synthesis of ATP by an F0F1 ATP Synthase in Membrane Vesicles from Halorubrum Saccharovorum

NASA Technical Reports Server (NTRS)

Faguy, David; Lawson, Darion; Hochstein, Lawrence I.; Chang, Sherwood (Technical Monitor)

1996-01-01

Vesicles prepared in a buffer containing ADP, Mg(2+) and Pi synthesized ATP at an initial rate of 2 nmols/min/mg protein after acidification of the bulk medium (pH 8 (right arrow) 4). The intravesicular ATP concentration reached a steady state after about 30 seconds and slowly declined thereafter. ATP synthesis was inhibited by low concentrations of dicyclohexylcarbodiimide and m-chlorophenylhydrazone indicating that synthesis took place in response to the proton gradient. NEM and PCMS, which inhibit vacuolar ATPases and the vacuolar-like ATPases of extreme halophiles, did not affect ATP synthesis, and, in fact, produced higher steady state levels of ATP. This suggested that two ATPase activities were present, one which catalyzed ATP synthesis and one that caused its hydrolysis. Azide, a specific inhibitor of F0F1 ATP Synthases, inhibited halobacterial ATP synthesis. The distribution of acridine orange as imposed by a delta pH demonstrated that azide inhibition was not due to the collapse of the proton gradient due to azide acting as a protonophore. Such an effect was observed, but only at azide concentrations higher than those that inhibited ATP synthesis. These results confirm the earler observations with cells of H. saccharovorum and other extreme halophiles that ATP synthesis is inconsistent with the operation of a vacuolar-like ATPase. Therefore, the observation that a vacuolar-like enzyme is responsible for ATP synthesis (and which serves as the basis for imputing ATP synthesis to the vacuolar-like ATPases of the extreme halophiles, and the Archaea in general) should be taken with some degree of caution.

Copper Transporter ATP7A Protects Against Endothelial Dysfunction in Type 1 Diabetic Mice by Regulating Extracellular Superoxide Dismutase

PubMed Central

Sudhahar, Varadarajan; Urao, Norifumi; Oshikawa, Jin; McKinney, Ronald D.; Llanos, Roxana M.; Mercer, Julian F.B.; Ushio-Fukai, Masuko; Fukai, Tohru

2013-01-01

Oxidative stress and endothelial dysfunction contribute to vascular complication in diabetes. Extracellular superoxide dismutase (SOD3) is one of the key antioxidant enzymes that obtains copper via copper transporter ATP7A. SOD3 is secreted from vascular smooth muscles cells (VSMCs) and anchors at the endothelial surface. The role of SOD3 and ATP7A in endothelial dysfunction in type 1 diabetes mellitus (T1DM) is entirely unknown. Here we show that the specific activity of SOD3, but not SOD1, is decreased, which is associated with increased O2•− production in aortas of streptozotocin-induced and genetically induced Ins2Akita T1DM mice. Exogenous copper partially rescued SOD3 activity in isolated T1DM vessels. Functionally, acetylcholine-induced, endothelium-dependent relaxation is impaired in T1DM mesenteric arteries, which is rescued by SOD mimetic tempol or gene transfer of SOD3. Mechanistically, ATP7A expression in T1DM vessels is dramatically decreased whereas other copper transport proteins are not altered. T1DM-induced endothelial dysfunction and decrease of SOD3 activity are rescued in transgenic mice overexpressing ATP7A. Furthermore, SOD3-deficient T1DM mice or ATP7A mutant T1DM mice augment endothelial dysfunction and vascular O2•− production versus T1DM mice. These effects are in part due to hypoinsulinemia in T1DM mice, since insulin treatment, but not high glucose, increases ATP7A expression in VSMCs and restores SOD3 activity in the organoid culture of T1DM vessels. In summary, a decrease in ATP7A protein expression contributes to impaired SOD3 activity, resulting in O2•− overproduction and endothelial dysfunction in blood vessels of T1DM. Thus, restoring copper transporter function is an essential therapeutic approach for oxidant stress–dependent vascular and metabolic diseases. PMID:23884884

EMERGING TECHNOLOGY REPORT: BENCH-SCALE TESTING OF PHOTOLYSIS, CHEMICAL OXIDATION AND BIODEGRADATION OF PCB CONTAMINATED SOILS AND PHOTOLYSIS OF TCDD CONTAMINATED SOILS

EPA Science Inventory

This report presents the results of bench-scale testing on degradation of 2,3,7,8-TCDD using W photolysis, and PCB degradation using UV photolysis, chemical oxidation and biological treatment. Bench-scale tests were conducted to investigate the feasibility of a two-phase detoxifi...

2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) DISRUPTS EARLY MORPHOGENETIC EVENTS THAT FORM THE LOWER REPRODUCTIVE TRACT IN FEMALE RAT FETUSES

EPA Science Inventory

In female rats, in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during critical periods of organogenesis causes a permanent thread of tissue across the vaginal opening, which consists of a core of mesenchyme surrounded by keratinized epithelia. The objective of t...

Volume-dependent ATP-conductive large-conductance anion channel as a pathway for swelling-induced ATP release.

PubMed

Sabirov, R Z; Dutta, A K; Okada, Y

2001-09-01

In mouse mammary C127i cells, during whole-cell clamp, osmotic cell swelling activated an anion channel current, when the phloretin-sensitive, volume-activated outwardly rectifying Cl(-) channel was eliminated. This current exhibited time-dependent inactivation at positive and negative voltages greater than around +/-25 mV. The whole-cell current was selective for anions and sensitive to Gd(3)+. In on-cell patches, single-channel events appeared with a lag period of approximately 15 min after a hypotonic challenge. Under isotonic conditions, cell-attached patches were silent, but patch excision led to activation of currents that consisted of multiple large-conductance unitary steps. The current displayed voltage- and time-dependent inactivation similar to that of whole-cell current. Voltage-dependent activation profile was bell-shaped with the maximum open probability at -20 to 0 mV. The channel in inside-out patches had the unitary conductance of approximately 400 pS, a linear current-voltage relationship, and anion selectivity. The outward (but not inward) single-channel conductance was suppressed by extracellular ATP with an IC(50) of 12.3 mM and an electric distance (delta) of 0.47, whereas the inward (but not outward) conductance was inhibited by intracellular ATP with an IC(50) of 12.9 mM and delta of 0.40. Despite the open channel block by ATP, the channel was ATP-conductive with P(ATP)/P(Cl) of 0.09. The single-channel activity was sensitive to Gd(3)+, SITS, and NPPB, but insensitive to phloretin, niflumic acid, and glibenclamide. The same pharmacological pattern was found in swelling-induced ATP release. Thus, it is concluded that the volume- and voltage-dependent ATP-conductive large-conductance anion channel serves as a conductive pathway for the swelling-induced ATP release in C127i cells.

The Structural Basis of ATP as an Allosteric Modulator

PubMed Central

Wang, Qi; Shen, Qiancheng; Li, Shuai; Nussinov, Ruth; Zhang, Jian

2014-01-01

Adenosine-5’-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems. PMID:25211773

Dual recognition unit strategy improves the specificity of the adenosine triphosphate (ATP) aptamer biosensor for cerebral ATP assay.

PubMed

Yu, Ping; He, Xiulan; Zhang, Li; Mao, Lanqun

2015-01-20

Adenosine triphosphate (ATP) aptamer has been widely used as a recognition unit for biosensor development; however, its relatively poor specificity toward ATP against adenosine-5'-diphosphate (ADP) and adenosine-5'-monophosphate (AMP) essentially limits the application of the biosensors in real systems, especially in the complex cerebral system. In this study, for the first time, we demonstrate a dual recognition unit strategy (DRUS) to construct a highly selective and sensitive ATP biosensor by combining the recognition ability of aptamer toward A nucleobase and of polyimidazolium toward phosphate. The biosensors are constructed by first confining the polyimidazolium onto a gold surface by surface-initiated atom transfer radical polymerization (SI-ATRP), and then the aptamer onto electrode surface by electrostatic self-assembly to form dual-recognition-unit-functionalized electrodes. The constructed biosensor based on DRUS not only shows an ultrahigh sensitivity toward ATP with a detection limit down to the subattomole level but also an ultrahigh selectivity toward ATP without interference from ADP and AMP. The constructed biosensor is used for selective and sensitive sensing of the extracellular ATP in the cerebral system by combining in vivo microdialysis and can be used as a promising neurotechnology to probing cerebral ATP concentration.

Thermodynamics of proton transport coupled ATP synthesis.

PubMed

Turina, Paola; Petersen, Jan; Gräber, Peter

2016-06-01

The thermodynamic H(+)/ATP ratio of the H(+)-ATP synthase from chloroplasts was measured in proteoliposomes after energization of the membrane by an acid base transition (Turina et al. 2003 [13], 418-422). The method is discussed, and all published data obtained with this system are combined and analyzed as a single dataset. This meta-analysis led to the following results. 1) At equilibrium, the transmembrane ΔpH is energetically equivalent to the transmembrane electric potential difference. 2) The standard free energy for ATP synthesis (reference reaction) is ΔG°(ref)=33.8±1.3kJ/mol. 3) The thermodynamic H(+)/ATP ratio, as obtained from the shift of the ATP synthesis equilibrium induced by changing the transmembrane ΔpH (varying either pH(in) or pH(out)) is 4.0±0.1. The structural H(+)/ATP ratio, calculated from the ratio of proton binding sites on the c-subunit-ring in F(0) to the catalytic nucleotide binding sites on the β-subunits in F(1), is c/β=14/3=4.7. We infer that the energy of 0.7 protons per ATP that flow through the enzyme, but do not contribute to shifting the ATP/(ADP·Pi) ratio, is used for additional processes within the enzyme, such as activation, and/or energy dissipation, due e.g. to internal uncoupling. The ratio between the thermodynamic and the structural H(+)/ATP values is 0.85, and we conclude that this value represents the efficiency of the chemiosmotic energy conversion within the chloroplast H(+)-ATP synthase. Copyright © 2016 Elsevier B.V. All rights reserved.

EXPRESSION OF EGFR AND ITS LIGANDS IN RESPONSE TO TCDD OR RETINOIC ACID IN EGF AND TGFALPHA KO FETAL MOUSE PALATE

EPA Science Inventory

EXPRESSION OF EGFR AND ITS LIGANDS IN RESPONSE TO TCDD OR RETINOIC ACID IN EGF AND TGF" KO FETAL MOUSE PALATE. Abbott, Barbara D.1; Boyd, Hadiya2; Wood, Carmen1; Held, Gary1. 1.EPA, ORD, NHEERL, RTD, US EPA, Research Triangle Park, NC, USA. 2MARC Program, NCCU, Durham, NC, USA. <...

Fluorescent ATP analog mant-ATP reports dynein activity in the isolated Chlamydomonas axoneme

NASA Astrophysics Data System (ADS)

Feofilova, Maria; Howard, Jonathon

Eukaryotic flagella are long rod-like extensions of cells, which play a fundamental role in single cell movement, as well as in fluid transport. Flagella contain a highly evolutionary conserved mechanical structure called the axoneme. The motion of the flagellum is generated by dynein motor proteins located all along the length of the axoneme. How the force production of motors is controlled spatially and temporally is still an open question. Therefore, monitoring dynein activity in the axonemal structure is expected to provide novel insights in regulation of the beat. We use high sensitivity fluorescence microscopy to monitor the binding and hydrolysis kinetics of the fluorescently labeled ATP analogue mant-ATP (2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate), which is known to support dynein activity. By studying the kinetics of mant-ATP fluorescence, we identified distinct mant-ATP binding sites in the axoneme. The application of this method to axonemes with reduced amounts of dynein, showed evidence that one of the sites is associated with binding to dynein. In the future, we would like to use this method to find the spatial distribution of dynein activity in the axoneme.

Atp1a3-deficient heterozygous mice show lower rank in the hierarchy and altered social behavior.

PubMed

Sugimoto, H; Ikeda, K; Kawakami, K

2018-06-01

Atp1a3 is the Na-pump alpha3 subunit gene expressed mainly in neurons of the brain. Atp1a3-deficient heterozygous mice (Atp1a3 +/- ) show altered neurotransmission and deficits of motor function after stress loading. To understand the function of Atp1a3 in a social hierarchy, we evaluated social behaviors (social interaction, aggression, social approach and social dominance) of Atp1a3 +/- and compared the rank and hierarchy structure between Atp1a3 +/- and wild-type mice within a housing cage using the round-robin tube test and barbering observations. Formation of a hierarchy decreases social conflict and promote social stability within the group. The hierarchical rank is a reflection of social dominance within a cage, which is heritable and can be regulated by specific genes in mice. Here we report: (1) The degree of social interaction but not aggression was lower in Atp1a3 +/- than wild-type mice, and Atp1a3 +/- approached Atp1a3 +/- mice more frequently than wild type. (2) The frequency of barbering was lower in the Atp1a3 +/- group than in the wild-type group, while no difference was observed in the mixed-genotype housing condition. (3) Hierarchy formation was not different between Atp1a3 +/- and wild type. (4) Atp1a3 +/- showed a lower rank in the mixed-genotype housing condition than that in the wild type, indicating that Atp1a3 regulates social dominance. In sum, Atp1a3 +/- showed unique social behavior characteristics of lower social interaction and preference to approach the same genotype mice and a lower ranking in the hierarchy. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Defects in mitochondrial localization and ATP synthesis in the mdx mouse model of Duchenne muscular dystrophy are not alleviated by PDE5 inhibition

PubMed Central

Percival, Justin M.; Siegel, Michael P.; Knowels, Gary; Marcinek, David J.

2013-01-01

Given the crucial roles for mitochondria in ATP energy supply, Ca2+ handling and cell death, mitochondrial dysfunction has long been suspected to be an important pathogenic feature in Duchenne muscular dystrophy (DMD). Despite this foresight, mitochondrial function in dystrophin-deficient muscles has remained poorly defined and unknown in vivo. Here, we used the mdx mouse model of DMD and non-invasive spectroscopy to determine the impact of dystrophin-deficiency on skeletal muscle mitochondrial localization and oxidative phosphorylation function in vivo. Mdx mitochondria exhibited significant uncoupling of oxidative phosphorylation (reduced P/O) and a reduction in maximal ATP synthesis capacity that together decreased intramuscular ATP levels. Uncoupling was not driven by increased UCP3 or ANT1 expression. Dystrophin was required to maintain subsarcolemmal mitochondria (SSM) pool density, implicating it in the spatial control of mitochondrial localization. Given that nitric oxide-cGMP pathways regulate mitochondria and that sildenafil-mediated phosphodiesterase 5 inhibition ameliorates dystrophic pathology, we tested whether sildenafil's benefits result from decreased mitochondrial dysfunction in mdx mice. Unexpectedly, sildenafil treatment did not affect mitochondrial content or oxidative phosphorylation defects in mdx mice. Rather, PDE5 inhibition decreased resting levels of ATP, phosphocreatine and myoglobin, suggesting that sildenafil improves dystrophic pathology through other mechanisms. Overall, these data indicate that dystrophin-deficiency disrupts SSM localization, promotes mitochondrial inefficiency and restricts maximal mitochondrial ATP-generating capacity. Together these defects decrease intramuscular ATP and the ability of mdx muscle mitochondria to meet ATP demand. These findings further understanding of how mitochondrial bioenergetic dysfunction contributes to disease pathogenesis in dystrophin-deficient skeletal muscle in vivo. PMID:23049075

Cervical anterior transpedicular screw fixation (ATPS)—Part II. Accuracy of manual insertion and pull-out strength of ATPS

PubMed Central

Acosta, Frank; Tauber, Mark; Fox, Michael; Martin, Hudelmaier; Forstner, Rosmarie; Augat, Peter; Penzkofer, Rainer; Pirich, Christian; Kässmann, H.; Resch, Herbert; Hitzl, Wolfgang

2008-01-01

Reconstruction after multilevel decompression of the cervical spine, especially in the weakened osteoporotic, neoplastic or infectious spine often requires circumferential stabilization and fusion. To avoid the additional posterior surgery in these cases while increasing rigidity of anterior-only screw-plate constructs, the authors introduce the concept of anterior transpedicular screw (ATPS) fixation. We demonstrated its morphological feasibility as well as its indications in a previous study in Part I of our project. Consequently, the objectives of the current study were to assess the ex vivo accuracy of placing ATPS into the cervical vertebra as well as the biomechanical performance of ATPS in comparison to traditional vertebral body screws (VBS) in terms of pull-out strength (POS). Twenty-three ATPS were inserted alternately to two screws into the pedicles and vertebral bodies, respectively, of six cadaveric specimens from C3–T1. For insertion of ATPS, a manual fluoroscopically assisted technique was used. Pre- and post insertional CT-scans were used to assess accuracy of ATPS insertion in the axial and sagittal planes. A newly designed grading system and accuracy score were used to delineate accuracy of ATPS insertion. Following insertion of screws, 23 ATPS and 22 VBS were subjected to pull-out testing (POT). The bone mineral density (BMD) of each specimen was assessed prior to POT. Statistical analysis showed that the incidence of correctly placed screws and non-critical pedicles breaches in axial plane was 78.3%, and 95.7% in sagittal plane. Hence, according to our definition of “critical” pedicle breach that exposes neurovascular structures at risk, 21.7% (n = 5) of all ATPS inserted showed a critical pedicle breach in axial plane. Notably, no critical pedicle perforation occurred at the C6 to T1 levels. Pull-out testing of ATPS and VBS revealed that pull-out resistance of ATPS was 2.5-fold that of VBS. Mean POS of 23 ATPS with a mean BMD of 0.566�

The interactome of the copper transporter ATP7A belongs to a network of neurodevelopmental and neurodegeneration factors

PubMed Central

Comstra, Heather S; McArthy, Jacob; Rudin-Rush, Samantha; Hartwig, Cortnie; Gokhale, Avanti; Zlatic, Stephanie A; Blackburn, Jessica B; Werner, Erica; Petris, Michael; D’Souza, Priya; Panuwet, Parinya; Barr, Dana Boyd; Lupashin, Vladimir; Vrailas-Mortimer, Alysia; Faundez, Victor

2017-01-01

Genetic and environmental factors, such as metals, interact to determine neurological traits. We reasoned that interactomes of molecules handling metals in neurons should include novel metal homeostasis pathways. We focused on copper and its transporter ATP7A because ATP7A null mutations cause neurodegeneration. We performed ATP7A immunoaffinity chromatography and identified 541 proteins co-isolating with ATP7A. The ATP7A interactome concentrated gene products implicated in neurodegeneration and neurodevelopmental disorders, including subunits of the Golgi-localized conserved oligomeric Golgi (COG) complex. COG null cells possess altered content and subcellular localization of ATP7A and CTR1 (SLC31A1), the transporter required for copper uptake, as well as decreased total cellular copper, and impaired copper-dependent metabolic responses. Changes in the expression of ATP7A and COG subunits in Drosophila neurons altered synapse development in larvae and copper-induced mortality of adult flies. We conclude that the ATP7A interactome encompasses a novel COG-dependent mechanism to specify neuronal development and survival. DOI: http://dx.doi.org/10.7554/eLife.24722.001 PMID:28355134

An ATP synthase harboring an atypical γ-subunit is involved in ATP synthesis in tomato fruit chromoplasts.

PubMed

Pateraki, Irini; Renato, Marta; Azcón-Bieto, Joaquín; Boronat, Albert

2013-04-01

Chromoplasts are non-photosynthetic plastids specialized in the synthesis and accumulation of carotenoids. During fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts in a process characterized by the degradation of the thylakoid membranes, and by the active synthesis and accumulation of carotenoids. This transition renders chromoplasts unable to photochemically synthesize ATP, and therefore these organelles need to obtain the ATP required for anabolic processes through alternative sources. It is widely accepted that the ATP used for biosynthetic processes in non-photosynthetic plastids is imported from the cytosol or is obtained through glycolysis. In this work, however, we show that isolated tomato (Solanum lycopersicum) fruit chromoplasts are able to synthesize ATP de novo through a respiratory pathway using NADPH as an electron donor. We also report the involvement of a plastidial ATP synthase harboring an atypical γ-subunit induced during ripening, which lacks the regulatory dithiol domain present in plant and algae chloroplast γ-subunits. Silencing of this atypical γ-subunit during fruit ripening impairs the capacity of isolated chromoplast to synthesize ATP de novo. We propose that the replacement of the γ-subunit present in tomato leaf and green fruit chloroplasts by the atypical γ-subunit lacking the dithiol domain during fruit ripening reflects evolutionary changes, which allow the operation of chromoplast ATP synthase under the particular physiological conditions found in this organelle. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

Hot flue-gas spiking and recovery study for tetrachlorodibenzodioxins (TCDD) using Modified Method 5 and SASS (Source Assessment Sampling System) sampling with a simulated incinerator. Final report, May 1981-February 1982

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cooke, M.; DeRoos, F.; Rising, B.

1984-10-01

The report gives results of an evaluation of the sampling and analysis of ultratrace levels of dibenzodioxins using EPA's recommended source sampling procedures (Modified Method 5 (MM5) train and the Source Assessment Sampling System--SASS). A gas-fired combustion system was used to simulate incineration flue gas, and a precision liquid injection system was designed for the program. The precision liquid injector was used to administer dilute solutions of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) directly into a hot--260C (500F)--flue gas stream. Injections occurred continuously during the sampling episode so that very low gas-phase concentrations of 1,2,3,4-TCDD were continuously mixed with the flue gases. Recoveries weremore » measured for eight burn experiments. For all but one, the recoveries could be considered quantitative, demonstrating efficient collection by the EPA sampling systems. In one study, the components and connecting lines from a sampling device were analyzed separately to show where the 1,2,3,4-TCDD deposited in the train.« less

A taste for ATP: neurotransmission in taste buds

PubMed Central

Kinnamon, Sue C.; Finger, Thomas E.

2013-01-01

Not only is ATP a ubiquitous source of energy but it is also used widely as an intercellular signal. For example, keratinocytes release ATP in response to numerous external stimuli including pressure, heat, and chemical insult. The released ATP activates purinergic receptors on nerve fibers to generate nociceptive signals. The importance of an ATP signal in epithelial-to-neuronal signaling is nowhere more evident than in the taste system. The receptor cells of taste buds release ATP in response to appropriate stimulation by tastants and the released ATP then activates P2X2 and P2X3 receptors on the taste nerves. Genetic ablation of the relevant P2X receptors leaves an animal without the ability to taste any primary taste quality. Of interest is that release of ATP by taste receptor cells occurs in a non-vesicular fashion, apparently via gated membrane channels. Further, in keeping with the crucial role of ATP as a neurotransmitter in this system, a subset of taste cells expresses a specific ectoATPase, NTPDase2, necessary to clear extracellular ATP which otherwise will desensitize the P2X receptors on the taste nerves. The unique utilization of ATP as a key neurotransmitter in the taste system may reflect the epithelial rather than neuronal origins of the receptor cells. PMID:24385952

Progesterone-dependent Regulation of Endometrial Cannabinoid Receptor Type 1 (CB1-R) Expression is Disrupted in Women with Endometriosis and in Isolated Stromal Cells Exposed to TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)

PubMed Central

Resuehr, David; Glore, Dana R.; Taylor, Hugh S.; Bruner-Tran, Kaylon L.; Osteen, Kevin G.

2012-01-01

Objective To examine the differentiation-related expression of CB1-R mRNA and protein in endometrial tissue obtained from women with and without endometriosis and to determine the impact of acute TCDD exposure on CB1-R gene expression in isolated endometrial stromal cells. Design Laboratory-based study Setting University-affiliated medical center Patients Women with and without endometriosis undergoing volunteer endometrial biopsies after informed consent. Interventions None Main Outcome Measures Analysis of in vivo CB1-R mRNA and protein expression in human endometrial tissues and mRNA expression in isolated stromal cells following exposure to TCDD or a progesterone receptor antagonist (Onapristone). Results CB1-R mRNA and protein expression was highest during the progesterone-dominated secretory phase in control women, while expression was minimal in endometrial tissues acquired from women with endometriosis, regardless of the cycle phase. Although progesterone was found to induce CB1-R mRNA expression in endometrial stromal cells from control donors, steroid-induced expression of this gene was inhibited by co-treatment with either TCDD or Onapristone. Conclusions Our studies reveal a role for the anti-inflammatory actions of progesterone in regulating endometrial cannabinoid signaling, which is disrupted in women with endometriosis. Significantly, our studies demonstrate, for the first time, that acute TCDD exposure disrupts cannabinoid signaling in the human endometrium. PMID:22789143

Dynamics of the metal binding domains and regulation of the human copper transporters ATP7B and ATP7A.

PubMed

Yu, Corey H; Dolgova, Natalia V; Dmitriev, Oleg Y

2017-04-01

Copper transporters ATP7A and ATP7B regulate copper levels in the human cells and deliver copper to the biosynthetic pathways. ATP7A and ATP7B belong to the P-type ATPases and share much of the domain architecture and the mechanism of ATP hydrolysis with the other, well-studied, enzymes of this type. A unique structural feature of the copper ATPases is the chain of six cytosolic metal-binding domains (MBDs), which are believed to be involved in copper-dependent regulation of the activity and intracellular localization of these enzymes. Although the structures of all the MBDs have been solved, the mechanism of copper-dependent regulation of ATP7B and ATP7A, the roles of individual MBDs, and the relationship between the regulatory and catalytic copper binding are still unknown. We describe the structure and dynamics of the MBDs, review the current knowledge about their functional roles and propose a mechanism of regulation of ATP7B by copper-dependent changes in the dynamics and conformation of the MBD chain. Transient interactions between the MBDs, rather than transitions between distinct static conformations are likely to form the structural basis of regulation of the ATP-dependent copper transporters in human cells. © 2016 IUBMB Life, 69(4):226-235, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

Alanine mutation of the catalytic sites of Pantothenate Synthetase causes distinct conformational changes in the ATP binding region.

PubMed

Pandey, Bharati; Grover, Sonam; Goyal, Sukriti; Kumari, Anchala; Singh, Aditi; Jamal, Salma; Kaur, Jagdeep; Grover, Abhinav

2018-01-17

The enzyme Pantothenate synthetase (PS) represents a potential drug target in Mycobacterium tuberculosis. Its X-ray crystallographic structure has demonstrated the significance and importance of conserved active site residues including His44, His47, Asn69, Gln72, Lys160 and Gln164 in substrate binding and formation of pantoyl adenylate intermediate. In the current study, molecular mechanism of decreased affinity of the enzyme for ATP caused by alanine mutations was investigated using molecular dynamics (MD) simulations and free energy calculations. A total of seven systems including wild-type + ATP, H44A + ATP, H47A + ATP, N69A + ATP, Q72A + ATP, K160A + ATP and Q164A + ATP were subjected to 50 ns MD simulations. Docking score, MM-GBSA and interaction profile analysis showed weak interactions between ATP (substrate) and PS (enzyme) in H47A and H160A mutants as compared to wild-type, leading to reduced protein catalytic activity. However, principal component analysis (PCA) and free energy landscape (FEL) analysis revealed that ATP was strongly bound to the catalytic core of the wild-type, limiting its movement to form a stable complex as compared to mutants. The study will give insight about ATP binding to the PS at the atomic level and will facilitate in designing of non-reactive analogue of pantoyl adenylate which will act as a specific inhibitor for PS.

ATP Synthesis in the Extremely Halophilic Bacteria

NASA Technical Reports Server (NTRS)

Hochstein, Lawrence I.; Morrison, David (Technical Monitor)

1994-01-01

The proton-translocating ATPases are multimeric enzymes that carry out a multitude of essential functions. Their origin and evolution represent a seminal event in the early evolution of life. Amino acid sequences of the two largest subunits from archaeal ATPases (A-ATPases), vacuolar ATPases (V-ATPases), and FOF1-ATP syntheses (FATPases) suggest these ATPases evolved from an ancestral vacuolar-like ATP syntheses. A necessary consequence of this notion is that the A-ATPases are ATP syntheses. With the possible exception of the A-ATPase from Halobacterium salinarium. no A-ATPase has been demonstrated to synthesize ATP. The evidence for this case is dubious since ATP synthesis occurs only when conditions are distinctively unphysiological. We demonstrated that ATP synthesis in H.saccharovorum is inconsistent with the operation of an A-type ATPase. In order to determine if this phenomenon was unique to H. saccharovorum, ATP synthesis was examined in various extremely halophilic bacteria with the goal of ascertaining if it resembled what occurred in a. saccharovorum, or was consistent with the operation of an A-type ATPase. A-, V-, and F-type ATPases respond singularly to certain inhibitors. Therefore, the effect of these inhibitors on ATP synthesis in several extreme halophiles was determined. Inhibitors that either blocked or collapsed proton-gradients inhibited the steady state synthesis of ATP thus verifying that synthesis took place at the expense of a proton gradient. Azide, an inhibitor of F-ATPases inhibited ATP synthesis. Since the arginine-dependent synthesis of ATP, which occurs by way of substrate-level phosphorylation, was unaffected by azide, it was unlikely that azide acted as an "uncoupler." N -ethylmaleimide and nitrate, which inhibit V- and A-ATPases, either did not inhibit ATP synthesis or resulted in higher steady-state levels of ATP. These results suggest there are two types of proton-motive ATPases in the extreme halophiles (and presumably in other

Snapshots of the maltose transporter during ATP hydrolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oldham, Michael L.; Chen, Jue

2011-12-05

ATP-binding cassette transporters are powered by ATP, but the mechanism by which these transporters hydrolyze ATP is unclear. In this study, four crystal structures of the full-length wild-type maltose transporter, stabilized by adenosine 5{prime}-({beta},{gamma}-imido)triphosphate or ADP in conjunction with phosphate analogs BeF{sub 3}{sup -}, VO{sub 4}{sup 3-}, or AlF{sub 4}{sup -}, were determined to 2.2- to 2.4-{angstrom} resolution. These structures led to the assignment of two enzymatic states during ATP hydrolysis and demonstrate specific functional roles of highly conserved residues in the nucleotide-binding domain, suggesting that ATP-binding cassette transporters catalyze ATP hydrolysis via a general base mechanism.

Rapid and precise determination of ATP using a modified photometer

USGS Publications Warehouse

Shultz, David J.; Stephens, Doyle W.

1980-01-01

An inexpensive delay timer was designed to modify a commercially available ATP photometer which allows a disposable tip pipette to be used for injecting either enzyme or sample into the reaction cuvette. The disposable tip pipette is as precise and accurate as a fixed-needle syringe but eliminates the problem of sample contamination and decreases analytical time. (USGS)

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

PubMed Central

Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K.; Dean, Dennis R.; Hoffman, Brian M.; Antony, Edwin; Seefeldt, Lance C.

2013-01-01

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein. PMID:24062462

ATP-sulfurylase, sulfur-compounds, and plant stress tolerance

PubMed Central

Anjum, Naser A.; Gill, Ritu; Kaushik, Manjeri; Hasanuzzaman, Mirza; Pereira, Eduarda; Ahmad, Iqbal; Tuteja, Narendra; Gill, Sarvajeet S.

2015-01-01

Sulfur (S) stands fourth in the list of major plant nutrients after N, P, and K. Sulfate (SO42-), a form of soil-S taken up by plant roots is metabolically inert. As the first committed step of S-assimilation, ATP-sulfurylase (ATP-S) catalyzes SO42--activation and yields activated high-energy compound adenosine-5′-phosphosulfate that is reduced to sulfide (S2-) and incorporated into cysteine (Cys). In turn, Cys acts as a precursor or donor of reduced S for a range of S-compounds such as methionine (Met), glutathione (GSH), homo-GSH (h-GSH), and phytochelatins (PCs). Among S-compounds, GSH, h-GSH, and PCs are known for their involvement in plant tolerance to varied abiotic stresses, Cys is a major component of GSH, h-GSH, and PCs; whereas, several key stress-metabolites such as ethylene, are controlled by Met through its first metabolite S-adenosylmethionine. With the major aim of briefly highlighting S-compound-mediated role of ATP-S in plant stress tolerance, this paper: (a) overviews ATP-S structure/chemistry and occurrence, (b) appraises recent literature available on ATP-S roles and regulations, and underlying mechanisms in plant abiotic and biotic stress tolerance, (c) summarizes ATP-S-intrinsic regulation by major S-compounds, and (d) highlights major open-questions in the present context. Future research in the current direction can be devised based on the discussion outcomes. PMID:25904923

Cerebrospinal fluid ATP metabolites in multiple sclerosis.

PubMed

Lazzarino, G; Amorini, A M; Eikelenboom, M J; Killestein, J; Belli, A; Di Pietro, V; Tavazzi, B; Barkhof, F; Polman, C H; Uitdehaag, B M J; Petzold, A

2010-05-01

Increased axonal energy demand and mitochondrial failure have been suggested as possible causes for axonal degeneration and disability in multiple sclerosis. Our objective was to test whether ATP depletion precedes clinical, imaging and biomarker evidence for axonal degeneration in multiple sclerosis. The method consisted of a longitudinal study which included 21 patients with multiple sclerosis. High performance liquid chromatography was used to quantify biomarkers of the ATP metabolism (oxypurines and purines) from the cerebrospinal fluid at baseline. The Expanded Disability Status Scale, MRI brain imaging measures for brain atrophy (ventricular and parenchymal fractions), and cerebrospinal fluid biomarkers for axonal damage (phosphorylated and hyperphosphorylated neurofilaments) were quantified at baseline and 3-year follow-up. Central ATP depletion (sum of ATP metabolites >19.7 micromol/litre) was followed by more severe progression of disability if compared to normal ATP metabolites (median 1.5 versus 0, p< 0.05). Baseline ATP metabolite levels correlated with change of Expanded Disability Status Scale in the pooled cohort (r= 0.66, p= 0.001) and subgroups (relapsing-remitting patients: r= 0.79, p< 0.05 and secondary progressive/primary progressive patients: r= 0.69, p< 0.01). There was no relationship between central ATP metabolites and either biomarker or MRI evidence for axonal degeneration. The data suggests that an increased energy demand in multiple sclerosis may cause a quantifiable degree of central ATP depletion. We speculate that the observed clinical disability may be related to depolarisation associated conduction block.

Responses of Rat P2X2 Receptors to Ultrashort Pulses of ATP Provide Insights into ATP Binding and Channel Gating

PubMed Central

Moffatt, Luciano; Hume, Richard I.

2007-01-01

To gain insight into the way that P2X2 receptors localized at synapses might function, we explored the properties of outside-out patches containing many of these channels as ATP was very rapidly applied and removed. Using a new method to calibrate the speed of exchange of solution over intact patches, we were able to reliably produce applications of ATP lasting <200 μs. For all concentrations of ATP, there was a delay of at least 80 μs between the time when ATP arrived at the receptor and the first detectable flow of inward current. In response to 200-μs pulses of ATP, the time constant of the rising phase of the current was ∼600 μs. Thus, most channel openings occurred when no free ATP was present. The current deactivated with a time constant of ∼60 ms. The amplitude of the peak response to a brief pulse of a saturating concentration of ATP was ∼70% of that obtained during a long application of the same concentration of ATP. Thus, ATP leaves fully liganded channels without producing an opening at least 30% of the time. Extensive kinetic modeling revealed three different schemes that fit the data well, a sequential model and two allosteric models. To account for the delay in opening at saturating ATP, it was necessary to incorporate an intermediate closed state into all three schemes. These kinetic properties indicate that responses to ATP at synapses that use homomeric P2X2 receptors would be expected to greatly outlast the duration of the synaptic ATP transient produced by a single presynaptic spike. Like NMDA receptors, P2X2 receptors provide the potential for complex patterns of synaptic integration over a time scale of hundreds of milliseconds. PMID:17664346

2,3,7,8-Tetrachlorodibenzo-p-dioxin activates the aryl hydrocarbon receptor and alters sex steroid hormone secretion without affecting growth of mouse antral follicles in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karman, Bethany N., E-mail: bklement@illinois.edu; Basavarajappa, Mallikarjuna S., E-mail: mbshivapur@gmail.com; Craig, Zelieann R., E-mail: zelieann@illinois.edu

The persistent environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an ovarian toxicant. These studies were designed to characterize the actions of TCDD on steroidogenesis and growth of intact mouse antral follicles in vitro. Specifically, these studies tested the hypothesis that TCDD exposure leads to decreased sex hormone production/secretion by antral follicles as well as decreased growth of antral follicles in vitro. Since TCDD acts through binding to the aryl hydrocarbon receptor (AHR), and the AHR has been identified as an important factor in ovarian function, we also conducted experiments to confirm the presence and activation of the AHR in our tissue culturemore » system. To do so, we exposed mouse antral follicles for 96 h to a series of TCDD doses previously shown to have effects on ovarian tissues and cells in culture, which also encompass environmentally relevant and pharmacological exposures (0.1–100 nM), to determine a dose response for TCDD in our culture system for growth, hormone production, and expression of the Ahr and Cyp1b1. The results indicate that TCDD decreases progesterone, androstenedione, testosterone, and estradiol levels in a non-monotonic dose response manner without altering growth of antral follicles. The addition of pregnenolone substrate (10 μM) restores hormone levels to control levels. Additionally, Cyp1b1 levels were increased by 3–4 fold regardless of the dose of TCDD exposure, evidence of AHR activation. Overall, these data indicate that TCDD may act prior to pregnenolone formation and through AHR transcriptional control of Cyp1b1, leading to decreased hormone levels without affecting growth of antral follicles. -- Highlights: ►TCDD disrupts sex steroid hormone levels, but not growth of antral follicles. ►Pregnenolone co-treatment by-passes TCDD-induced steroid hormone disruption. ►TCDD affects steroid hormone levels through an AHR pathway in antral follicles.« less

Effects of aging on resistance to Trichinella spiralis infection in rodents exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

PubMed

Luebke, R W; Copeland, C B; Andrews, D L

1999-08-13

Immune function, including resistance to infection, decreases as humans and rodents age. We have shown that preinfection exposure of young (9-11 weeks) mice or rats to TCDD decreased resistance to Trichinella spiralis (Ts) infection, expressed as delayed onset or completion of parasite elimination and as increased muscle burdens of larvae. It has also been shown that aged mice express lower constitutive levels of resistance to Ts infection, compared to young adult animals. This study tested the hypothesis that the age-related decrease in constitutive levels of resistance to Ts infection exacerbates the decreased resistance to infection that follows TCDD exposure. This hypothesis addresses the concern that TCDD may pose a greater threat to the elderly than to the population at large. Animals were given a single oral dose of 1, 10, or 30 microg TCDD/kg, 7 days before infection. Eleven days later, young (approximately 10 weeks) control rodents had eliminated a greater proportion of the original parasite burden from the intestine than aged control animals. Nevertheless, parasite elimination was decreased by TCDD exposure only in young rodents. The effect of TCDD exposure on numbers of encysted larvae was evaluated only in rats. Increased larvae burdens occurred in young rats at 30 microg TCDD/kg and at 10 or 30 microg TCDD/kg in aged rats. Parasite-specific splenocyte and lymph node cell proliferation was suppressed following dioxin exposure in young mice; cells from aged mice were markedly less responsive to stimulation, yet less sensitive to TCDD exposure. The response to parasite antigens was not affected in aged rats exposed to TCDD, although elevated mitogen-driven B-cell proliferation was observed. These results indicate that age-related constitutive immunosuppression did not exacerbate TCDD-induced suppression of T-cell mediated adult parasite expulsion; rather, advanced age provided some degree of protection. On the other hand, a lower dose of TCDD was required

Hypophosphatemia promotes lower rates of muscle ATP synthesis

PubMed Central

Pesta, Dominik H.; Tsirigotis, Dimitrios N.; Befroy, Douglas E.; Caballero, Daniel; Jurczak, Michael J.; Rahimi, Yasmeen; Cline, Gary W.; Dufour, Sylvie; Birkenfeld, Andreas L.; Rothman, Douglas L.; Carpenter, Thomas O.; Insogna, Karl; Petersen, Kitt Falk; Bergwitz, Clemens; Shulman, Gerald I.

2016-01-01

Hypophosphatemia can lead to muscle weakness and respiratory and heart failure, but the mechanism is unknown. To address this question, we noninvasively assessed rates of muscle ATP synthesis in hypophosphatemic mice by using in vivo saturation transfer [31P]-magnetic resonance spectroscopy. By using this approach, we found that basal and insulin-stimulated rates of muscle ATP synthetic flux (VATP) and plasma inorganic phosphate (Pi) were reduced by 50% in mice with diet-induced hypophosphatemia as well as in sodium-dependent Pi transporter solute carrier family 34, member 1 (NaPi2a)-knockout (NaPi2a−/−) mice compared with their wild-type littermate controls. Rates of VATP normalized in both hypophosphatemic groups after restoring plasma Pi concentrations. Furthermore, VATP was directly related to cellular and mitochondrial Pi uptake in L6 and RC13 rodent myocytes and isolated muscle mitochondria. Similar findings were observed in a patient with chronic hypophosphatemia as a result of a mutation in SLC34A3 who had a 50% reduction in both serum Pi content and muscle VATP. After oral Pi repletion and normalization of serum Pi levels, muscle VATP completely normalized in the patient. Taken together, these data support the hypothesis that decreased muscle ATP synthesis, in part, may be caused by low blood Pi concentrations, which may explain some aspects of muscle weakness observed in patients with hypophosphatemia.—Pesta, D. H., Tsirigotis, D. N., Befroy, D. E., Caballero, D., Jurczak, M. J., Rahimi, Y., Cline, G. W., Dufour, S., Birkenfeld, A. L., Rothman, D. L., Carpenter, T. O., Insogna, K., Petersen, K. F., Bergwitz, C., Shulman, G. I. Hypophosphatemia promotes lower rates of muscle ATP synthesis. PMID:27338702

ATP synthesis in Halobacterium saccharovorum: evidence that synthesis may be catalysed by an F0F1-ATP synthase

NASA Technical Reports Server (NTRS)

Hochstein, L. I.

1992-01-01

Halobacterium saccharovorum synthesized ATP in response to a pH shift from 8 to 6.2. Synthesis was inhibited by carbonyl cyanide m-chloro-phenylhydrazone, dicyclohexylcarbodiimide, and azide. Nitrate, an inhibitor of the membrane-bound ATPase previously isolated from this organism, did not inhibit ATP synthesis. N-Ethymaleimide, which also inhibited this ATPase, stimulated the production of ATP. These observations suggested that H. saccharovorum synthesized and hydrolysed ATP using different enzymes and that the vacuolar-like ATPase activity previously described in H. saccharovorum was an ATPase whose function is yet to be identified.

Inhibitors of the 5-lipoxygenase arachidonic acid pathway induce ATP release and ATP-dependent organic cation transport in macrophages.

PubMed

da Silva-Souza, Hercules Antônio; Lira, Maria Nathalia de; Costa-Junior, Helio Miranda; da Cruz, Cristiane Monteiro; Vasconcellos, Jorge Silvio Silva; Mendes, Anderson Nogueira; Pimenta-Reis, Gabriela; Alvarez, Cora Lilia; Faccioli, Lucia Helena; Serezani, Carlos Henrique; Schachter, Julieta; Persechini, Pedro Muanis

2014-07-01

We have previously described that arachidonic acid (AA)-5-lipoxygenase (5-LO) metabolism inhibitors such as NDGA and MK886, inhibit cell death by apoptosis, but not by necrosis, induced by extracellular ATP (ATPe) binding to P2X7 receptors in macrophages. ATPe binding to P2X7 also induces large cationic and anionic organic molecules uptake in these cells, a process that involves at least two distinct transport mechanisms: one for cations and another for anions. Here we show that inhibitors of the AA-5-LO pathway do not inhibit P2X7 receptors, as judged by the maintenance of the ATPe-induced uptake of fluorescent anionic dyes. In addition, we describe two new transport phenomena induced by these inhibitors in macrophages: a cation-selective uptake of fluorescent dyes and the release of ATP. The cation uptake requires secreted ATPe, but, differently from the P2X7/ATPe-induced phenomena, it is also present in macrophages derived from mice deficient in the P2X7 gene. Inhibitors of phospholipase A2 and of the AA-cyclooxygenase pathway did not induce the cation uptake. The uptake of non-organic cations was investigated by measuring the free intracellular Ca(2+) concentration ([Ca(2+)]i) by Fura-2 fluorescence. NDGA, but not MK886, induced an increase in [Ca(2+)]i. Chelating Ca(2+) ions in the extracellular medium suppressed the intracellular Ca(2+) signal without interfering in the uptake of cationic dyes. We conclude that inhibitors of the AA-5-LO pathway do not block P2X7 receptors, trigger the release of ATP, and induce an ATP-dependent uptake of organic cations by a Ca(2+)- and P2X7-independent transport mechanism in macrophages. Copyright © 2014 Elsevier B.V. All rights reserved.

Fatigue and changes of ATP, creatine phosphate, and lactate during the 400-m sprint.

PubMed

Hirvonen, J; Nummela, A; Rusko, H; Rehunen, S; Härkönen, M

1992-06-01

Fatigue during the 400-m sprint was studied by measuring muscle ATP, creatine phosphate (CP), lactate (M-La), and blood lactate (B-La) in six male runners before and after four experimental sprints (100, 200, 300, and 400 m). During the first 100 m, muscle CP decreased from 15.8 +/- 1.7 to 8.3 +/- 0.3 mmol/kg while M-La increased to 3.6 +/- 0.4 mmol/kg. After 200 m the CP had decreased to 6.5 +/- 0.5 mmol/kg and M-La had increased to 8.3 +/- 1.1 mmol/kg. At the end of the 400 meters, ATP and CP concentrations had decreased by 27% and 89%, respectively, and M-La had increased to 17.3 +/- 0.9 mmol/kg. It was concluded that after 200 m the speed of running decreased, although CP was not depleted and lactate concentration was not at maximum level. Complete fatigue occurred when CP stores were depleted and B-La and M-La attained an individual maximum.

PERSISTENT SUPPRESSION OF CONTACT HYPERSENSITIVITY, AND ALTERED T-CELL PARAMETERS IN F344 RATS EXPOSED PERINATALLY TO 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD)

EPA Science Inventory

Abstract
The outcome of perinatal low-level TCDD exposure on the T cell-mediated contact hypersensitivity response (CHS) in adult F344 rats was investigated. Suppression of the 2,4- dinitrofluorobenzene (DNFB)-specific contact hypersensitivity reponse occurred in mature off...

ATP and Mg2+ Promote the Reversible Oligomerization and Aggregation of Chloroplast 2-Cys Peroxiredoxin*

PubMed Central

Aran, Martín; Ferrero, Diego; Wolosiuk, Alejandro; Mora-García, Santiago; Wolosiuk, Ricardo A.

2011-01-01

2-Cys peroxiredoxins (2-Cys Prxs) are ubiquitous peroxidases with important roles in cellular antioxidant defense and hydrogen peroxide-mediated signaling. Post-translational modifications of conserved cysteines cause the transition from low to high molecular weight oligomers, triggering the functional change from peroxidase to molecular chaperone. However, it remains unclear how non-covalent interactions of 2-Cys Prx with metabolites modulate the quaternary structure. Here, we disclose that ATP and Mg2+ (ATP/Mg) promote the self-polymerization of chloroplast 2-Cys Prx (polypeptide 23.5 kDa) into soluble higher order assemblies (>2 MDa) that proceed to insoluble aggregates beyond 5 mm ATP. Remarkably, the withdrawal of ATP or Mg2+ brings soluble oligomers and insoluble aggregates back to the native conformation without compromising the associated functions. As confirmed by transmission electron microscopy, ATP/Mg drive the toroid-like decamers (diameter 13 nm) to the formation of large sphere-like particles (diameter ∼30 nm). Circular dichroism studies on ATP-labeled 2-Cys Prx reveal that ATP/Mg enhance the proportion of β-sheets with the concurrent decrease in the content of α-helices. In line with this observation, the formation of insoluble aggregates is strongly prevented by 2,2,2-trifluoroethanol, a cosolvent employed to induce α-helical conformations. We further find that the response of self-polymerization to ATP/Mg departs abruptly from that of the associated peroxidase and chaperone activities when two highly conserved residues, Arg129 and Arg152, are mutated. Collectively, our data uncover that non-covalent interactions of ATP/Mg with 2-Cys Prx modulate dynamically the quaternary structure, thereby coupling the non-redox chemistry of cell energy with redox transformations at cysteine residues. PMID:21525006

Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae.

PubMed

Milne, N; Luttik, M A H; Cueto Rojas, H F; Wahl, A; van Maris, A J A; Pronk, J T; Daran, J M

2015-07-01

In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni(2+)-dependent enzyme and Saccharomyces cerevisiae does not express native Ni(2+)-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01 µmol min(-1) mg protein(-1) and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni(2+) concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni(2+)-dependent enzymes can be functionally expressed in S. cerevisiae. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Inhibition of aldolase A blocks biogenesis of ATP and attenuates Japanese encephalitis virus production.

PubMed

Tien, Chih-Feng; Cheng, Shih-Ching; Ho, Yen-Peng; Chen, Yi-Shiuan; Hsu, Jung-Hsin; Chang, Ruey-Yi

2014-01-10

Viral replication depends on host proteins to supply energy and replication accessories for the sufficient production of viral progeny. In this study, we identified fructose-bisphosphate aldolase A as a binding partner of Japanese encephalitis virus (JEV) untranslated regions (UTRs) on the antigenome via RNA affinity capture and mass spectrometry. Direct interaction of aldolase A with JEV RNAs was confirmed by gel mobility shift assay and colocalization with active replication of double-stranded RNA in JEV-infected cells. Infection of JEV caused an increase in aldolase A expression of up to 33%. Knocking down aldolase A reduced viral translation, genome replication, and viral production significantly. Furthermore, JEV infection consumed 50% of cellular ATP, and the ATP level decreased by 70% in the aldolase A-knockdown cells. Overexpression of aldolase A in aldolase A-knockdown cells increased ATP levels significantly. Taken together, these results indicate that JEV replication requires aldolase A and consumes ATP. This is the first report of direct involvement of a host metabolic enzyme, aldolase A protein, in JEV replication. Copyright © 2013 Elsevier Inc. All rights reserved.

[Molecular and functional diversity of ATP-sensitive K+ channels: the pathophysiological roles and potential drug targets].

PubMed

Nakaya, Haruaki; Miki, Takashi; Seino, Susumu; Yamada, Katsuya; Inagaki, Nobuya; Suzuki, Masashi; Sato, Toshiaki; Yamada, Mitsuhiko; Matsushita, Kenji; Kurachi, Yoshihisa; Arita, Makoto

2003-09-01

ATP-sensitive K(+) (K(ATP)) channels comprise the pore-forming subunit (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea receptors (SUR1 or SUR2). K(ATP) channels with different combinations of these subunits are present in various tissues and regulate cellular functions. From the analysis of mouse models with targeted deletion of the gene encoding the pore-forming subunit Kir6.1 or Kir6.2, functional roles of K(ATP) channels in various organs have been clarified. Kir6.1(-/-) mice showed sudden death associated with ST elevation and atrioventricular block in ECG, a phenotype resembling Prinzmetal angina in humans. Kir6.2(-/-) mice were more susceptible to generalized seizure during hypoxia than wild-type (WT) mice, suggesting that neuronal K(ATP) channels, probably composed of Kir6.2 and SUR1, play a crucial role for the protection of the brain against lethal damage due to seizure. In Kir6.2(-/-) mice lacking the sarcolemmal K(ATP) channel activity in cardiac cells, ischemic preconditioning failed to reduce the infarct size, suggesting that sarcolemmal K(ATP) channels play an important role in cardioprotection against ischemia/reperfusion injuries in the heart. Mitochondrial K(ATP) channels have been also proposed to play a crucial role in cardioprotection, although the molecular identity of the channel has not been established. Nicorandil and minoxidil, K(+) channel openers activating mitochondrial K(ATP) channels, decreased the mitochondrial membrane potential, thereby preventing the Ca(2+) overload in the mitochondria of guinea-pig ventricular cells. SURs are the receptors for K(+) channel openers and the activating effects on sarcolemmal K(ATP) channels in cardiovascular tissues could be modulated by the interaction of nucleotides. Due to the molecular diversity of the accessory and pore subunits of K(ATP) channels, there would be considerable differences in the tissue selectivity of K(ATP) channel-acting drugs. Studies of Kir6.1 and Kir6.2 knockout

HNP-3 enhanced the antimicrobial activity of CIP by promoting ATP efflux from P. aeruginosa cells.

PubMed

Wang, Hao; Dong, Birong; Lou, Li

2011-04-01

To establish a novel strategy of P. aeruginosa control, we acquired recombination HNP-3 by gene recombination. Then we examined HNP-3 bio-activities and the influences of antimicrobial peptide on the efflux of ATP. Consequently, we obtained target protein with a molecular mass of 3,000 D consistent with the Anticipation. FIC index of Ciprofloxacin added HNP-3 was less than 0.5 and HNP3 synergistically cooperated with CIP to suppress P. aeruginosa colony formation revealed that there was significant synergy. ATP efflux was however up-regulated by low concentrations of HNP-3, although CIP did not exert any influence on ATP efflux. Conclusively, recombination protein HNP-3 displayed antimicrobial and synergic effects. HNP-3 enhanced the antimicrobial activity of CIP by promoting ATP efflux from P. aeruginosa cells and decreasing efflux of the drugs, which could have useful clinical applications.

The ADP/ATP Carrier and Its Relationship to Oxidative Phosphorylation in Ancestral Protist Trypanosoma brucei

PubMed Central

Gnipová, Anna; Šubrtová, Karolína; Panicucci, Brian; Horváth, Anton; Lukeš, Julius

2015-01-01

The highly conserved ADP/ATP carrier (AAC) is a key energetic link between the mitochondrial (mt) and cytosolic compartments of all aerobic eukaryotic cells, as it exchanges the ATP generated inside the organelle for the cytosolic ADP. Trypanosoma brucei, a parasitic protist of medical and veterinary importance, possesses a single functional AAC protein (TbAAC) that is related to the human and yeast ADP/ATP carriers. However, unlike previous studies performed with these model organisms, this study showed that TbAAC is most likely not a stable component of either the respiratory supercomplex III+IV or the ATP synthasome but rather functions as a physically separate entity in this highly diverged eukaryote. Therefore, TbAAC RNA interference (RNAi) ablation in the insect stage of T. brucei does not impair the activity or arrangement of the respiratory chain complexes. Nevertheless, RNAi silencing of TbAAC caused a severe growth defect that coincides with a significant reduction of mt ATP synthesis by both substrate and oxidative phosphorylation. Furthermore, TbAAC downregulation resulted in a decreased level of cytosolic ATP, a higher mt membrane potential, an elevated amount of reactive oxygen species, and a reduced consumption of oxygen in the mitochondria. Interestingly, while TbAAC has previously been demonstrated to serve as the sole ADP/ATP carrier for ADP influx into the mitochondria, our data suggest that a second carrier for ATP influx may be present and active in the T. brucei mitochondrion. Overall, this study provides more insight into the delicate balance of the functional relationship between TbAAC and the oxidative phosphorylation (OXPHOS) pathway in an early diverged eukaryote. PMID:25616281

A Transient Rise in Free Mg2+ Ions Released from ATP-Mg Hydrolysis Contributes to Mitotic Chromosome Condensation.

PubMed

Maeshima, Kazuhiro; Matsuda, Tomoki; Shindo, Yutaka; Imamura, Hiromi; Tamura, Sachiko; Imai, Ryosuke; Kawakami, Syoji; Nagashima, Ryosuke; Soga, Tomoyoshi; Noji, Hiroyuki; Oka, Kotaro; Nagai, Takeharu

2018-02-05

For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [1-5], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appropriate chromosome shape [6-17], free divalent cations such as Mg 2+ and Ca 2+ , which condense chromatin or chromosomes in vitro [18-28], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like "beads on a string" by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion [29]. However, technical limitations to measure intracellular free divalent cations, but not total cations [30], especially Mg 2+ , have prevented us from elucidating their function. Here, we developed a Förster resonance energy transfer (FRET)-based Mg 2+ indicator that monitors free Mg 2+ dynamics throughout the cell cycle. By combining this indicator with Ca 2+ [31] and adenosine triphosphate (ATP) [32] indicators, we demonstrate that the levels of free Mg 2+ , but not Ca 2+ , increase during mitosis. The Mg 2+ increase is coupled with a decrease in ATP, which is normally bound to Mg 2+ in the cell [33]. ATP inhibited Mg 2+ -dependent chromatin condensation in vitro. Chelating Mg 2+ induced mitotic cell arrest and chromosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP-bound Mg 2+ is released by ATP hydrolysis and contributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg 2+ -ATP balance. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Minoxidil opens mitochondrial K(ATP) channels and confers cardioprotection.

PubMed

Sato, Toshiaki; Li, Yulong; Saito, Tomoaki; Nakaya, Haruaki

2004-01-01

1. ATP-sensitive potassium channel in the mitochondrial inner membrane (mitoK(ATP) channel) rather than in the sarcolemma (sarcK(ATP) channel) appears to play an important role in cardioprotection. We examined the effect of minoxidil, a potent antihypertensive agent and hair growth stimulator, on sarcK(ATP) and mitoK(ATP) channels in guinea-pig ventricular myocytes. 2. Minoxidil activated a glybenclamide-sensitive sarcK(ATP) channel current in the whole-cell recording mode with an EC(50) of 182.6 microm. Minoxidil reversibly increased the flavoprotein oxidation, an index of mitoK(ATP) channel activity, in a concentration-dependent manner. The EC(50) for mitoK(ATP) channel activation was estimated to be 7.3 microm; this value was notably approximately 25-fold lower than that for sarcK(ATP) channel activation. 3. Minoxidil (10 microm) significantly attenuated the ouabain-induced increase of mitochondrial Ca(2+) concentration, which was measured by loading cells with rhod-2 fluorescence. Furthermore, pretreatment with minoxidil (10 microm) before 20-min no-flow ischaemia significantly improved the recovery of developed tension measured after 60 min of reperfusion in coronary perfused guinea-pig ventricular muscles. These cardioprotective effects of minoxidil were completely abolished by the mitoK(ATP) channel blocker 5-hydroxydecanoate (500 microm). 4. Our results indicate that minoxidil exerts a direct cardioprotective effect on heart muscle cells, an effect mediated by the selective activation of mitoK(ATP) channels.

LACK OF EXPRESSION OF EGF AND TGF-ALPHA IN THE FETAL MOUSE ALTERS FORMATION OF PROSTATIC EPITHELIAL BUDS AND INFLUENCES THE RESPONSE TO TCDD

EPA Science Inventory

Lack of Expression of EGF and TGF in the Fetal Mouse Alters Formation of Prostatic Epithelial Buds and Responsiveness to TCDD-Induced Impairment of Prostatic Bud Formation.

Barbara D. Abbott, Tien-Min Lin, Nathan T. Rasmussen, Robert W. Moore,
Ralph M. Albrecht, Judi...

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus , a New Twist on ATP Formation

DOE PAGES

James, Kimberly L.; Ríos-Hernández, Luis A.; Wofford, Neil Q.; ...

2016-08-16

Syntrophus aciditrophicusis a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation byS. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome ofS. aciditrophicusleaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show thatS. aciditrophicususes AMP-forming, acetyl-CoA synthetase (Acs1)more » for ATP synthesis from acetyl-CoA.acs1mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, ofS. aciditrophicusgrown in pure culture and coculture. Cell extracts ofS. aciditrophicushad low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified fromS. aciditrophicusand recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) inS. aciditrophicuscells support the operation of Acs1 in the acetate-forming direction. Thus,S. aciditrophicushas a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. We find bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA.Syntrophus aciditrophicusapparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus , a New Twist on ATP Formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

James, Kimberly L.; Ríos-Hernández, Luis A.; Wofford, Neil Q.

Syntrophus aciditrophicusis a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation byS. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome ofS. aciditrophicusleaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show thatS. aciditrophicususes AMP-forming, acetyl-CoA synthetase (Acs1)more » for ATP synthesis from acetyl-CoA.acs1mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, ofS. aciditrophicusgrown in pure culture and coculture. Cell extracts ofS. aciditrophicushad low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified fromS. aciditrophicusand recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) inS. aciditrophicuscells support the operation of Acs1 in the acetate-forming direction. Thus,S. aciditrophicushas a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. We find bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA.Syntrophus aciditrophicusapparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as

Cyclophilin D Promotes Brain Mitochondrial F1FO ATP Synthase Dysfunction in Aging Mice

PubMed Central

Gauba, Esha; Guo, Lan; Du, Heng

2017-01-01

Brain aging is the known strongest risk factor for Alzheimer’s disease (AD). In recent years, mitochondrial deficits have been proposed to be a common mechanism linking brain aging to AD. Therefore, to elucidate the causative mechanisms of mitochondrial dysfunction in aging brains is of paramount importance for our understanding of the pathogenesis of AD, in particular its sporadic form. Cyclophilin D (CypD) is a specific mitochondrial protein. Recent studies have shown that F1FO ATP synthase oligomycin sensitivity conferring protein (OSCP) is a binding partner of CypD. The interaction of CypD with OSCP modulates F1FO ATP synthase function and mediates mitochondrial permeability transition pore (mPTP) opening. Here, we have found that increased CypD expression, enhanced CypD/OSCP interaction, and selective loss of OSCP are prominent brain mitochondrial changes in aging mice. Along with these changes, brain mitochondria from the aging mice demonstrated decreased F1FO ATP synthase activity and defective F1FO complex coupling. In contrast, CypD deficient mice exhibited substantially mitigated brain mitochondrial F1FO ATP synthase dysfunction with relatively preserved mitochondrial function during aging. Interestingly, the aging-related OSCP loss was also dramatically attenuated by CypD depletion. Therefore, the simplest interpretation of this study is that CypD promotes F1FO ATP synthase dysfunction and the resultant mitochondrial deficits in aging brains. In addition, in view of CypD and F1FO ATP synthase alterations seen in AD brains, the results further suggest that CypD-mediated F1FO ATP synthase deregulation is a shared mechanism linking mitochondrial deficits in brain aging and AD. PMID:27834780

Cyclophilin D Promotes Brain Mitochondrial F1FO ATP Synthase Dysfunction in Aging Mice.

PubMed

Gauba, Esha; Guo, Lan; Du, Heng

2017-01-01

Brain aging is the known strongest risk factor for Alzheimer's disease (AD). In recent years, mitochondrial deficits have been proposed to be a common mechanism linking brain aging to AD. Therefore, to elucidate the causative mechanisms of mitochondrial dysfunction in aging brains is of paramount importance for our understanding of the pathogenesis of AD, in particular its sporadic form. Cyclophilin D (CypD) is a specific mitochondrial protein. Recent studies have shown that F1FO ATP synthase oligomycin sensitivity conferring protein (OSCP) is a binding partner of CypD. The interaction of CypD with OSCP modulates F1FO ATP synthase function and mediates mitochondrial permeability transition pore (mPTP) opening. Here, we have found that increased CypD expression, enhanced CypD/OSCP interaction, and selective loss of OSCP are prominent brain mitochondrial changes in aging mice. Along with these changes, brain mitochondria from the aging mice demonstrated decreased F1FO ATP synthase activity and defective F1FO complex coupling. In contrast, CypD deficient mice exhibited substantially mitigated brain mitochondrial F1FO ATP synthase dysfunction with relatively preserved mitochondrial function during aging. Interestingly, the aging-related OSCP loss was also dramatically attenuated by CypD depletion. Therefore, the simplest interpretation of this study is that CypD promotes F1FO ATP synthase dysfunction and the resultant mitochondrial deficits in aging brains. In addition, in view of CypD and F1FO ATP synthase alterations seen in AD brains, the results further suggest that CypD-mediated F1FO ATP synthase deregulation is a shared mechanism linking mitochondrial deficits in brain aging and AD.

Uncoupling of oxidative phosphorylation and ATP synthase reversal within the hyperthermic heart

PubMed Central

Power, Amelia; Pearson, Nicholas; Pham, Toan; Cheung, Carlos; Phillips, Anthony; Hickey, Anthony

2014-01-01

Abstract Heart failure is a common cause of death with hyperthermia, and the exact cause of hyperthermic heart failure appears elusive. We hypothesize that the energy supply (ATP) of the heart may become impaired due to increased inner‐mitochondrial membrane permeability and inefficient oxidative phosphorylation (OXPHOS). Therefore, we assessed isolated working heart and mitochondrial function. Ex vivo working rat hearts were perfused between 37 and 43.5°C and showed break points in all functional parameters at ~40.5°C. Mitochondrial high‐resolution respirometry coupled to fluorometry was employed to determine the effects of hyperthermia on OXPHOS and mitochondrial membrane potential (ΔΨ) in vitro using a comprehensive metabolic substrate complement with isolated mitochondria. Relative to 37 and 40°C, 43°C elevated Leak O2 flux and depressed OXPHOS O2 flux and ∆Ψ. Measurement of steady‐state ATP production from mitochondria revealed decreased ATP synthesis capacity, and a negative steady‐state P:O ratio at 43°C. This approach offers a more powerful analysis of the effects of temperature on OXPHOS that cannot be measured using simple measures such as the traditional respiratory control ratio (RCR) or P:O ratio, which, respectively, can only approach 1 or 0 with inner‐membrane failure. At 40°C there was only a slight enhancement of the Leak O2 flux and this did not significantly affect ATP production rate. Therefore, during mild hyperthermia (40°C) there is no enhancement of ATP supply by mitochondria, to accompany increasing cardiac energy demands, while between this and critical hyperthermia (43°C), mitochondria become net consumers of ATP. This consumption may contribute to cardiac failure or permanent damage during severe hyperthermia. PMID:25263202

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

PubMed

Chin, Randall M; Fu, Xudong; Pai, Melody Y; Vergnes, Laurent; Hwang, Heejun; Deng, Gang; Diep, Simon; Lomenick, Brett; Meli, Vijaykumar S; Monsalve, Gabriela C; Hu, Eileen; Whelan, Stephen A; Wang, Jennifer X; Jung, Gwanghyun; Solis, Gregory M; Fazlollahi, Farbod; Kaweeteerawat, Chitrada; Quach, Austin; Nili, Mahta; Krall, Abby S; Godwin, Hilary A; Chang, Helena R; Faull, Kym F; Guo, Feng; Jiang, Meisheng; Trauger, Sunia A; Saghatelian, Alan; Braas, Daniel; Christofk, Heather R; Clarke, Catherine F; Teitell, Michael A; Petrascheck, Michael; Reue, Karen; Jung, Michael E; Frand, Alison R; Huang, Jing

2014-06-19

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing

Uncoupling of oxidative phosphorylation and ATP synthase reversal within the hyperthermic heart.

PubMed

Power, Amelia; Pearson, Nicholas; Pham, Toan; Cheung, Carlos; Phillips, Anthony; Hickey, Anthony

2014-09-01

Heart failure is a common cause of death with hyperthermia, and the exact cause of hyperthermic heart failure appears elusive. We hypothesize that the energy supply (ATP) of the heart may become impaired due to increased inner-mitochondrial membrane permeability and inefficient oxidative phosphorylation (OXPHOS). Therefore, we assessed isolated working heart and mitochondrial function. Ex vivo working rat hearts were perfused between 37 and 43.5°C and showed break points in all functional parameters at ~40.5°C. Mitochondrial high-resolution respirometry coupled to fluorometry was employed to determine the effects of hyperthermia on OXPHOS and mitochondrial membrane potential (ΔΨ) in vitro using a comprehensive metabolic substrate complement with isolated mitochondria. Relative to 37 and 40°C, 43°C elevated Leak O2 flux and depressed OXPHOS O2 flux and ∆Ψ. Measurement of steady-state ATP production from mitochondria revealed decreased ATP synthesis capacity, and a negative steady-state P:O ratio at 43°C. This approach offers a more powerful analysis of the effects of temperature on OXPHOS that cannot be measured using simple measures such as the traditional respiratory control ratio (RCR) or P:O ratio, which, respectively, can only approach 1 or 0 with inner-membrane failure. At 40°C there was only a slight enhancement of the Leak O2 flux and this did not significantly affect ATP production rate. Therefore, during mild hyperthermia (40°C) there is no enhancement of ATP supply by mitochondria, to accompany increasing cardiac energy demands, while between this and critical hyperthermia (43°C), mitochondria become net consumers of ATP. This consumption may contribute to cardiac failure or permanent damage during severe hyperthermia. © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Stretch-induced Ca2+ independent ATP release in hippocampal astrocytes.

PubMed

Xiong, Yingfei; Teng, Sasa; Zheng, Lianghong; Sun, Suhua; Li, Jie; Guo, Ning; Li, Mingli; Wang, Li; Zhu, Feipeng; Wang, Changhe; Rao, Zhiren; Zhou, Zhuan

2018-02-28

Similar to neurons, astrocytes actively participate in synaptic transmission via releasing gliotransmitters. The Ca 2+ -dependent release of gliotransmitters includes glutamate and ATP. Following an 'on-cell-like' mechanical stimulus to a single astrocyte, Ca 2+ independent single, large, non-quantal, ATP release occurs. Astrocytic ATP release is inhibited by either selective antagonist treatment or genetic knockdown of P2X7 receptor channels. Our work suggests that ATP can be released from astrocytes via two independent pathways in hippocampal astrocytes; in addition to the known Ca 2+ -dependent vesicular release, larger non-quantal ATP release depends on P2X7 channels following mechanical stretch. Astrocytic ATP release is essential for brain functions such as synaptic long-term potentiation for learning and memory. However, whether and how ATP is released via exocytosis remains hotly debated. All previous studies of non-vesicular ATP release have used indirect assays. By contrast, two recent studies report vesicular ATP release using more direct assays. In the present study, using patch clamped 'ATP-sniffer cells', we re-investigated astrocytic ATP release at single-vesicle resolution in hippocampal astrocytes. Following an 'on-cell-like' mechanical stimulus of a single astrocyte, a Ca 2+ independent single large non-quantal ATP release occurred, in contrast to the Ca 2+ -dependent multiple small quantal ATP release in a chromaffin cell. The mechanical stimulation-induced ATP release from an astrocyte was inhibited by either exposure to a selective antagonist or genetic knockdown of P2X7 receptor channels. Functional P2X7 channels were expressed in astrocytes in hippocampal brain slices. Thus, in addition to small quantal ATP release, larger non-quantal ATP release depends on P2X7 channels in astrocytes. © 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.

Kinetic mechanism of the dimeric ATP sulfurylase from plants

PubMed Central

Ravilious, Geoffrey E.; Herrmann, Jonathan; Goo Lee, Soon; Westfall, Corey S.; Jez, Joseph M.

2013-01-01

In plants, sulfur must be obtained from the environment and assimilated into usable forms for metabolism. ATP sulfurylase catalyses the thermodynamically unfavourable formation of a mixed phosphosulfate anhydride in APS (adenosine 5′-phosphosulfate) from ATP and sulfate as the first committed step of sulfur assimilation in plants. In contrast to the multi-functional, allosterically regulated ATP sulfurylases from bacteria, fungi and mammals, the plant enzyme functions as a mono-functional, non-allosteric homodimer. Owing to these differences, here we examine the kinetic mechanism of soybean ATP sulfurylase [GmATPS1 (Glycine max (soybean) ATP sulfurylase isoform 1)]. For the forward reaction (APS synthesis), initial velocity methods indicate a single-displacement mechanism. Dead-end inhibition studies with chlorate showed competitive inhibition versus sulfate and non-competitive inhibition versus APS. Initial velocity studies of the reverse reaction (ATP synthesis) demonstrate a sequential mechanism with global fitting analysis suggesting an ordered binding of substrates. ITC (isothermal titration calorimetry) showed tight binding of APS to GmATPS1. In contrast, binding of PPi (pyrophosphate) to GmATPS1 was not detected, although titration of the E•APS complex with PPi in the absence of magnesium displayed ternary complex formation. These results suggest a kinetic mechanism in which ATP and APS are the first substrates bound in the forward and reverse reactions, respectively. PMID:23789618

An RNA motif that binds ATP

NASA Technical Reports Server (NTRS)

Sassanfar, M.; Szostak, J. W.

1993-01-01

RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.

EGF AND TGF ALPHA EXPRESSION INFLUENCE THE DEVELOPMENTAL TOXICITY OF TCDD: DOSE RESPONSE AND AHR PHENOTYPE IN EGF, TGF ALPHA AND EGF+TGF ALPHA KNOCKOUT MICE

EPA Science Inventory

Abstract
The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces cleft palate (CP) and hydronephrosis (HN) in mice. The etiology of these defects involves hyperproliferation of epithelial cells of the secondary palatal shelf and ureter, respectively. ...

A High-Throughput TNP-ATP Displacement Assay for Screening Inhibitors of ATP-Binding in Bacterial Histidine Kinases

PubMed Central

Guarnieri, Michael T.; Blagg, Brian S. J.

2011-01-01

Abstract Bacterial histidine kinases (HK) are members of the GHKL superfamily, which share a unique adenosine triphosphate (ATP)-binding Bergerat fold. Our previous studies have shown that Gyrase, Hsp90, MutL (GHL) inhibitors bind to the ATP-binding pocket of HK and may provide lead compounds for the design of novel antibiotics targeting these kinases. In this article, we developed a competition assay using the fluorescent ATP analog, 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate. The method can be used for high-throughput screening of compound libraries targeting HKs or other ATP-binding proteins. We utilized the assay to screen a library of GHL inhibitors targeting the bacterial HK PhoQ, and discuss the applications of the 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate competition assay beyond GHKL inhibitor screening. PMID:21050069

Theoretical studies of the ATP hydrolysis mechanism of myosin.

PubMed

Okimoto, N; Yamanaka, K; Ueno, J; Hata, M; Hoshino, T; Tsuda, M

2001-11-01

The ATP hydrolysis mechanism of myosin was studied using quantum chemical (QM) and molecular dynamics calculations. The initial model compound for QM calculations was constructed on the basis of the energy-minimized structure of the myosin(S1dc)-ATP complex, which was determined by molecular mechanics calculations. The result of QM calculations suggested that the ATP hydrolysis mechanism of myosin consists of a single elementary reaction in which a water molecule nucleophilically attacked gamma-phosphorus of ATP. In addition, we performed molecular dynamics simulations of the initial and final states of the ATP hydrolysis reaction, that is, the myosin-ATP and myosin-ADP.Pi complexes. These calculations revealed roles of several amino acid residues (Lys185, Thr186, Ser237, Arg238, and Glu459) in the ATPase pocket. Lys185 maintains the conformation of beta- and gamma-phosphate groups of ATP by forming the hydrogen bonds. Thr186 and Ser237 are coordinated to a Mg(2+) ion, which interacts with the phosphates of ATP and therefore contributes to the stabilization of the ATP structure. Arg238 and Glu459, which consisted of the gate of the ATPase pocket, retain the water molecule acting on the hydrolysis at the appropriate position for initiating the hydrolysis.

The function of mitochondrial F(O)F(1) ATP-synthase from the whiteleg shrimp Litopenaeus vannamei muscle during hypoxia.

PubMed

Martinez-Cruz, O; Calderon de la Barca, A M; Uribe-Carvajal, S; Muhlia-Almazan, A

2012-08-01

The effect of hypoxia and re-oxygenation on the mitochondrial complex F(O)F(1)-ATP synthase was investigated in the whiteleg shrimp Litopenaeus vannamei. A 660 kDa protein complex isolated from mitochondria of the shrimp muscle was identified as the ATP synthase complex. After 10h at hypoxia (1.5-2.0 mg oxygen/L), the concentration of L-lactate in plasma increased significantly, but the ATP amount and the concentration of ATPβ protein remained unaffected. Nevertheless, an increase of 70% in the ATPase activity was detected, suggesting that the enzyme may be regulated at a post-translational level. Thus, during hypoxia shrimp are able to maintain ATP amounts probably by using some other energy sources as phosphoarginine when an acute lack of energy occurs. During re-oxygenation, the ATPase activity decreased significantly and the ATP production continued via the electron transport chain and oxidative phosphorylation. The results obtained showed that shrimp faces hypoxia partially by hydrolyzing the ATP through the reaction catalyzed by the mitochondrial ATPase which increases its activity. Copyright © 2012 Elsevier Inc. All rights reserved.

ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites*

PubMed Central

Randak, Christoph O.; Dong, Qian; Ver Heul, Amanda R.; Elcock, Adrian H.; Welsh, Michael J.

2013-01-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5′-triphosphate (8-N3-ATP) and 8-azidoadenosine 5′-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P1,P5-di(adenosine-5′) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2. PMID:23921386

Prostatic relaxation induced by agmatine is decreased in spontaneously hypertensive rats.

PubMed

Lee, Liang-Ming; Tsai, Tsung-Chin; Chung, Hsien-Hui; Tong, Yat-Ching; Cheng, Juei-Tang

2012-09-01

What's known on the subject? and What does the study add? Neurotransmitters are known to control prostate contractility. Agmatine is one of them and induces relaxation through imidazoline receptors. The paper shows that the action of agmatine is reduced in hypertensive rats, and that this change is related to the decrease of ATP-sensitive potassium channels in the prostate. The findings can increase our understanding of the possible underlying mechanism for the development of clinical benign prostatic hyperplasia. To compare agmatine-induced prostatic relaxation in hypertensive and control rats. To investigate the responsible mechanism(s) and the role of the ATP-sensitive potassium channel. Prostate strips were isolated from male spontaneously hypertensive (SH) rats and normal Wistar-Kyoto (WKY) rats for measurement of isometric tension. The strips were precontracted with 1 µmol/L phenylephrine or 50 mmol/L KCl. Dose-dependent relaxation of the prostatic strips was studied by cumulative administration of agmatine, 1 to 100 µmol/L, into the organ bath. Effects of specific antagonists on agmatine-induced relaxation were studied. Western blotting analysis was used to measure the gene expression of the ATP-sensitive potassium channel in the rat prostate. Prostatic relaxation induced by agmatine was markedly reduced in SH rats compared with WKY rats. The relaxation caused by agmatine was abolished by BU224, a selective imidazoline I(2)-receptor antagonist, but was not modified by efaroxan at a dose sufficient to block imidazoline I(1)-receptors. The relaxation induced by diazoxide at a concentration sufficient to activate ATP-sensitive potassium channels was markedly reduced in the SH rat prostate. Expressions of ATP-sensitive potassium channel sulphonylurea receptor and inwardly rectifying potassium channel (Kir) 6.2 subunits were both decreased in the prostate of SH rats. The decrease of agmatine-induced prostatic relaxation in SH rats is related to the change in

Effect of LLLT on the level of ATP and ROS from organ of corti cells

NASA Astrophysics Data System (ADS)

Rhee, ChungKu; Chang, So-Young; Ahn, Jin-Chul; Suh, Myung-Whan; Jung, Jae Yun

2014-03-01

It is well established that ototoxic antibiotics and acoustic trauma can damage cochlear hair cells and cause hearing loss. Previous studies using transcanal LLLT (Low level laser therapy) showed that LLLT can promote recovery of hearing thresholds and cochlear hair cells. However, its mechanism has not been studied. Aim: The aim of this study is to investigate the mechanism of hearing recovery from gentamicin induced ototoxic hearing loss by LLLT. Methods: HEI- OC1 (House ear institute organ of Corti) cells were cultured for 18 hours and ototoxicity was induced by gentamicin (GM) treatment to the cells. Cultured cells were divided into 6 groups, No treatment control, LLLT only, GM 6.6 mM and GM 13.1 mM, GM 6.6 mM+LLLT and GM 13.1 mM+LLLT cells. LD laser 808 nm, 15 mW, was irradiated to the cultured cells for 15 min, at 4 hours after GM treatment to the cells. ATP was assayed using the ATP assay Kit. ROS was measured using confocal microscope after application of H2DCFDA dye. Results: ATP was decreased in GM 13.1 mM cells and increased in LLLT only cells and GM 13.1 mM+LLLT cells compared to control and 13.1 mM cells. ROS was increased in GM 6.6 mM and GM 13.1 mM cells, and decreased in GM 6.6 mM+LLLT and GM 13.1 mM+LLLT cells compared to GM 6.6 and 13.1 mM cells immediately after laser irradiation. Conclusion: This study demonstrated that LLLT on GM treated HEI-OC1 cells increased ATP and decreased ROS that may contribute to the recovery of hearing.

The adenosine salvage pathway as an alternative to mitochondrial production of ATP in maturing mammalian oocytes.

PubMed

Scantland, Sara; Tessaro, Irene; Macabelli, Carolina H; Macaulay, Angus D; Cagnone, Gaël; Fournier, Éric; Luciano, Alberto M; Robert, Claude

2014-09-01

Although the oocyte is the largest cell in the body and an unavoidable phase in life, its physiology is still poorly understood, and other cell types provide little insight into its unique nature. Even basic cellular functions in the oocyte such as energy metabolism are not yet fully understood. It is known that the mitochondria of the female gamete exhibit an immature form characterized by limited energy production from glucose and oxidative phosphorylation. We show that the bovine oocyte uses alternative means to maintain ATP production during maturation, namely, the adenosine salvage pathway. Meiosis resumption is triggered by destruction of cyclic AMP by phosphodiesterases producing adenosine monophosphate that is converted into ATP by adenylate kinases and creatine kinases. Inhibition of these enzymes decreased ATP production, and addition of their substrates restored ATP production in denuded oocytes. Addition of phosphocreatine to the oocyte maturation medium influenced the phenotype of the resulting blastocysts. We propose a model in which adenylate kinases and creatine kinases act as drivers of ATP production from added AMP during oocyte maturation. © 2014 by the Society for the Study of Reproduction, Inc.

Customized ATP towpreg. [Automated Tow Placement

NASA Technical Reports Server (NTRS)

Sandusky, Donald A.; Marchello, Joseph M.; Baucom, Robert M.; Johnston, Norman J.

1992-01-01

Automated tow placement (ATP) utilizes robotic technology to lay down adjacent polymer-matrix-impregnated carbon fiber tows on a tool surface. Consolidation and cure during ATP requires that void elimination and polymer matrix adhesion be accomplished in the short period of heating and pressure rolling that follows towpreg ribbon placement from the robot head to the tool. This study examined the key towpreg ribbon properties and dimensions which play a significant role in ATP. Analysis of the heat transfer process window indicates that adequate heating can be achieved at lay down rates as high as 1 m/sec. While heat transfer did not appear to be the limiting factor, resin flow and fiber movement into tow lap gaps could be. Accordingly, consideration was given to towpreg ribbon having uniform yet non-rectangular cross sections. Dimensional integrity of the towpreg ribbon combined with customized ribbon architecture offer great promise for processing advances in ATP of high performance composites.

Torque generation mechanism of ATP synthase

NASA Astrophysics Data System (ADS)

Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.

2010-03-01

ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes

PubMed Central

Leal Denis, M. Florencia; Alvarez, H. Ariel; Lauri, Natalia; Alvarez, Cora L.; Chara, Osvaldo; Schwarzbaum, Pablo J.

2016-01-01

Introduction The peptide mastoparan 7 (MST7) triggered in human erythrocytes (rbcs) the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe), interacting with P (purinergic) receptors, can affect cell volume (Vr), we explored the dynamic regulation between Vr and ATPe. Methods and Treatments We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors. Results In rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40–50% and swelling by 40–60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%. Analysis and Discussion Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop

Electrical Stimuli Are Anti-Apoptotic in Skeletal Muscle via Extracellular ATP. Alteration of This Signal in Mdx Mice Is a Likely Cause of Dystrophy

PubMed Central

Valladares, Denisse; Almarza, Gonzalo; Contreras, Ariel; Pavez, Mario; Buvinic, Sonja; Jaimovich, Enrique; Casas, Mariana

2013-01-01

ATP signaling has been shown to regulate gene expression in skeletal muscle and to be altered in models of muscular dystrophy. We have previously shown that in normal muscle fibers, ATP released through Pannexin1 (Panx1) channels after electrical stimulation plays a role in activating some signaling pathways related to gene expression. We searched for a possible role of ATP signaling in the dystrophy phenotype. We used muscle fibers from flexor digitorum brevis isolated from normal and mdx mice. We demonstrated that low frequency electrical stimulation has an anti-apoptotic effect in normal muscle fibers repressing the expression of Bax, Bim and PUMA. Addition of exogenous ATP to the medium has a similar effect. In dystrophic fibers, the basal levels of extracellular ATP were higher compared to normal fibers, but unlike control fibers, they do not present any ATP release after low frequency electrical stimulation, suggesting an uncoupling between electrical stimulation and ATP release in this condition. Elevated levels of Panx1 and decreased levels of Cav1.1 (dihydropyridine receptors) were found in triads fractions prepared from mdx muscles. Moreover, decreased immunoprecipitation of Cav1.1 and Panx1, suggest uncoupling of the signaling machinery. Importantly, in dystrophic fibers, exogenous ATP was pro-apoptotic, inducing the transcription of Bax, Bim and PUMA and increasing the levels of activated Bax and cytosolic cytochrome c. These evidence points to an involvement of the ATP pathway in the activation of mechanisms related with cell death in muscular dystrophy, opening new perspectives towards possible targets for pharmacological therapies. PMID:24282497

Electrical stimuli are anti-apoptotic in skeletal muscle via extracellular ATP. Alteration of this signal in Mdx mice is a likely cause of dystrophy.

PubMed

Valladares, Denisse; Almarza, Gonzalo; Contreras, Ariel; Pavez, Mario; Buvinic, Sonja; Jaimovich, Enrique; Casas, Mariana

2013-01-01

ATP signaling has been shown to regulate gene expression in skeletal muscle and to be altered in models of muscular dystrophy. We have previously shown that in normal muscle fibers, ATP released through Pannexin1 (Panx1) channels after electrical stimulation plays a role in activating some signaling pathways related to gene expression. We searched for a possible role of ATP signaling in the dystrophy phenotype. We used muscle fibers from flexor digitorum brevis isolated from normal and mdx mice. We demonstrated that low frequency electrical stimulation has an anti-apoptotic effect in normal muscle fibers repressing the expression of Bax, Bim and PUMA. Addition of exogenous ATP to the medium has a similar effect. In dystrophic fibers, the basal levels of extracellular ATP were higher compared to normal fibers, but unlike control fibers, they do not present any ATP release after low frequency electrical stimulation, suggesting an uncoupling between electrical stimulation and ATP release in this condition. Elevated levels of Panx1 and decreased levels of Cav1.1 (dihydropyridine receptors) were found in triads fractions prepared from mdx muscles. Moreover, decreased immunoprecipitation of Cav1.1 and Panx1, suggest uncoupling of the signaling machinery. Importantly, in dystrophic fibers, exogenous ATP was pro-apoptotic, inducing the transcription of Bax, Bim and PUMA and increasing the levels of activated Bax and cytosolic cytochrome c. These evidence points to an involvement of the ATP pathway in the activation of mechanisms related with cell death in muscular dystrophy, opening new perspectives towards possible targets for pharmacological therapies.

Real-Time Noninvasive Assessment of Pancreatic ATP Levels During Cold Preservation

PubMed Central

Scott, W.E.; Matsumoto, S.; Tanaka, T.; Avgoustiniatos, E.S.; Graham, M.L.; Williams, P.C.; Tempelman, L.A.; Sutherland, D.E.; Hering, B.J.; Hammer, B.E.; Papas, K.K.

2008-01-01

31P-NMR spectroscopy was utilized to investigate rat and porcine pancreatic ATP:Pi ratios to assess the efficacy of existing protocols for cold preservation (CP) in maintaining organ quality. Following sacrifice, rat pancreata were immediately excised or left enclosed in the body for 15 minutes of warm ischemia (WI). After excision, rat pancreata were stored at 6°C to 8°C using histidine-tryptophan-ketoglutarate solution (HTK) presaturated with air (S1), HTK presaturated with O2 (S2), or the HTK/perfluorodecalin two-layer method (TLM) with both liquids presaturated with O2 (S3). 31P-NMR spectra were sequentially collected at 3, 6, 9, 12, and 24 hours of CP from pancreata stored with each of the three protocols examined. The ATP:Pi ratio for rat pancreata exposed to 15 minutes of WI and stored with S3 increased during the first 9 hours of CP, approaching values observed for organs procured with no WI. A marked reduction in the ATP:Pi ratio was observed beyond 12 hours of CP with S3. After 6 hours of CP, the ATP:Pi ratio was highest for S3, substantially decreased for S2, and below detection for S1. In sharp contrast to the rat model, ATP was barely detectable in porcine pancreata exposed to minimal warm ischemia (<15 minutes) stored with the TLM regardless of CP time. We conclude that 31P-NMR spectroscopy is a powerful tool that can be used to (1) noninvasively evaluate pancreata prior to islet isolation, (2) assess the efficacy of different preservation protocols, (3) precisely define the timing of reversible versus irreversible damage, and (4) assess whether intervention will extend this timing. PMID:18374082

Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters retinoid homeostasis in maternal and perinatal tissues of the Holtzman rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kransler, Kevin M.; Tonucci, David A.; McGarrigle, Barbara P.

2007-10-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), one of the most widely studied environmental contaminants, causes a variety of adverse health effects including teratogenesis and altered development which may be related to disruptions in retinoid homeostasis. The purpose of this study was to determine the effect that gestational administration of TCDD has on retinoid homeostasis in both pregnant Holtzman rats and developing fetuses and neonates. A single oral dose of TCDD (0, 1.5, 3, or 6 {mu}g/kg) was administered to pregnant rats on gestation day 10, with fetuses analyzed on gestation days 17 and 20, and neonates analyzed on post natal day 7. Exposure tomore » TCDD generally produced decreases in the concentrations of retinyl esters, such as retinyl palmitate, and retinol in maternal and perinatal liver and lung, while increasing levels in the maternal kidney. Additionally, perinatal hepatic retinol binding protein 1-dependent retinyl ester hydrolysis was also decrease by TCDD. Sensitivity of the developing perinates to TCDD appeared to have an age-related component demonstrated by an increased rate of mortality and significant alterations to body weight and length on post natal day 7 relative to that observed at gestation day 20. A unique observation made in this study was a significant decrease in lung weight observed in the perinates exposed to TCDD. Taken together, these data demonstrate that TCDD significantly alters retinoid homeostasis in tissues of the developing fetus and neonate, suggesting that their unique sensitivity to TCDD may at least be in part the result of altered retinoid homeostasis.« less

ATP Synthase Diseases of Mitochondrial Genetic Origin

PubMed Central

Dautant, Alain; Meier, Thomas; Hahn, Alexander; Tribouillard-Tanvier, Déborah; di Rago, Jean-Paul; Kucharczyk, Roza

2018-01-01

Devastating human neuromuscular disorders have been associated to defects in the ATP synthase. This enzyme is found in the inner mitochondrial membrane and catalyzes the last step in oxidative phosphorylation, which provides aerobic eukaryotes with ATP. With the advent of structures of complete ATP synthases, and the availability of genetically approachable systems such as the yeast Saccharomyces cerevisiae, we can begin to understand these molecular machines and their associated defects at the molecular level. In this review, we describe what is known about the clinical syndromes induced by 58 different mutations found in the mitochondrial genes encoding membrane subunits 8 and a of ATP synthase, and evaluate their functional consequences with respect to recently described cryo-EM structures. PMID:29670542

Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors*

PubMed Central

Sielaff, Hendrik; Martin, James; Singh, Dhirendra; Biuković, Goran; Grüber, Gerhard; Frasch, Wayne D.

2016-01-01

The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A3B3DF, from the Methanosarcina mazei Gö1 A-ATP synthase, and the thermophilic motor α3β3γ, from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase, are resolved at 5 μs resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and they differ only in the magnitude of their velocities. M. mazei A3B3DF power strokes occurred in 120° steps at saturating ATP concentrations like the F-type motors. However, because ATP-binding dwells did not interrupt the 120° steps at limiting ATP, ATP binding to A3B3DF must occur during the catalytic dwell. Elevated concentrations of ADP did not increase dwells occurring 40° after the catalytic dwell. In F-type motors, elevated ADP induces dwells 40° after the catalytic dwell and slows the overall velocity. The similarities in these power stroke profiles are consistent with a common rotational mechanism for A-type and F-type rotary motors, in which the angular velocity is limited by the rotary position at which ATP binding occurs and by the drag imposed on the axle as it rotates within the ring of stator subunits. PMID:27729450

Lysosomal Storage of Subunit c of Mitochondrial ATP Synthase in Brain-Specific Atp13a2-Deficient Mice.

PubMed

Sato, Shigeto; Koike, Masato; Funayama, Manabu; Ezaki, Junji; Fukuda, Takahiro; Ueno, Takashi; Uchiyama, Yasuo; Hattori, Nobutaka

2016-12-01

Kufor-Rakeb syndrome (KRS) is an autosomal recessive form of early-onset parkinsonism linked to the PARK9 locus. The causative gene for KRS is Atp13a2, which encodes a lysosomal type 5 P-type ATPase. We recently showed that KRS/PARK9-linked mutations lead to several lysosomal alterations, including reduced proteolytic processing of cathepsin D in vitro. However, it remains unknown how deficiency of Atp13a2 is connected to lysosomal impairments. To address this issue, we analyzed brain tissues of Atp13a2 conditional-knockout mice, which exhibited characteristic features of neuronal ceroid lipofuscinosis, including accumulation of lipofuscin positive for subunit c of mitochondrial ATP synthase, suggesting that a common pathogenic mechanism underlies both neuronal ceroid lipofuscinosis and Parkinson disease. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Induction of energy metabolism related enzymes in yeast Saccharomyces cerevisiae exposed to ibogaine is adaptation to acute decrease in ATP energy pool.

PubMed

Paskulin, Roman; Jamnik, Polona; Obermajer, Natasa; Slavić, Marija; Strukelj, Borut

2010-02-10

Ibogaine has been extensively studied in the last decades in relation to its anti-addictive properties that have been repeatedly reported as being addiction interruptive and craving eliminative. In our previous study we have already demonstrated induction of energy related enzymes in rat brains treated with ibogaine at a dose of 20mg/kg i.p. 24 and 72 h prior to proteomic analysis. In this study a model organism yeast Saccharomyces cerevisiae was cultivated with ibogaine in a concentration of 1mg/l. Energy metabolism cluster enzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, enolase and alcohol dehydrogenase were induced after 5h of exposure. This is a compensation of demonstrated ATP pool decrease after ibogaine. Yeast in a stationary growth phase is an accepted model for studies of housekeeping metabolism of eukaryotes, including humans. Study showed that ibogaine's influence on metabolism is neither species nor tissue specific. Effect is not mediated by binding of ibogaine to receptors, as previously described in literature since they are lacking in this model. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Modulation of nucleotide sensitivity of ATP-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase

PubMed Central

Shyng, S.-L.; Barbieri, A.; Gumusboga, A.; Cukras, C.; Pike, L.; Davis, J. N.; Stahl, P. D.; Nichols, C. G.

2000-01-01

ATP-sensitive potassium channels (KATP channels) regulate cell excitability in response to metabolic changes. KATP channels are formed as a complex of a sulfonylurea receptor (SURx), a member of the ATP-binding cassette protein family, and an inward rectifier K+ channel subunit (Kir6.x). Membrane phospholipids, in particular phosphatidylinositol (PI) 4,5-bisphosphate (PIP2), activate KATP channels and antagonize ATP inhibition of KATP channels when applied to inside-out membrane patches. To examine the physiological relevance of this regulatory mechanism, we manipulated membrane PIP2 levels by expressing either the wild-type or an inactive form of PI-4-phosphate 5-kinase (PIP5K) in COSm6 cells and examined the ATP sensitivity of coexpressed KATP channels. Channels from cells expressing the wild-type PIP5K have a 6-fold lower ATP sensitivity (K1/2, the half maximal inhibitory concentration, ≈ 60 μM) than the sensitivities from control cells (K1/2 ≈ 10 μM). An inactive form of the PIP5K had little effect on the K1/2 of wild-type channels but increased the ATP-sensitivity of a mutant KATP channel that has an intrinsically lower ATP sensitivity (from K1/2 ≈ 450 μM to K1/2 ≈ 100 μM), suggesting a decrease in membrane PIP2 levels as a consequence of a dominant-negative effect of the inactive PIP5K. These results show that PIP5K activity, which regulates PIP2 and PI-3,4,5-P3 levels, is a significant determinant of the physiological nucleotide sensitivity of KATP channels. PMID:10639183

Identification of a new Mpl-interacting protein, Atp5d.

PubMed

Liu, Hongyan; Zhao, Zhenhu; Zhong, Yuxu; Shan, Yajun; Sun, Xiaohong; Mao, Bingzhi; Cong, Yuwen

2014-06-01

Thrombopoietin (TPO) can regulate hematopoiesis and megakaryopoiesis via activation of its receptor, c-Mpl, and multiple downstream signal transduction pathways. Using the cytoplasmic domain of Mpl as bait, we performed yeast two-hybrid screening, and found that the protein Atp5d might associate with Mpl. Atp5d is known as the δ subunit of mitochondrial ATP synthase, but little is known about the function of dissociative Atp5d. The interaction between Mpl and Atp5d was confirmed by the yeast two-hybrid system, mammalian two-hybrid assay, pull-down experiment, and co-immunoprecipitation study in vivo and in vitro. An additional immunofluorescence assay showed that the two proteins can colocalize along the plasma membrane in the cytoplasm. Using the yeast two-hybrid system, we tested a series of cytoplasmic truncated mutations for their ability to bind Atp5d and found an association between Atp5d and the Aa98-113 domain of Mpl. The dissociation of Atp5d from Mpl after TPO stimulation suggests that Atp5d may be a new component of TPO signaling.

ATP synthesis in the energy metabolism pathway: a new perspective for manipulating CdSe quantum dots biosynthesized in Saccharomyces cerevisiae.

PubMed

Zhang, Rong; Shao, Ming; Han, Xu; Wang, Chuan; Li, Yong; Hu, Bin; Pang, Daiwen; Xie, Zhixiong

2017-01-01

Due to a growing trend in their biomedical application, biosynthesized nanomaterials are of great interest to researchers nowadays with their biocompatible, low-energy consumption, economic, and tunable characteristics. It is important to understand the mechanism of biosynthesis in order to achieve more efficient applications. Since there are only rare studies on the influences of cellular energy levels on biosynthesis, the influence of energy is often overlooked. Through determination of the intracellular ATP concentrations during the biosynthesis process, significant changes were observed. In addition, ATP synthesis deficiency caused great decreases in quantum dots (QDs) biosynthesis in the Δ atp1 , Δ atp2 , Δ atp14 , and Δ atp17 strains. With inductively coupled plasma-atomic emission spectrometry and atomic absorption spectroscopy analyses, it was found that ATP affected the accumulation of the seleno-precursor and helped with the uptake of Cd and the formation of QDs. We successfully enhanced the fluorescence intensity 1.5 or 2 times through genetic modification to increase ATP or SeAM (the seleno analog of S -adenosylmethionine, the product that would accumulate when ATP is accrued). This work explains the mechanism for the correlation of the cellular energy level and QDs biosynthesis in living cells, demonstrates control of the biosynthesis using this mechanism, and thus provides a new manipulation strategy for the biosynthesis of other nanomaterials to widen their applications.

ATP synthesis in the energy metabolism pathway: a new perspective for manipulating CdSe quantum dots biosynthesized in Saccharomyces cerevisiae

PubMed Central

Zhang, Rong; Shao, Ming; Han, Xu; Wang, Chuan; Li, Yong; Hu, Bin; Pang, Daiwen; Xie, Zhixiong

2017-01-01

Due to a growing trend in their biomedical application, biosynthesized nanomaterials are of great interest to researchers nowadays with their biocompatible, low-energy consumption, economic, and tunable characteristics. It is important to understand the mechanism of biosynthesis in order to achieve more efficient applications. Since there are only rare studies on the influences of cellular energy levels on biosynthesis, the influence of energy is often overlooked. Through determination of the intracellular ATP concentrations during the biosynthesis process, significant changes were observed. In addition, ATP synthesis deficiency caused great decreases in quantum dots (QDs) biosynthesis in the Δatp1, Δatp2, Δatp14, and Δatp17 strains. With inductively coupled plasma-atomic emission spectrometry and atomic absorption spectroscopy analyses, it was found that ATP affected the accumulation of the seleno-precursor and helped with the uptake of Cd and the formation of QDs. We successfully enhanced the fluorescence intensity 1.5 or 2 times through genetic modification to increase ATP or SeAM (the seleno analog of S-adenosylmethionine, the product that would accumulate when ATP is accrued). This work explains the mechanism for the correlation of the cellular energy level and QDs biosynthesis in living cells, demonstrates control of the biosynthesis using this mechanism, and thus provides a new manipulation strategy for the biosynthesis of other nanomaterials to widen their applications. PMID:28579774

Evidence that Na+/H+ exchanger 1 is an ATP-binding protein.

PubMed

Shimada-Shimizu, Naoko; Hisamitsu, Takashi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

2013-03-01

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1. © 2013 The Authors Journal compilation © 2013 FEBS.

Formation of hydrotalcite in aqueous solutions and intercalation of ATP by anion exchange.

PubMed

Tamura, Hiroki; Chiba, Jun; Ito, Masahiro; Takeda, Takashi; Kikkawa, Shinichi; Mawatari, Yasuteru; Tabata, Masayoshi

2006-08-15

The formation reaction and the intercalation of adenosine triphosphate (ATP) were studied for hydrotalcite (HT), a layered double hydroxide (LDH) of magnesium and aluminum. Hydrotalcite with nitrate ions in the interlayer (HT-NO(3)) was formed (A) by dropwise addition of a solution of magnesium and aluminum nitrates (pH ca. 3) to a sodium hydroxide solution (pH ca. 14) until the pH decreased from 14 to 10 and (B) by dropwise addition of the NaOH solution to the solution of magnesium and aluminum nitrates with pH increasing from 3 to 10. The precipitate obtained with method B was contaminated with aluminum hydroxide and the crystallinity of the product was low, possibly because aluminum hydroxide precipitates at pH 4 or 5 and remains even after HT-NO(3) forms at pH above 8. With method A, however, the precipitate was pure HT-NO(3) with increased crystallinity, since the solubility of aluminum hydroxide at pH above and around 10 is high as dissolved aluminate anions are stable in this high pH region, and there was no aluminum hydroxide contamination. The formed HT-NO(3) had a composition of [Mg(0.71)Al(0.29)(OH)(2)](NO(3))(0.29).0.58H(2)O. To intercalate ATP anions into the HT-NO(3), HT-NO(3) was dispersed in an ATP solution at pH 7. It was found that the interlayer nitrate ions were completely exchanged with ATP anions by ion exchange, and the interlayer distance expanded almost twice with a free space distance of 1.2 nm. The composition of HT-ATP was established as [Mg(0.68)Al(0.32)(OH)(2)](ATP)(0.080)0.88H(2)O. The increased distance could be explained with a calculated molecular configuration of the ATP as follows: An ATP molecule is bound to an interlayer surface with the triphosphate group, the adenosine group bends owing to its bond angles and projects into the interlayer to a height of 1 nm, and the adenosine groups aligned in the interlayer support the interlayer distance.

Impaired ATP release from red blood cells promotes their adhesion to endothelial cells: A mechanism of hypoxemia after transfusion

PubMed Central

Zhu, Hongmei; Zennadi, Rahima; Xu, Bruce X.; Eu, Jerry P.; Torok, Jordan A.; Telen, Marilyn J.; McMahon, Timothy J.

2011-01-01

Objective Transfusion of red blood cells (RBCs) has been linked to disappointing clinical outcomes in the critically ill, but specific mechanisms of organ dysfunction after transfusion remain poorly understood. We tested the hypothesis that RBC storage impairs the ability of RBCs to release ATP and that impaired ATP-release was injurious in vivo, in part through increased RBC adhesion. Design Prospective, controlled, mechanistic study. Setting University research laboratory. Subjects Human and mouse blood donors; nude mouse transfusion recipients. Interventions Manipulation of ATP release, supplemental ATP, and antibodies to RBC and endothelial adhesion receptors were used in vitro and in vivo to probe the roles of released ATP and adhesion in responses to (transfused) RBCs. Measurements and main results The ability of stored RBCs to release ATP declined markedly within 14 days after collection, despite relatively stable levels of ATP within the RBCs. Inhibiting ATP release promoted the adhesion of stored RBCs to endothelial cells in vitro and RBC sequestration in the lungs of transfused mice in vivo. Unlike transfusion of fresh human RBCs, stored-RBC transfusion in mice decreased blood oxygenation and increased extravasation of RBCs into the lung’s alveolar airspaces. Similar findings were seen with transfusion of fresh RBCs treated with the ATP-release inhibitors glibenclamide and carbenoxolone. These findings were prevented by either co-infusion of an ATP analog or pre-transfusion incubation of the RBCs with an antibody against the erythrocyte adhesion receptor LW (Landsteiner-Wiener; ICAM-4). Conclusions The normal flow of RBCs in pulmonary microvessels depends in part on the release of anti-adhesive ATP from RBCs, and storage-induced deficiency in ATP release from transfused RBCs may promote or exacerbate microvascular pathophysiology in the lung, in part through increased RBC adhesion. PMID:21765360

Possible involvement of ATP-sensitive potassium channels in the antidepressant-like effects of gabapentin in mouse forced swimming test.

PubMed

Ostadhadi, Sattar; Akbarian, Reyhaneh; Norouzi-Javidan, Abbas; Nikoui, Vahid; Zolfaghari, Samira; Chamanara, Mohsen; Dehpour, Ahmad-Reza

2017-07-01

Gabapentin as an anticonvulsant drug also has beneficial effects in treatment of depression. Previously, we showed that acute administration of gabapentin produced an antidepressant-like effect in the mouse forced swimming test (FST) by a mechanism that involves the inhibition of nitric oxide (NO). Considering the involvement of NO in adenosine triphosphate (ATP)-sensitive potassium channels (K ATP ), in the present study we investigated the involvement of K ATP channels in antidepressant-like effect of gabapentin. Gabapentin at different doses (5-10 mg/kg) and fluoxetine (20 mg/kg) were administrated by intraperitoneal route, 60 and 30 min, respectively, before the test. To clarify the probable involvement of K ATP channels, mice were pretreated with K ATP channel inhibitor or opener. Gabapentin at dose 10 mg/kg significantly decreased the immobility behavior of mice similar to fluoxetine (20 mg/kg). Co-administration of subeffective dose (1 mg/kg) of glibenclamide (inhibitor of K ATP channels) with gabapentin (3 mg/kg) showed a synergistic antidepressant-like effect. Also, subeffective dose of cromakalim (opener of K ATP channels, 0.1 mg/kg) inhibited the antidepressant-like effect of gabapentin (10 mg/kg). None of the treatments had any impact on the locomotor movement. Our study, for the first time, revealed that antidepressant-like effect of gabapentin in mice is mediated by blocking the K ATP channels.

Purinergic modulation of preBötzinger complex inspiratory rhythm in rodents: the interaction between ATP and adenosine

PubMed Central

Zwicker, J D; Rajani, V; Hahn, L B; Funk, G D

2011-01-01

Abstract ATP signalling in the CNS is mediated by a three-part system comprising the actions of ATP (and ADP) at P2 receptors (P2Rs), adenosine (ADO) at P1 receptors (P1Rs), and ectonucleotidases that degrade ATP into ADO. ATP excites preBötzinger complex (preBötC) inspiratory rhythm-generating networks where its release attenuates the hypoxic depression of breathing. Its metabolite, ADO, inhibits breathing through unknown mechanisms that may involve the preBötC. Our objective is to understand the dynamics of this signalling system and its influence on preBötC networks. We show that the preBötC of mouse and rat is sensitive to P2Y1 purinoceptor (P2Y1R) activation, responding with a >2-fold increase in frequency. Remarkably, the mouse preBötC is insensitive to ATP. Only after block of A1 ADORs is the ATP-evoked, P2Y1R-mediated frequency increase observed. This demonstrates that ATP is rapidly degraded to ADO, which activates inhibitory A1Rs, counteracting the P2Y1R-mediated excitation. ADO sensitivity of mouse preBötC was confirmed by a frequency decrease that was absent in rat. Differential ectonucleotidase activities are likely to contribute to the negligible ATP sensitivity of mouse preBötC. Real-time PCR analysis of ectonucleotidase isoforms in preBötC punches revealed TNAP (degrades ATP to ADO) or ENTPDase2 (favours production of excitatory ADP) as the primary constituent in mouse and rat, respectively. These data further establish the sensitivity of this vital network to P2Y1R-mediated excitation, emphasizing that individual components of the three-part signalling system dramatically alter network responses to ATP. Data also suggest therapeutic potential may derive from methods that alter the ATP–ADO balance to favour the excitatory actions of ATP. PMID:21788352

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria.

PubMed

Bhattacharjee, Ashima; Yang, Haojun; Duffy, Megan; Robinson, Emily; Conrad-Antoville, Arianrhod; Lu, Ya-Wen; Capps, Tony; Braiterman, Lelita; Wolfgang, Michael; Murphy, Michael P; Yi, Ling; Kaler, Stephen G; Lutsenko, Svetlana; Ralle, Martina

2016-08-05

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia). © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhattacharjee, Ashima; Yang, Haojun; Duffy, Megan

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria,more » whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).« less

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria*

PubMed Central

Bhattacharjee, Ashima; Yang, Haojun; Duffy, Megan; Robinson, Emily; Conrad-Antoville, Arianrhod; Lu, Ya-Wen; Capps, Tony; Braiterman, Lelita; Wolfgang, Michael; Murphy, Michael P.; Yi, Ling; Kaler, Stephen G.; Lutsenko, Svetlana; Ralle, Martina

2016-01-01

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia). PMID:27226607

Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors.

PubMed

Sielaff, Hendrik; Martin, James; Singh, Dhirendra; Biuković, Goran; Grüber, Gerhard; Frasch, Wayne D

2016-12-02

The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A 3 B 3 DF, from the Methanosarcina mazei Gö1 A-ATP synthase, and the thermophilic motor α 3 β 3 γ, from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase, are resolved at 5 μs resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and they differ only in the magnitude of their velocities. M. mazei A 3 B 3 DF power strokes occurred in 120° steps at saturating ATP concentrations like the F-type motors. However, because ATP-binding dwells did not interrupt the 120° steps at limiting ATP, ATP binding to A 3 B 3 DF must occur during the catalytic dwell. Elevated concentrations of ADP did not increase dwells occurring 40° after the catalytic dwell. In F-type motors, elevated ADP induces dwells 40° after the catalytic dwell and slows the overall velocity. The similarities in these power stroke profiles are consistent with a common rotational mechanism for A-type and F-type rotary motors, in which the angular velocity is limited by the rotary position at which ATP binding occurs and by the drag imposed on the axle as it rotates within the ring of stator subunits. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Critical role of ATP-induced ATP release for Ca2+ signaling in nonsensory cell networks of the developing cochlea

PubMed Central

Ceriani, Federico; Pozzan, Tullio; Mammano, Fabio

2016-01-01

Spatially and temporally coordinated variations of the cytosolic free calcium concentration ([Ca2+]c) play a crucial role in a variety of tissues. In the developing sensory epithelium of the mammalian cochlea, elevation of extracellular adenosine trisphosphate concentration ([ATP]e) triggers [Ca2+]c oscillations and propagation of intercellular inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ waves. What remains uncertain is the relative contribution of gap junction channels and connexin hemichannels to these fundamental mechanisms, defects in which impair hearing acquisition. Another related open question is whether [Ca2+]c oscillations require oscillations of the cytosolic IP3 concentration ([IP3]c) in this system. To address these issues, we performed Ca2+ imaging experiments in the lesser epithelial ridge of the mouse cochlea around postnatal day 5 and constructed a computational model in quantitative adherence to experimental data. Our results indicate that [Ca2+]c oscillations are governed by Hopf-type bifurcations within the experimental range of [ATP]e and do not require [IP3]c oscillations. The model replicates accurately the spatial extent and propagation speed of intercellular Ca2+ waves and predicts that ATP-induced ATP release is the primary mechanism underlying intercellular propagation of Ca2+ signals. The model also uncovers a discontinuous transition from propagating regimes (intercellular Ca2+ wave speed > 11 μm⋅s−1) to propagation failure (speed = 0), which occurs upon lowering the maximal ATP release rate below a minimal threshold value. The approach presented here overcomes major limitations due to lack of specific connexin channel inhibitors and can be extended to other coupled cellular systems. PMID:27807138

The ATP required for potentiation of skeletal muscle contraction is released via pannexin hemichannels.

PubMed

Riquelme, Manuel A; Cea, Luis A; Vega, José L; Boric, Mauricio P; Monyer, Hannah; Bennett, Michael V L; Frank, Marina; Willecke, Klaus; Sáez, Juan C

2013-12-01

During repetitive stimulation of skeletal muscle, extracellular ATP levels raise, activating purinergic receptors, increasing Ca2+ influx, and enhancing contractile force, a response called potentiation. We found that ATP appears to be released through pannexin1 hemichannels (Panx1 HCs). Immunocytochemical analyses and function were consistent with pannexin1 localization to T-tubules intercalated with dihydropyridine and ryanodine receptors in slow (soleus) and fast (extensor digitorum longus, EDL) muscles. Isolated myofibers took up ethidium (Etd+) and released small molecules (as ATP) during electrical stimulation. Consistent with two glucose uptake pathways, induced uptake of 2-NBDG, a fluorescent glucose derivative, was decreased by inhibition of HCs or glucose transporter (GLUT4), and blocked by dual blockade. Adult skeletal muscles apparently do not express connexins, making it unlikely that connexin hemichannels contribute to the uptake and release of small molecules. ATP release, Etd+ uptake, and potentiation induced by repetitive electrical stimulation were blocked by HC blockers and did not occur in muscles of pannexin1 knockout mice. MRS2179, a P2Y1R blocker, prevented potentiation in EDL, but not soleus muscles, suggesting that in fast muscles ATP activates P2Y1 but not P2X receptors. Phosphorylation on Ser and Thr residues of pannexin1 was increased during potentiation, possibly mediating HC opening. Opening of Panx1 HCs during repetitive activation allows efflux of ATP, influx of glucose and possibly Ca2+ too, which are required for potentiation of contraction. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'. Copyright © 2013 Elsevier Ltd. All rights reserved.

Unexpected Role of the Copper Transporter ATP7A in PDGF-Induced Vascular Smooth Muscle Cell Migration

PubMed Central

Ashino, Takashi; Sudhahar, Varadarajan; Urao, Norifumi; Oshikawa, Jin; Chen, Gin-Fu; Wang, Huan; Huo, Yuqing; Finney, Lydia; Vogt, Stefan; McKinney, Ronald D.; Maryon, Edward B.; Kaplan, Jack H.; Ushio-Fukai, Masuko; Fukai, Tohru

2010-01-01

Rationale Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1, but also by the copper exporter ATP7A (Menke ATPase) whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. Objective To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. Methods and Results Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A siRNA or CTR siRNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor pro-lysyl oxidase (Pro-LOX) in lipid raft fraction as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based X-ray fluorescence microscopy at neointimal VSMCs in wire injury model. Conclusions These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis. PMID:20671235

Unexpected role of the copper transporter ATP7A in PDGF-induced vascular smooth muscle cell migration.

PubMed

Ashino, Takashi; Sudhahar, Varadarajan; Urao, Norifumi; Oshikawa, Jin; Chen, Gin-Fu; Wang, Huan; Huo, Yuqing; Finney, Lydia; Vogt, Stefan; McKinney, Ronald D; Maryon, Edward B; Kaplan, Jack H; Ushio-Fukai, Masuko; Fukai, Tohru

2010-09-17

Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1 (copper transporter 1) but also by the copper exporter ATP7A (Menkes ATPase), whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts, which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A small interfering RNA or CTR small interfering RNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor prolysyl oxidase (Pro-LOX) in lipid raft fraction, as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based x-ray fluorescence microscopy at neointimal VSMCs in wire injury model. These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge, as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis.

Sarcolemmal cardiac K(ATP) channels as a target for the cardioprotective effects of the fluorine-containing pinacidil analogue, flocalin.

PubMed

Voitychuk, Oleg I; Strutynskyi, Ruslan B; Yagupolskii, Lev M; Tinker, Andrew; Moibenko, Olexiy O; Shuba, Yaroslav M

2011-02-01

A class of drugs known as K(ATP) -channel openers induce cardioprotection. This study examined the effects of the novel K(ATP) -channel opener, the fluorine-containing pinacidil derivative, flocalin, on cardiac-specific K(ATP) -channels, excitability of native cardiac myocytes and on the ischaemic heart. The action of flocalin was investigated on: (i) membrane currents through cardiac-specific K(ATP) -channels (I(KATP) ) formed by K(IR) 6.2/SUR2A heterologously expressed in HEK-293 cells (HEK-293(₆.₂/₂A) ); (ii) excitability and intracellular Ca²(+) ([Ca²(+) ](i) ) transients of cultured rat neonatal cardiac myocytes; and (iii) functional and ultrastructural characteristics of isolated guinea-pig hearts subjected to ischaemia-reperfusion. Flocalin concentration-dependently activated a glibenclamide-sensitive I(KATP) in HEK-293(₆.₂/₂A) cells with an EC₅₀= 8.1 ± 0.4 µM. In cardiac myocytes, flocalin (5 µM) hyperpolarized resting potential by 3-5 mV, markedly shortened action potential duration, reduced the amplitude of [Ca²(+) ](i) transients by 2-3-fold and suppressed contraction. The magnitude and extent of reversibility of these effects depended on the type of cardiac myocytes. In isolated hearts, perfusion with 5 µmol·L⁻¹ flocalin, before inducing ischaemia, facilitated restoration of contraction during reperfusion, decreased the number of extrasystoles, prevented the appearance of coronary vasoconstriction and reduced damage to the cardiac tissue at the ultrastructural level (state of myofibrils, membrane integrity, mitochondrial cristae structure). Flocalin induced potent cardioprotection by activating cardiac-type K(ATP) -channels with all the benefits of the presence of fluorine group in the drug structure: higher lipophilicity, decreased toxicity, resistance to oxidation and thermal degradation, decreased metabolism in the organism and prolonged therapeutic action. © 2011 The Authors. British Journal of Pharmacology © 2011 The

Vascular ATP-sensitive potassium channels are over-expressed and partially regulated by nitric oxide in experimental septic shock.

PubMed

Collin, Solène; Sennoun, Nacira; Dron, Anne-Gaëlle; de la Bourdonnaye, Mathilde; Montemont, Chantal; Asfar, Pierre; Lacolley, Patrick; Meziani, Ferhat; Levy, Bruno

2011-05-01

To study the activation and expression of vascular (aorta and small mesenteric arteries) potassium channels during septic shock with or without modulation of the NO pathway. Septic shock was induced in rats by peritonitis. Selective inhibitors of vascular K(ATP) (PNU-37883A) or BK(Ca) [iberiotoxin (IbTX)] channels were used to demonstrate their involvement in vascular hyporeactivity. Vascular response to phenylephrine was measured on aorta and small mesenteric arteries mounted on a wire myograph. Vascular expression of potassium channels was studied by PCR and Western blot, in the presence or absence of 1400W, an inducible NO synthase (iNOS) inhibitor. Aortic activation of the transcriptional factor nuclear factor-kappaB (NF-κB) was assessed by electrophoretic mobility shift assay. Arterial pressure as well as in vivo and ex vivo vascular reactivity were reduced by sepsis and improved by PNU-37883A but not by IbTX. Sepsis was associated with an up-regulation of mRNA and protein expression of vascular K(ATP) channels, while expression of vascular BK(Ca) channels remained unchanged. Selective iNOS inhibition blunted the sepsis-induced increase in aortic NO, decreased NF-κB activation, and down-regulated vascular K(ATP) channel expression. Vascular K(ATP) but not BK(Ca) channels are activated, over-expressed, and partially regulated by NO via NF-κB activation during septic shock. Their selective inhibition restores arterial pressure and vascular reactivity and decreases lactate concentration. The present data suggest that selective vascular K(ATP) channel inhibitors offer potential therapeutic perspectives for septic shock.

Modeling the effects of hypoxia on ATP turnover in exercising muscle

NASA Technical Reports Server (NTRS)

Arthur, P. G.; Hogan, M. C.; Bebout, D. E.; Wagner, P. D.; Hochachka, P. W.

1992-01-01

Most models of metabolic control concentrate on the regulation of ATP production and largely ignore the regulation of ATP demand. We describe a model, based on the results of Hogan et al. (J. Appl. Physiol. 73: 728-736, 1992), that incorporates the effects of ATP demand. The model is developed from the premise that a unique set of intracellular conditions can be measured at each level of ATP turnover and that this relationship is best described by energetic state. Current concepts suggest that cells are capable of maintaining oxygen consumption in the face of declines in the concentration of oxygen through compensatory changes in cellular metabolites. We show that these compensatory changes can cause significant declines in ATP demand and result in a decline in oxygen consumption and ATP turnover. Furthermore we find that hypoxia does not directly affect the rate of anaerobic ATP synthesis and associated lactate production. Rather, lactate production appears to be related to energetic state, whatever the PO2. The model is used to describe the interaction between ATP demand and ATP supply in determining final ATP turnover.

7 CFR 3300.88 - Fees for U.S. ATP certificates.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 15 2014-01-01 2014-01-01 false Fees for U.S. ATP certificates. 3300.88 Section 3300... EQUIPMENT TO BE USED FOR SUCH CARRIAGE (ATP); INSPECTION, TESTING, AND CERTIFICATION OF SPECIAL EQUIPMENT Other Provisions § 3300.88 Fees for U.S. ATP certificates. The fee schedule for issuance of U.S. ATP...

7 CFR 3300.88 - Fees for U.S. ATP certificates.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 15 2012-01-01 2012-01-01 false Fees for U.S. ATP certificates. 3300.88 Section 3300... EQUIPMENT TO BE USED FOR SUCH CARRIAGE (ATP); INSPECTION, TESTING, AND CERTIFICATION OF SPECIAL EQUIPMENT Other Provisions § 3300.88 Fees for U.S. ATP certificates. The fee schedule for issuance of U.S. ATP...

7 CFR 3300.88 - Fees for U.S. ATP certificates.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 15 2013-01-01 2013-01-01 false Fees for U.S. ATP certificates. 3300.88 Section 3300... EQUIPMENT TO BE USED FOR SUCH CARRIAGE (ATP); INSPECTION, TESTING, AND CERTIFICATION OF SPECIAL EQUIPMENT Other Provisions § 3300.88 Fees for U.S. ATP certificates. The fee schedule for issuance of U.S. ATP...

7 CFR 3300.88 - Fees for U.S. ATP certificates.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 15 2010-01-01 2010-01-01 false Fees for U.S. ATP certificates. 3300.88 Section 3300... EQUIPMENT TO BE USED FOR SUCH CARRIAGE (ATP); INSPECTION, TESTING, AND CERTIFICATION OF SPECIAL EQUIPMENT Other Provisions § 3300.88 Fees for U.S. ATP certificates. The fee schedule for issuance of U.S. ATP...

7 CFR 3300.88 - Fees for U.S. ATP certificates.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 15 2011-01-01 2011-01-01 false Fees for U.S. ATP certificates. 3300.88 Section 3300... EQUIPMENT TO BE USED FOR SUCH CARRIAGE (ATP); INSPECTION, TESTING, AND CERTIFICATION OF SPECIAL EQUIPMENT Other Provisions § 3300.88 Fees for U.S. ATP certificates. The fee schedule for issuance of U.S. ATP...

Quantum dots electrochemical aptasensor based on three-dimensionally ordered macroporous gold film for the detection of ATP.

PubMed

Zhou, Jinjun; Huang, Haiping; Xuan, Jie; Zhang, Jianrong; Zhu, Jun-Jie

2010-10-15

A sensitive electrochemical aptasensor was successfully fabricated for the detection of adenosine triphosphate (ATP) by combining three-dimensionally ordered macroporous (3DOM) gold film and quantum dots (QDs). The 3DOM gold film was electrochemically fabricated with an inverted opal template, making the active surface area of the electrode up to 9.52 times larger than that of a classical bare flat one. 5′-thiolated ATP-binding aptamer (ABA) was first assembled onto the 3DOM gold film via sulfur–gold affinity. Then, 5′-biotinated complementary strand (BCS) was immobilized via hybridization reaction to form the DNA/DNA duplex. Since the tertiary structure of the aptamer was stabilized in the presence of target ATP, the duplex can be denatured to liberate BCS. The reaction was monitored by electrochemical stripping analysis of dissolved QDs which were bound to the residual BCS through biotin-streptavidin system. The decrease of peak current was proportional to the amount of ATP. The unique interconnected structure in 3DOM gold film along with the "built-in" preconcentration remarkably improved the sensitivity. ATP detection with high selectivity, wide linear dynamic range of 4 orders of magnitude and high sensitivity down to 0.01 nm were achieved. The results demonstrated that the novel strategy was feasible for sensitive ATP assay and provided a promising model for the detection of small molecules.

ROLES OF EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-ALPHA (TGF-A) IN MEDIATION OF DIOXIN (TCDD)-INDUCED DELAYS IN DEVELOPMENT OF THE MOUSE MAMMARY GLAND

EPA Science Inventory

Roles of Epidermal Growth Factor (EGF) and Transforming Growth Factor-alpha (TGF-a) in Mediation of Dioxin (TCDD)-Induced Delays in Development of the Mouse Mammary Gland.
Suzanne E. Fenton, Barbara Abbott, Lamont Bryant, and Angela Buckalew. U.S. EPA, NHEERL, Reproductive Tox...

Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor

PubMed Central

Conley, Jason M.

2017-01-01

Extracellular adenosine triphosphate (ATP) is a key purinergic signal that mediates cell-to-cell communication both within and between organ systems. We address the need for a robust and minimally invasive approach to measuring extracellular ATP by re-engineering the ATeam ATP sensor to be expressed on the cell surface. Using this approach, we image real-time changes in extracellular ATP levels with a sensor that is fully genetically-encoded and does not require an exogenous substrate. In addition, the sensor is ratiometric to allow for reliable quantitation of extracellular ATP fluxes. Using live-cell microscopy, we characterize sensor performance when expressed on cultured Neuro2A cells, and we measure both stimulated release of ATP and its clearance by ectonucleotidases. Thus, this proof-of-principle demonstrates a first-generation sensor to report extracellular ATP dynamics that may be useful for studying purinergic signaling in living specimens. PMID:29121644

Deletion of a unique loop in the mycobacterial F-ATP synthase γ subunit sheds light on its inhibitory role in ATP hydrolysis-driven H(+) pumping.

PubMed

Hotra, Adam; Suter, Manuel; Biuković, Goran; Ragunathan, Priya; Kundu, Subhashri; Dick, Thomas; Grüber, Gerhard

2016-05-01

The F1 FO -ATP synthase is one of the enzymes that is essential to meet the energy requirement of both the proliferating aerobic and hypoxic dormant stages of the life cycle of mycobacteria. Most F-ATP synthases consume ATP in the α3 :β3 headpiece to drive the γ subunit, which couples ATP cleavage with proton pumping in the c ring of FO via the bottom of the γ subunit. ATPase-driven H(+) pumping is latent in mycobacteria. The presence of a unique 14 amino acid residue loop of the mycobacterial γ subunit has been described and aligned in close vicinity to the c-ring loop Priya R et al. (2013) J Bioenerg Biomembr 45, 121-129 Here, we used inverted membrane vesicles (IMVs) of fast-growing Mycobacterium smegmatis and a variety of covalent and non-covalent inhibitors to characterize the ATP hydrolysis activity of the F-ATP synthase inside IMVs. These vesicles formed a platform to investigate the function of the unique mycobaterial γ loop by deleting the respective loop-encoding sequence (γ166-179 ) in the genome of M. smegmatis. ATP hydrolysis-driven H(+) pumping was observed in IMVs containing the Δγ166-179 mutant protein but not for IMVs containing the wild-type F-ATP synthase. In addition, when compared to the wild-type enzyme, IMVs containing the Δγ166-179 mutant protein showed increased ATP cleavage and lower levels of ATP synthesis, demonstrating that the loop affects ATPase activity, ATPase-driven H(+) pumping and ATP synthesis. These results further indicate that the loop may affect coupling of ATP hydrolysis and synthesis in a different mode. © 2016 Federation of European Biochemical Societies.

Effects of ultraviolet B irradiation, proinflammatory cytokines and raised extracellular calcium concentration on the expression of ATP2A2 and ATP2C1.

PubMed

Mayuzumi, N; Ikeda, S; Kawada, H; Fan, P S; Ogawa, H

2005-04-01

Darier disease (DD) and Hailey-Hailey disease (HHD) are autosomal dominantly inherited skin disorders that histologically share the characteristics of suprabasal separation and acantholysis of epidermal keratinocytes. Various mutations in the DD gene (ATP2A2) and the HHD gene (ATP2C1) (respectively encoding the calcium pumps of the sarco/endoplasmic reticulum and the Golgi apparatus) have recently been described in multiple families with DD and HHD. Mutations in ATP2A2 or ATP2C1 have been suggested as causing the conditions via the mechanism of haploinsufficiency. Ultraviolet (UV) B irradiation is thought to be an aggravating factor in both diseases. To examine the effects of various stimuli on ATP2A2 and ATP2C1 mRNA expression, and to examine the role of calcium pumps during keratinocyte differentiation. The effects of UVB irradiation, of UVB-inducible inflammatory cytokines produced by keratinocytes and of high-calcium medium (1.8 mmol L(-1) as opposed to 0.08 mmol L(-1) Ca2+) on ATP2A2 and ATP2C1 mRNA expression were quantified in cultured normal human keratinocytes using reverse transcription-polymerase chain reaction. Expression of ATP2A2 and ATP2C1 mRNA was suppressed immediately after exposure to UVB irradiation, and modulation of mRNA expression was achieved in keratinocytes cultured with proinflammatory cytokines. The mRNA expression of both genes was increased significantly after the shift to high extracellular Ca2+ concentration. The results suggest that modulation of ATP2A2 and ATP2C1 mRNA expression by UV or cytokines might contribute to the clinical presentations unique to DD and HHD, and that the controlled expression of these genes plays an important role in keratinocyte homeostasis, function and differentiation.

Mechanisms for the control of local tissue blood flow during thermal interventions: influence of temperature‐dependent ATP release from human blood and endothelial cells

PubMed Central

Chiesa, Scott T.; Trangmar, Steven J.; Ali, Leena; Lotlikar, Makrand D.; González‐Alonso, José

2017-01-01

New Findings What is the central question of this study? Skin and muscle blood flow increases with heating and decreases with cooling, but the temperature‐sensitive mechanisms underlying these responses are not fully elucidated. What is the main finding and its importance? We found that local tissue hyperaemia was related to elevations in ATP release from erythrocytes. Increasing intravascular ATP augmented skin and tissue perfusion to levels equal or above thermal hyperaemia. ATP release from isolated erythrocytes was altered by heating and cooling. Our findings suggest that erythrocytes are involved in thermal regulation of blood flow via modulation of ATP release. Local tissue perfusion changes with alterations in temperature during heating and cooling, but the thermosensitivity of the vascular ATP signalling mechanisms for control of blood flow during thermal interventions remains unknown. Here, we tested the hypotheses that the release of the vasodilator mediator ATP from human erythrocytes, but not from endothelial cells or other blood constituents, is sensitive to both increases and reductions in temperature and that increasing intravascular ATP availability with ATP infusion would potentiate thermal hyperaemia in limb tissues. We first measured blood temperature, brachial artery blood flow and plasma [ATP] during passive arm heating and cooling in healthy men and found that they increased by 3.0 ± 1.2°C, 105 ± 25 ml min−1 °C−1 and twofold, respectively, (all P < 0.05) with heating, but decreased or remained unchanged with cooling. In additional men, infusion of ATP into the brachial artery increased skin and deep tissue perfusion to levels equal or above thermal hyperaemia. In isolated erythrocyte samples exposed to different temperatures, ATP release increased 1.9‐fold from 33 to 39°C (P < 0.05) and declined by ∼50% at 20°C (P < 0.05), but no changes were observed in cultured human endothelial cells, plasma or serum samples. In

Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

PubMed Central

Barr, Travis P.; Albrecht, Phillip J.; Hou, Quanzhi; Mongin, Alexander A.; Strichartz, Gary R.; Rice, Frank L.

2013-01-01

Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease. PMID:23457608

Air-stimulated ATP release from keratinocytes occurs through connexin hemichannels.

PubMed

Barr, Travis P; Albrecht, Phillip J; Hou, Quanzhi; Mongin, Alexander A; Strichartz, Gary R; Rice, Frank L

2013-01-01

Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease.

Inhibitory effect of protopine on K(ATP) channel subunits expressed in HEK-293 cells.

PubMed

Jiang, Bo; Cao, Kun; Wang, Rui

2004-12-15

Protopine is an isoquinoline alkaloid purified from Corydalis tubers and other families of medicinal plants. The purpose of the present study was to investigate the effects of protopine on K(ATP) channels and big conductance (BKCa) channels. Protopine concentration-dependently inhibited K(ATP) channel currents in human embryonic kidney cells (HEK-293) which were cotransfected with Kir6.1 and sulfonylurea receptor 1 (SUR1) subunits, but not that with Kir6.1 cDNA transfection alone. At 25 muM, protopine reversibly decreased Kir6.1/SUR1 currents densities from -17.4+/-3 to -13.2+/-2.4 pA/pF at -60 mV (n=5, P<0.05). The heterologously expressed mSlo-encoded BK(Ca) channel currents in HEK-293 cells were not affected by protopine (25 muM), although iberiotoxin (100 nM) significantly inhibited the expressed BK(Ca) currents (n=5, P<0.05). In summary, protopine selectively inhibited K(ATP) channels by targeting on SUR1 subunit. This discovery may help design specific agents to selectively modulate the function of Kir6.1/SUR1 channel complex and facilitate the understanding of the structure-function relationship of specific subtype of K(ATP) channels.

Synergistic cellular effects including mitochondrial destabilization, autophagy and apoptosis following low-level exposure to a mixture of lipophilic persistent organic pollutants.

PubMed

Rainey, Nathan E; Saric, Ana; Leberre, Alexandre; Dewailly, Etienne; Slomianny, Christian; Vial, Guillaume; Zeliger, Harold I; Petit, Patrice X

2017-07-05

Humans are exposed to multiple exogenous environmental pollutants. Many of these compounds are parts of mixtures that can exacerbate harmful effects of the individual mixture components. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is primarily produced via industrial processes including incineration and the manufacture of herbicides. Both endosulfan and TCDD are persistent organic pollutants which elicit cytotoxic effects by inducing reactive oxygen species generation. Sublethal concentrations of mixtures of TCDD and endosulfan increase oxidative stress, as well as mitochondrial homeostasis disruption, which is preceded by a calcium rise and, in fine, induce cell death. TCDD+Endosulfan elicit a complex signaling sequence involving reticulum endoplasmic destalilization which leads to Ca 2+ rise, superoxide anion production, ATP drop and late NADP(H) depletion associated with a mitochondrial induced apoptosis concomitant early autophagic processes. The ROS scavenger, N-acetyl-cysteine, blocks both the mixture-induced autophagy and death. Calcium chelators act similarly and mitochondrially targeted anti-oxidants also abrogate these effects. Inhibition of the autophagic fluxes with 3-methyladenine, increases mixture-induced cell death. These findings show that subchronic doses of pollutants may act synergistically. They also reveal that the onset of autophagy might serve as a protective mechanism against ROS-triggered cytotoxic effects of a cocktail of pollutants in Caco-2 cells and increase their tumorigenicity.

Structural Characterization of Two Metastable ATP-Bound States of P-Glycoprotein

PubMed Central

O’Mara, Megan L.; Mark, Alan E.

2014-01-01

ATP Binding Cassette (ABC) transporters couple the binding and hydrolysis of ATP to the transport of substrate molecules across the membrane. The mechanism by which ATP binding and/or hydrolysis drives the conformational changes associated with substrate transport has not yet been characterized fully. Here, changes in the conformation of the ABC export protein P-glycoprotein on ATP binding are examined in a series of molecular dynamics simulations. When one molecule of ATP is placed at the ATP binding site associated with each of the two nucleotide binding domains (NBDs), the membrane-embedded P-glycoprotein crystal structure adopts two distinct metastable conformations. In one, each ATP molecule interacts primarily with the Walker A motif of the corresponding NBD. In the other, the ATP molecules interacts with both Walker A motif of one NBD and the Signature motif of the opposite NBD inducing the partial dimerization of the NBDs. This interaction is more extensive in one of the two ATP binding site, leading to an asymmetric structure. The overall conformation of the transmembrane domains is not altered in either of these metastable states, indicating that the conformational changes associated with ATP binding observed in the simulations in the absence of substrate do not lead to the outward-facing conformation and thus would be insufficient in themselves to drive transport. Nevertheless, the metastable intermediate ATP-bound conformations observed are compatible with a wide range of experimental cross-linking data demonstrating the simulations do capture physiologically important conformations. Analysis of the interaction between ATP and its cofactor Mg2+ with each NBD indicates that the coordination of ATP and Mg2+ differs between the two NBDs. The role structural asymmetry may play in ATP binding and hydrolysis is discussed. Furthermore, we demonstrate that our results are not heavily influenced by the crystal structure chosen for initiation of the simulations

Psychophysical evaluation of the image quality of a dynamic flat-panel digital x-ray image detector using the threshold contrast detail detectability (TCDD) technique

NASA Astrophysics Data System (ADS)

Davies, Andrew G.; Cowen, Arnold R.; Bruijns, Tom J. C.

1999-05-01

We are currently in an era of active development of the digital X-ray imaging detectors that will serve the radiological communities in the new millennium. The rigorous comparative physical evaluations of such devices are therefore becoming increasingly important from both the technical and clinical perspectives. The authors have been actively involved in the evaluation of a clinical demonstration version of a flat-panel dynamic digital X-ray image detector (or FDXD). Results of objective physical evaluation of this device have been presented elsewhere at this conference. The imaging performance of FDXD under radiographic exposure conditions have been previously reported, and in this paper a psychophysical evaluation of the FDXD detector operating under continuous fluoroscopic conditions is presented. The evaluation technique employed was the threshold contrast detail detectability (TCDD) technique, which enables image quality to be measured on devices operating in the clinical environment. This approach addresses image quality in the context of both the image acquisition and display processes, and uses human observers to measure performance. The Leeds test objects TO[10] and TO[10+] were used to obtain comparative measurements of performance on the FDXD and two digital spot fluorography (DSF) systems, one utilizing a Plumbicon camera and the other a state of the art CCD camera. Measurements were taken at a range of detector entrance exposure rates, namely 6, 12, 25 and 50 (mu) R/s. In order to facilitate comparisons between the systems, all fluoroscopic image processing such as noise reduction algorithms, were disabled during the experiments. At the highest dose rate FDXD significantly outperformed the DSF comparison systems in the TCDD comparisons. At 25 and 12 (mu) R/s all three-systems performed in an equivalent manner and at the lowest exposure rate FDXD was inferior to the two DSF systems. At standard fluoroscopic exposures, FDXD performed in an equivalent

Chloroplast ATP Synthase Modulation of the Thylakoid Proton Motive Force: Implications for Photosystem I and Photosystem II Photoprotection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kanazawa, Atsuko; Ostendorf, Elisabeth; Kohzuma, Kaori

In wild type plants, decreasing CO 2 lowers the activity of the chloroplast ATP synthase, slowing proton efflux from the thylakoid lumen resulting in buildup of thylakoid proton motive force (pmf). The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b 6f complex. Here in this paper, we show that the cfq mutant of Arabidopsis, harboring single point mutation in its γ-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO 2. The increased thylakoid proton conductivitymore » (g H +) in cfq results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO 2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage.« less

Chloroplast ATP Synthase Modulation of the Thylakoid Proton Motive Force: Implications for Photosystem I and Photosystem II Photoprotection

DOE PAGES

Kanazawa, Atsuko; Ostendorf, Elisabeth; Kohzuma, Kaori; ...

2017-05-03

In wild type plants, decreasing CO 2 lowers the activity of the chloroplast ATP synthase, slowing proton efflux from the thylakoid lumen resulting in buildup of thylakoid proton motive force (pmf). The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b 6f complex. Here in this paper, we show that the cfq mutant of Arabidopsis, harboring single point mutation in its γ-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO 2. The increased thylakoid proton conductivitymore » (g H +) in cfq results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO 2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage.« less

Three-Dimensional Structures Reveal Multiple ADP/ATP Binding Modes

DOE Office of Scientific and Technical Information (OSTI.GOV)

C Simmons; C Magee; D Smith

The creation of synthetic enzymes with predefined functions represents a major challenge in future synthetic biology applications. Here, we describe six structures of de novo proteins that have been determined using protein crystallography to address how simple enzymes perform catalysis. Three structures are of a protein, DX, selected for its stability and ability to tightly bind ATP. Despite the addition of ATP to the crystallization conditions, the presence of a bound but distorted ATP was found only under excess ATP conditions, with ADP being present under equimolar conditions or when crystallized for a prolonged period of time. A bound ADPmore » cofactor was evident when Asp was substituted for Val at residue 65, but ATP in a linear configuration is present when Phe was substituted for Tyr at residue 43. These new structures complement previously determined structures of DX and the protein with the Phe 43 to Tyr substitution [Simmons, C. R., et al. (2009) ACS Chem. Biol. 4, 649-658] and together demonstrate the multiple ADP/ATP binding modes from which a model emerges in which the DX protein binds ATP in a configuration that represents a transitional state for the catalysis of ATP to ADP through a slow, metal-free reaction capable of multiple turnovers. This unusual observation suggests that design-free methods can be used to generate novel protein scaffolds that are tailor-made for catalysis.« less

Myosin Motors: The Chemical Restraints Imposed by ATP

NASA Astrophysics Data System (ADS)

Rayment, I.; Allingham, J.

Most molecular motors use ATP. This is not surprising since this is the universal currency of energy for most of life's processes. The question to be addressed in this chapter is how does the fundamental chemistry of ATP hydrolysis in- fluence the observed organization of linear molecular motors seen today with a focus on myosin. This chapter is written to ask in simple terms what can be gained by reconsidering the chemistry of ATP hydrolysis.

The chloroplast ATP synthase features the characteristic redox regulation machinery.

PubMed

Hisabori, Toru; Sunamura, Ei-Ichiro; Kim, Yusung; Konno, Hiroki

2013-11-20

Regulation of the activity of the chloroplast ATP synthase is largely accomplished by the chloroplast thioredoxin system, the main redox regulation system in chloroplasts, which is directly coupled to the photosynthetic reaction. We review the current understanding of the redox regulation system of the chloroplast ATP synthase. The thioredoxin-targeted portion of the ATP synthase consists of two cysteines located on the central axis subunit γ. The redox state of these two cysteines is under the influence of chloroplast thioredoxin, which directly controls rotation during catalysis by inducing a conformational change in this subunit. The molecular mechanism of redox regulation of the chloroplast ATP synthase has recently been determined. Regulation of the activity of the chloroplast ATP synthase is critical in driving efficiency into the ATP synthesis reaction in chloroplasts. The molecular architecture of the chloroplast ATP synthase, which confers redox regulatory properties requires further investigation, in light of the molecular structure of the enzyme complex as well as the physiological significance of the regulation system.

Effects of vitamin E on reproductive hormones and testis structure in chronic dioxin-treated mice.

PubMed

Yin, Hai-Ping; Xu, Jian-Ping; Zhou, Xian-Qing; Wang, Ying

2012-03-01

The purpose of this study was to investigate the effects of vitamin E on reproductive hormones and testis structure in mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Five experimental groups of a combination of TCDD and vitamin E were designed as follows: 0 ng/kg/d and 0 mg/kg/d (control group), 100 ng/kg/d and 0 mg/kg/d (Group I), 100 ng/kg/d and 20 mg/kg/d (Group II), 100 ng/kg/d and 100 mg/kg/d (Group III), and 100 ng/kg/d and 500 mg/kg/d (Group IV) respectively. Vitamin E and TCDD were given by oral gavage for 7 weeks. The results demonstrated that TCDD decreased the levels of brain gonadotropin releasing hormone (GnRH), testis luteinizing hormone (LH) and follicle stimulating hormone (FSH), serum testosterone and testis spermatozoa number, and damaged testis structure. Vitamin E at 20 mg/kg alleviated the decrease of GnRH; vitamin E at 20, 100, and 500 mg/kg antagonized the decline of LH and FSH; vitamin E at 20 and 100 mg/kg reversed the decrease of testosterone and spermatozoa number; and vitamin E at 100 mg/kg decreased the damage of the testis structure caused by TCDD. The results indicate that vitamin E antagonizes the reproductive endocrine toxicity and alleviates the changes in testicular structure caused by TCDD.

UTILIZATION OF A PBPK MODEL TO PREDICT THE DISTRIBUTION OF 2, 3, 7-8 TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN HUMANS DURING CRITICAL WINDOWS OF DEVELOPMENT

EPA Science Inventory

Utilization of A PBPK model to predict the distribution of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in humans during critical windows of development.
C Emond1, MJ DeVito2 and LS Birnbaum2
1National Research Council, US EPA, ORD, NHEERL, (ETD, PK), RTP, NC, 27711, USA 2 US...

Neurokinin B potentiates ATP-activated currents in rat DRG neurons.

PubMed

Wang, M J; Xiong, S H; Li, Z W

2001-12-27

This study aimed to explore whether NKB could modulate the responses mediated by ATP receptor (P2X purinoceptor). Whole-cell patch clamp and repatch experiments were performed on cultured rat DRG neurons. The majority of neurons examined were sensitive both to ATP and to NKB (77.1%, 54/70). NKB preapplied could potentiate ATP-activated currents (I(ATP)) markedly; this effect was concentration-dependent and could be blocked by SR 142801, an NK3 receptor antagonist. Preapplication of 0.001, 0.01, 0.1 and 1.0 microM NKB increased ATP-activated currents by 55.1+/-18.8, 75.2+/-17.4, 84.1+/-18.8 and 81.0+/-21.7%, respectively. The concentration-response curves for ATP with and without preapplication of NKB show that: (1) preapplication of NKB shifted the curve upwards; (2) the maximal amplitude of I(ATP) with NKB preapplication increased by 78.5%, while the threshold value remained unchanged; (3) the EC(50) values of the two curves were very close (44 vs. 42 microM). Intracellular dialysis of H-7 by using repatch clamp technique could block the potentiation of I(ATP) by NKB. It suggests that this potentiating effect was caused by phosphorylation of ATP receptor, which resulted from the activation of G protein coupled NK3 receptor and consequential intracellular signal transduction cascade.

Inhibition of ATP Hydrolysis by Thermoalkaliphilic F1Fo-ATP Synthase Is Controlled by the C Terminus of the ɛ Subunit

PubMed Central

Keis, Stefanie; Stocker, Achim; Dimroth, Peter; Cook, Gregory M.

2006-01-01

The F1Fo-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile Bacillus sp. strain TA2.A1, this activity is intrinsic to the F1 moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F1 complexes from this thermoalkaliphile. Like the native F1Fo-ATP synthase, the recombinant TA2F1 was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide. To determine if the C-terminal domain of the ɛ subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F1 mutant with either a truncated ɛ subunit [i.e., TA2F1(ɛΔC)] or substitution of basic residues in the second α-helix of ɛ with nonpolar alanines [i.e., TA2F1(ɛ6A)] was constructed. Both mutants showed ATP hydrolysis activity at low and high concentrations of ATP. Treatment of the purified F1Fo-ATP synthase and TA2F1(ɛWT) complex with proteases revealed that the ɛ subunit was resistant to proteolytic digestion. In contrast, the ɛ subunit of TA2F1(ɛ6A) was completely degraded by trypsin, indicating that the C-terminal arm was in a conformation where it was no longer protected from proteolytic digestion. In addition, ATPase activity was not further activated by protease treatment when compared to the untreated control, supporting the observation that ɛ was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the ɛ subunit in the entire holoenzyme, we reconstituted recombinant TA2F1 complexes with F1-stripped native membranes of strain TA2.A1. The reconstituted TA2FoF1(ɛWT) was blocked in ATP hydrolysis and exhibited low levels of ATP-driven proton pumping consistent with the F1Fo-ATP synthase in native membranes. Reconstituted TA2FoF1(ɛ6A) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the �

ATP7B detoxifies silver in ciliated airway epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ibricevic, Aida, E-mail: aidaibricevic@hotmail.co; Brody, Steven L., E-mail: sbrody@dom.wustl.ed; Youngs, Wiley J., E-mail: youngs@uakron.ed

2010-03-15

Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compoundsmore » but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B{sup -/-} mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag{sup +}/Cu{sup +} transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.« less

Emodin Inhibits ATP-Induced Proliferation and Migration by Suppressing P2Y Receptors in Human Lung Adenocarcinoma Cells.

PubMed

Wang, Xia; Li, Long; Guan, Ruijuan; Zhu, Danian; Song, Nana; Shen, Linlin

2017-01-01

Extracellular ATP performs multiple important functions via activation of P2 receptors on the cell surface. P2Y receptors play critical roles in ATP evoked response in human lung adenocarcinoma cells (A549 cells). Emodin is an anthraquinone derivative originally isolated from Chinese rhubarb, possesses anticancer properties. In this study we examined the inhibiting effects of emodin on proliferation, migration and epithelial-mesenchymal transition (EMT) by suppressing P2Y receptors-dependent Ca2+ increase and nuclear factor-κB (NF-KB) signaling in A549 cells. A549 cells were pretreated with emodin before stimulation with ATP for the indicated time. Then, intracellular Ca2+ concentration ([Ca2+]i) was measured by Fluo-8/AM staining. Cell proliferation and cell cycle progression were tested by CCK8 assay and flow cytometry In addition, wound healing and western blot were performed to determine cell migration and related protein levels (Bcl-2, Bax, claudin-1, NF-κB). Emodin blunted ATP/UTP-induced increase of [Ca2+]i and cell proliferation concentration-dependently Meanwhile, it decreased ATP-induced cells accumulation in the S phase. Furthermore, emodin altered protein abundance of Bcl-2, Bax and claudin-1 and attenuated EMT caused by ATP. Such ATP-induced cellular reactions were also inhibited by a nonselective P2Y receptors antagonist, suramin, in a similar way to emodin. Besides, emodin could inhibit activation of NF-κB, thus suppressed ATP-induced proliferation, migration and EMT. Our results demonstrated that emodin inhibits ATP-induced proliferation, migration, EMT by suppressing P2Y receptors-mediated [Ca2+]i increase and NF-κB signaling in A549 cells. © 2017 The Author(s). Published by S. Karger AG, Basel.

Functional analysis of mutations in the ATP loop of the Wilson disease copper transporter, ATP7B.

PubMed

Luoma, Leiah M; Deeb, Taha M M; Macintyre, Georgina; Cox, Diane W

2010-05-01

Wilson disease (WND) is an autosomal recessive disorder resulting from mutation of ATP7B. Transport of copper by ATP7B from the trans-Golgi of hepatocytes into apical membrane-trafficked vesicles for excretion in the bile is the major means of copper elimination from the body. Although copper is an essential nutrient, homeostasis must be carefully maintained. If homeostasis is disrupted, copper can accumulate within the liver, kidney, cornea, and/or brain. The range of organs affected leads to clinical heterogeneity and difficulty in WND diagnosis. Sequencing of ATP7B is an important adjunct for diagnosis but has led to the discovery of many novel missense variants. Although prediction programs are available, functional characterization is essential for determining the consequence of novel variants. We have tested 12 missense variants localized to the ATP loop of ATP7B and compared three predictive programs (SIFT, PolyPhen, and Align-GVGD). We found p.L1043P, p.G1000R, p.G1101R, p.I1102T, p.V1239G, and p.D1267V deleterious; p.G1176E and p.G1287S intermediate; p.E1173G temperature sensitive; p.T991M and p.I1148T mild; and p.R1228T functioning as wild type. We found that SIFT most often agreed with functional data (92%), compared with PolyPhen (83%) and Align-GVGD (67%). We conclude that variants found to negatively affect function likely contribute to the WND phenotype in patients. (c) 2010 Wiley-Liss, Inc.

2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in yellow perch (Perca flavescens)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spitsbergen, J.M.; Kleeman, J.M.; Peterson, R.E.

1988-01-01

Growth, mortality and morphologic lesions in juvenile, hatchery-reared yellow perch, Perca flavescens, were studied after treatment with graded single doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 1-125 ..mu..g/kg, intraperitoneally). TCDD doses of 25 and 125 ..mu..g/kg caused 95% mortality by 28 d after treatment, without decreasing body weight. A TCDD dose of 5 ..mu..g/kg resulted in progressive loss of body weight with cumulative mortality of 80% by 80 d posttreatment. Periodic handling stress did not affect the time course of mortality or cumulative percent lethality in TCDD-treated perch. Fin necrosis, petechial cutaneous hemorrhage, and ascites occurred in perch treated with 5 ..mu..g/kg ormore » more of TCDD. Thymic atrophy, decreased hematopoiesis in the head kidney, fibrinous pericarditis, focal myocardial necrosis, submucosal gastric edema, and hyperplasia of the epithelium of gill filaments and lamellae occurred in perch dosed with 25 or 125 ..mu..g/kg. Dose-related splenic lymphoid depletion occurred in perch receiving 5 ..mu..g/kg or more TCDD, and hepatocycte lipidosis occurred in groups treated with doses of 1 ..mu..g/kg or more TCDD. Thus yellow perch are as responsive to the acute toxic effects of TCDD as some of the more sensitive mammalian species, and neither loss of body weight nor histologic lesions in TCDD-treated perch are sufficient to explain mortality.« less

Fast transient currents in Na,K-ATPase induced by ATP concentration jumps from the P3-[1-(3',5'-dimethoxyphenyl)-2-phenyl-2-oxo]ethyl ester of ATP.

PubMed Central

Sokolov, V S; Apell, H J; Corrie, J E; Trentham, D R

1998-01-01

Electrogenic ion transport by Na,K-ATPase was investigated by analysis of transient currents in a model system of protein-containing membrane fragments adsorbed to planar lipid bilayers. Sodium transport was triggered by ATP concentration jumps in which ATP was released from an inactive precursor by an intense near-UV light flash. The method has been used previously with the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP), from which the relatively slow rate of ATP release limits analysis of processes in the pump mechanism controlled by rate constants greater than 100 s(-1) at physiological pH. Here Na,K-ATPase was reinvestigated using the P3-[1-(3,5-dimethoxyphenyl)-2-phenyl-2-oxo]ethyl ester of ATP (DMB-caged ATP), which has an ATP release rate of >10(5) s(-1). Under otherwise identical conditions, photorelease of ATP from DMB-caged ATP showed faster kinetics of the transient current compared to that from NPE-caged ATP. With DMB-caged ATP, transient currents had rate profiles that were relatively insensitive to pH and the concentration of caged compound. Rate constants of ATP binding and of the E1 to E2 conformational change were compatible with earlier studies. Rate constants of enzyme phosphorylation and ADP-dependent dephosphorylation were 600 s(-1) and 1.5 x 10(6) M(-1) s(-1), respectively, at pH 7.2 and 22 degrees C. PMID:9591656

Copper(II) ions interactions in the systems with triamines and ATP. Potentiometric and spectroscopic studies.

PubMed

Hoffmann, S K; Goslar, J; Bregier-Jarzebowska, R; Gasowska, A; Zalewska, A; Lomozik, L

2017-12-01

The mode of interaction and thermodynamic stability of complexes formed in binary and ternary Cu(II)/ATP/triamines systems were studied using potentiometric and spectroscopic (NMR, EPR, UV-Vis) methods. It was found that in binary metal-free systems ATP/H x PA species are formed (PA: Spd=spermidine or 3,3-tri=1,7-diamino-4-azaheptane) where the phosphate groups from nucleotides are preferred negative centers and protonated amine groups of amines are positive centers of reaction. In the ternary systems Cu/ATP/H x (PA) as well as Cu/(ATP)(PA) species are formed. The type of the formed Cu(II) complexes depends on pH of the solution. For a low pH value the complexation appears between Cu(II) and ATP molecules via oxygen atoms of phosphate groups. For a very high pH value, where ATP is hydrolyzed, the Cu(II) ions are bound to the nitrogen atoms of polyamine molecules. We did not detect any direct coordination of the N7 nitrogen atom of adenosine to Cu(II) ions. It means that the CuN7 interaction is an indirect type and can be due to noncovalent interplay including water molecule. EPR studies were performed at glassy state (77K) after a fast freezing both for binary and ternary systems. The glassy state EPR spectra do not reflect species identified in titration studies indicating significant effect of rapid temperature decrease on equilibrium of Cu(II) complexes. We propose the molecular structure of all the studied complexes at the glassy state deduced from EPR and optical spectroscopy results. Copyright © 2017 Elsevier Inc. All rights reserved.

Regulation of ATP production: dependence on calcium concentration and respiratory state.

PubMed

Fink, Brian D; Bai, Fan; Yu, Liping; Sivitz, William I

2017-08-01

Nanomolar free calcium enhances oxidative phosphorylation. However, the effects over a broad concentration range, at different respiratory states, or on specific energy substrates are less clear. We examined the action of varying [Ca 2+ ] over respiratory states ranging 4 to 3 on skeletal muscle mitochondrial respiration, potential, ATP production, and H 2 O 2 production using ADP recycling to clamp external [ADP]. Calcium at 450 nM enhanced respiration in mitochondria energized by the complex I substrates, glutamate/malate (but not succinate), at [ADP] of 4-256 µM, but more substantially at intermediate respiratory states and not at all at state 4. Using varied [Ca 2+ ], we found that the stimulatory effects on respiration and ATP production were most prominent at nanomolar concentrations, but inhibitory at 10 µM or higher. ATP production decreased more than respiration at 10 µM calcium. However, potential continued to increase up to 10 µM; suggesting a calcium-induced inability to utilize potential for phosphorylation independent of opening of the mitochondrial permeability transition pore (MTP). This effect of 10 µM calcium was confirmed by direct determination of ATP production over a range of potential created by differing substrate concentrations. Consistent with past reports, nanomolar [Ca 2+ ] had a stimulatory effect on utilization of potential for phosphorylation. Increasing [Ca 2+ ] was positively and continuously associated with H 2 O 2 production. In summary, the stimulatory effect of calcium on mitochondrial function is substrate dependent and most prominent over intermediate respiratory states. Calcium stimulates or inhibits utilization of potential for phosphorylation dependent on concentration with inhibition at higher concentration independent of MTP opening.

The metal chaperone Atox1 regulates the activity of the human copper transporter ATP7B by modulating domain dynamics.

PubMed

Yu, Corey H; Yang, Nan; Bothe, Jameson; Tonelli, Marco; Nokhrin, Sergiy; Dolgova, Natalia V; Braiterman, Lelita; Lutsenko, Svetlana; Dmitriev, Oleg Y

2017-11-03

The human transporter ATP7B delivers copper to the biosynthetic pathways and maintains copper homeostasis in the liver. Mutations in ATP7B cause the potentially fatal hepatoneurological disorder Wilson disease. The activity and intracellular localization of ATP7B are regulated by copper, but the molecular mechanism of this regulation is largely unknown. We show that the copper chaperone Atox1, which delivers copper to ATP7B, and the group of the first three metal-binding domains (MBD1-3) are central to the activity regulation of ATP7B. Atox1-Cu binding to ATP7B changes domain dynamics and interactions within the MBD1-3 group and activates ATP hydrolysis. To understand the mechanism linking Atox1-MBD interactions and enzyme activity, we have determined the MBD1-3 conformational space using small angle X-ray scattering and identified changes in MBD dynamics caused by apo -Atox1 and Atox1-Cu by solution NMR. The results show that copper transfer from Atox1 decreases domain interactions within the MBD1-3 group and increases the mobility of the individual domains. The N-terminal segment of MBD1-3 was found to interact with the nucleotide-binding domain of ATP7B, thus physically coupling the domains involved in copper binding and those involved in ATP hydrolysis. Taken together, the data suggest a regulatory mechanism in which Atox1-mediated copper transfer activates ATP7B by releasing inhibitory constraints through increased freedom of MBD1-3 motions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A Therapeutic Connection between Dietary Phytochemicals and ATP Synthase.

PubMed

Ahmad, Zulfiqar; Hassan, Sherif S; Azim, Sofiya

2017-11-20

For centuries, phytochemicals have been used to prevent and cure multiple health ailments. Phytochemicals have been reported to have antioxidant, antidiabetic, antitussive, antiparasitic, anticancer, and antimicrobial properties. Generally, the therapeutic use of phytochemicals is based on tradition or word of mouth with few evidence-based studies. Moreover, molecular level interactions or molecular targets for the majority of phytochemicals are unknown. In recent years, antibiotic resistance by microbes has become a major healthcare concern. As such, the use of phytochemicals with antimicrobial properties has become pertinent. Natural compounds from plants, vegetables, herbs, and spices with strong antimicrobial properties present an excellent opportunity for preventing and combating antibiotic resistant microbial infections. ATP synthase is the fundamental means of cellular energy. Inhibition of ATP synthase may deprive cells of required energy leading to cell death, and a variety of dietary phytochemicals are known to inhibit ATP synthase. Structural modifications of phytochemicals have been shown to increase the inhibitory potency and extent of inhibition. Sitedirected mutagenic analysis has elucidated the binding site(s) for some phytochemicals on ATP synthase. Amino acid variations in and around the phytochemical binding sites can result in selective binding and inhibition of microbial ATP synthase. In this review, the therapeutic connection between dietary phytochemicals and ATP synthase is summarized based on the inhibition of ATP synthase by dietary phytochemicals. Research suggests selective targeting of ATP synthase is a valuable alternative molecular level approach to combat antibiotic resistant microbial infections. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A Therapeutic Connection between Dietary Phytochemicals and ATP Synthase

PubMed Central

Ahmad, Zulfiqar; Hassan, Sherif S.; Azim, Sofiya

2017-01-01

For centuries, phytochemicals have been used to prevent and cure multiple health ailments. Phytochemicals have been reported to have antioxidant, antidiabetic, antitussive, antiparasitic, anticancer, and antimicrobial properties. Generally, the therapeutic use of phy-tochemicals is based on tradition or word of mouth with few evidence-based studies. Moreo-ver, molecular level interactions or molecular targets for the majority of phytochemicals are unknown. In recent years, antibiotic resistance by microbes has become a major healthcare concern. As such, the use of phytochemicals with antimicrobial properties has become perti-nent. Natural compounds from plants, vegetables, herbs, and spices with strong antimicrobial properties present an excellent opportunity for preventing and combating antibiotic resistant microbial infections. ATP synthase is the fundamental means of cellular energy. Inhibition of ATP synthase may deprive cells of required energy leading to cell death, and a variety of die-tary phytochemicals are known to inhibit ATP synthase. Structural modifications of phyto-chemicals have been shown to increase the inhibitory potency and extent of inhibition. Site-directed mutagenic analysis has elucidated the binding site(s) for some phytochemicals on ATP synthase. Amino acid variations in and around the phytochemical binding sites can re-sult in selective binding and inhibition of microbial ATP synthase. In this review, the therapeu-tic connection between dietary phytochemicals and ATP synthase is summarized based on the inhibition of ATP synthase by dietary phytochemicals. Research suggests selective target-ing of ATP synthase is a valuable alternative molecular level approach to combat antibiotic resistant microbial infections. PMID:28831918

Dietary protein deficiency reduces lysosomal and nonlysosomal ATP-dependent proteolysis in muscle

NASA Technical Reports Server (NTRS)

Tawa, N. E. Jr; Kettelhut, I. C.; Goldberg, A. L.

1992-01-01

When rats are fed a protein deficient (PD) diet for 7 days, rates of proteolysis in skeletal muscle decrease by 40-50% (N. E. Tawa, Jr., and A. L. Goldberg. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E317-325, 1992). To identify the underlying biochemical adaptations, we measured different proteolytic processes in incubated muscles. The capacity for intralysosomal proteolysis, as shown by sensitivity to methylamine or lysosomal protease inhibitors, fell 55-75% in muscles from PD rats. Furthermore, extracts of muscles of PD rats showed 30-70% lower activity of many lysosomal proteases, including cathepsins B, H, and C, and carboxypeptidases A and C, as well as other lysosomal hydrolases. The fall in cathepsin B and proteolysis was evident by 3 days on the PD diet, and both returned to control levels 3 days after refeeding of the normal diet. In muscles maintained under optimal conditions, 80-90% of protein breakdown occurs by nonlysosomal pathways. In muscles of PD rats, this ATP-dependent process was also 40-60% slower. Even though overall proteolysis decreased in muscles of PD rats, their capacity for Ca(2+)-dependent proteolysis increased (by 66%), as did the activity of the calpains (+150-250%). Thus the lysosomal and the ATP-dependent processes decrease coordinately and contribute to the fall in muscle proteolysis in PD animals.

Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg2+ in cell respiration

PubMed Central

Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

2014-01-01

In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg2+ concentrations must be considered as well. Here we developed in vivo/in vitro techniques using 31P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg2+ concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg2+ in the mitochondrial matrix, where [Mg2+] is tenfold higher. In contrast, owing to a much higher affinity for Mg2+, ATP is mostly complexed by Mg2+ in both compartments. Mg2+ starvation used to alter cytosolic and mitochondrial [Mg2+] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg2+ concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis. PMID:25313036

Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

PubMed

Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

2014-10-28

In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

Hypo-and hyperthyroidism affect the ATP, ADP and AMP hydrolysis in rat hippocampal and cortical slices.

PubMed

Bruno, Alessandra Nejar; Diniz, Gabriela Placoná; Ricachenevsky, Felipe Klein; Pochmann, Daniela; Bonan, Carla Denise; Barreto-Chaves, Maria Luiza M; Sarkis, João José Freitas

2005-05-01

The presence of severe neurological symptoms in thyroid diseases has highlighted the importance of thyroid hormones in the normal functioning of the mature brain. Since, ATP is an important excitatory neurotransmitter and adenosine acts as a neuromodulatory structure inhibiting neurotransmitters release in the central nervous system (CNS), the ectonucleotidase cascade that hydrolyzes ATP to adenosine, is also involved in the control of brain functions. Thus, we investigated the influence of hyper-and hypothyroidism on the ATP, ADP and AMP hydrolysis in hippocampal and cortical slices from adult rats. Hyperthyroidism was induced by daily injections of l-thyroxine (T4) 25 microg/100 g body weight, for 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) added to their drinking water for 14 days. Hypothyroid rats were hormonally replaced by daily injections of T4 (5 microg/100 g body weight, i.p.) for 5 days. Hyperthyroidism significantly inhibited the ATP, ADP and AMP hydrolysis in hippocampal slices. In brain cortical slices, hyperthyroidism inhibited the AMP hydrolysis. In contrast, hypothyroidism increased the ATP, ADP and AMP hydrolysis in both hippocampal and cortical slices and these effects were reverted by T4 replacement. Furthermore, hypothyroidism increased the expression of NTPDase1 and 5'-nucleotidase, whereas hyperthyroidism decreased the expression of 5'-nucleotidase in hippocampus of adult rats. These findings demonstrate that thyroid disorders may influence the enzymes involved in the complete degradation of ATP to adenosine and possibly affects the responses mediated by adenine nucleotides in the CNS of adult rats.

Uridylylation of Herbaspirillum seropedicae GlnB and GlnK proteins is differentially affected by ATP, ADP and 2-oxoglutarate in vitro.

PubMed

Bonatto, Ana C; Souza, Emanuel M; Oliveira, Marco A S; Monteiro, Rose A; Chubatsu, Leda S; Huergo, Luciano F; Pedrosa, Fábio O

2012-08-01

PII are signal-transducing proteins that integrate metabolic signals and transmit this information to a large number of proteins. In proteobacteria, PII are modified by GlnD (uridylyltransferase/uridylyl-removing enzyme) in response to the nitrogen status. The uridylylation/deuridylylation cycle of PII is also regulated by carbon and energy signals such as ATP, ADP and 2-oxoglutarate (2-OG). These molecules bind to PII proteins and alter their tridimensional structure/conformation and activity. In this work, we determined the effects of ATP, ADP and 2-OG levels on the in vitro uridylylation of Herbaspirillum seropedicae PII proteins, GlnB and GlnK. Both proteins were uridylylated by GlnD in the presence of ATP or ADP, although the uridylylation levels were higher in the presence of ATP and under high 2-OG levels. Under excess of 2-OG, the GlnB uridylylation level was higher in the presence of ATP than with ADP, while GlnK uridylylation was similar with ATP or ADP. Moreover, in the presence of ADP/ATP molar ratios varying from 10/1 to 1/10, GlnB uridylylation level decreased as ADP concentration increased, whereas GlnK uridylylation remained constant. The results suggest that uridylylation of both GlnB and GlnK responds to 2-OG levels, but only GlnB responds effectively to variation on ADP/ATP ratio.

The Chloroplast ATP Synthase Features the Characteristic Redox Regulation Machinery

PubMed Central

Sunamura, Ei-Ichiro; Kim, Yusung; Konno, Hiroki

2013-01-01

Abstract Significance: Regulation of the activity of the chloroplast ATP synthase is largely accomplished by the chloroplast thioredoxin system, the main redox regulation system in chloroplasts, which is directly coupled to the photosynthetic reaction. We review the current understanding of the redox regulation system of the chloroplast ATP synthase. Recent Advances: The thioredoxin-targeted portion of the ATP synthase consists of two cysteines located on the central axis subunit γ. The redox state of these two cysteines is under the influence of chloroplast thioredoxin, which directly controls rotation during catalysis by inducing a conformational change in this subunit. The molecular mechanism of redox regulation of the chloroplast ATP synthase has recently been determined. Critical Issues: Regulation of the activity of the chloroplast ATP synthase is critical in driving efficiency into the ATP synthesis reaction in chloroplasts. Future Directions: The molecular architecture of the chloroplast ATP synthase, which confers redox regulatory properties requires further investigation, in light of the molecular structure of the enzyme complex as well as the physiological significance of the regulation system. Antioxid. Redox Signal. 19, 1846–1854. PMID:23145525

Functional studies of ATP sulfurylase from Penicillium chrysogenum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seubert, P.A.

1985-01-01

ATP sulfurylase from Penicillium chrysogenum has a specific activity (V/sub max/) of 6-7 units x mg protein/sup -1/ determined with the physiological substrates of MgATP and SO/sub 4//sup 2 -/ and assayed by (A) initial velocity measurements with APS kinase and inorganic pyrophosphatase present and (B) analysis of nonlinear reaction progress curves. The fact both assays give the same results show the intrinsic activity of ATP sulfurylase is much higher than previously reported. In initial velocity dead-end inhibition studies, the sulfate analog S/sub 2/O/sub 3//sup 2 -/ is a competitive inhibitor of SO/sub 42/..sqrt.. and a noncompetitive inhibitor of MgATP.more » Monovalent oxyanions such as NO/sub 3//sup -/, ClO/sub 3//sup -/, ClO/sub 4//sup -/, and FSO/sub 3//sup -/ behave as uncompetitive inhibitors of MgATP and thus seem not to be true sulfate analogs. The reverse reaction was assayed by the pyrophosphate dependent release of /sup 35/SO/sub 4//sup 2 -/ from AP/sup 35/S. Product inhibition by MgATP or SO/sub 4//sup 2 -/ is competitive with APS and mixed-type with PP/sub i/. Imidodiphosphate can serve as an alternative substrate for PP/sub i/. ATP sulfurylase binds (but does not hydrolyze) APS. A Scatchard plot of the APS binding is nonlinear, suggesting at least two types of sites. The cumulative results are qualitatively consistent with the random addition of MgATP and SO/sub 4//sup 2 -/ and the ordered release of first MgPP/sub i/ then APS, with APS release being partially rate limiting. Certain quantitative discrepancies suggest either an unknown variable (e.g. enzyme concentration) complicates the analysis or, in light of binding studies that the actual mechanism is more complicated (e.g. alternating sites) than any of the conventional models examined.« less

Adenosine triphosphate (ATP) as a possible indicator of extraterrestrial biology

NASA Technical Reports Server (NTRS)

Chappelle, E. W.; Picciolo, G. L.

1974-01-01

The ubiquity of adenosine triphosphate (ATP) in terrestrial organisms provides the basis for proposing the assay of this vital metabolic intermediate for detecting extraterrestrial biological activity. If an organic carbon chemistry is present on the planets, the occurrence of ATP is possible either from biosynthetic or purely chemical reactions. However, ATP's relative complexity minimizes the probability of abiogenic synthesis. A sensitive technique for the quantitative detection of ATP was developed using the firefly bioluminescent reaction. The procedure was used successfully for the determination of the ATP content of soil and bacteria. This technique is also being investigated from the standpoint of its application in clinical medicine.

Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages

PubMed Central

Graziano, Francesca; Desdouits, Marion; Garzetti, Livia; Podini, Paola; Alfano, Massimo; Rubartelli, Anna; Furlan, Roberto; Benaroch, Philippe; Poli, Guido

2015-01-01

HIV type 1 (HIV-1) infects CD4+ T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as “Trojan horses” carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages. PMID:26056317

Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages.

PubMed

Graziano, Francesca; Desdouits, Marion; Garzetti, Livia; Podini, Paola; Alfano, Massimo; Rubartelli, Anna; Furlan, Roberto; Benaroch, Philippe; Poli, Guido

2015-06-23

HIV type 1 (HIV-1) infects CD4(+) T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as "Trojan horses" carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages.

Fo-driven Rotation in the ATP Synthase Direction against the Force of F1 ATPase in the FoF1 ATP Synthase*

PubMed Central

Martin, James; Hudson, Jennifer; Hornung, Tassilo; Frasch, Wayne D.

2015-01-01

Living organisms rely on the FoF1 ATP synthase to maintain the non-equilibrium chemical gradient of ATP to ADP and phosphate that provides the primary energy source for cellular processes. How the Fo motor uses a transmembrane electrochemical ion gradient to create clockwise torque that overcomes F1 ATPase-driven counterclockwise torque at high ATP is a major unresolved question. Using single FoF1 molecules embedded in lipid bilayer nanodiscs, we now report the observation of Fo-dependent rotation of the c10 ring in the ATP synthase (clockwise) direction against the counterclockwise force of ATPase-driven rotation that occurs upon formation of a leash with Fo stator subunit a. Mutational studies indicate that the leash is important for ATP synthase activity and support a mechanism in which residues aGlu-196 and cArg-50 participate in the cytoplasmic proton half-channel to promote leash formation. PMID:25713065

ATP-induced lipid membrane reordering in the myelinated nerve fiber identified using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Kutuzov, N. P.; Brazhe, A. R.; Yusipovich, A. I.; Maksimov, G. V.; Dracheva, O. E.; Lyaskovskiy, V. L.; Bulygin, F. V.; Rubin, A. B.

2013-07-01

We demonstrate a successful application of Raman spectroscopy to the problem of lipid ordering with microscopic resolution in different regions of the myelinated nerve fiber. Simultaneous collection of Raman spectra of lipids and carotenoids has enabled us to characterize membrane fluidity and the degree of lipid ordering based on intensity ratios for the 1527/1160 and 2940/2885 cm-1 bands. We show that the intensity profiles of the major Raman bands vary significantly between the three major regions of myelinated nerve fiber: internode, paranode and the node of Ranvier. Mapping Raman peak intensities over these areas suggested that the carotenoid molecules are localized in the myelin membranes of nerve cells. Paranodal membranes were sensitive to extracellular ATP. ATP solutions (7 mM) influenced the 1527/1160 and 2940/2885 cm-1 intensity ratios. Changes in both carotenoid and lipid Raman spectra were in accord and indicated an increase in lipid ordering degree and decrease in membrane fluidity under ATP administration. The collected data provide evidence for the existence of a regulatory purinergic signaling pathway in the peripheral nervous system.

ATP transport through VDAC and the VDAC-tubulin complex probed by equilibrium and nonequilibrium MD simulations.

PubMed

Noskov, Sergei Yu; Rostovtseva, Tatiana K; Bezrukov, Sergey M

2013-12-23

Voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane, serves as a principal pathway for ATP, ADP, and other respiratory substrates across this membrane. Using umbrella-sampling simulations, we established the thermodynamic and kinetic components governing ATP transport across the VDAC1 channel. We found that there are several low-affinity binding sites for ATP along the translocation pathway and that the main barrier for ATP transport is located around the center of the channel and is formed predominantly by residues in the N-terminus. The binding affinity of ATP to an open channel was found to be in the millimolar to micromolar range. However, we show that this weak binding increases the ATP translocation probability by about 10-fold compared with the VDAC pore in which attractive interactions were artificially removed. Recently, it was found that free dimeric tubulin induces a highly efficient, reversible blockage of VDAC reconstituted into planar lipid membranes. It was proposed that by blocking VDAC permeability for ATP/ADP and other mitochondrial respiratory substrates tubulin controls mitochondrial respiration. Using the Rosetta protein-protein docking algorithm, we established a tentative structure of the VDAC-tubulin complex. An extensive set of equilibrium and nonequilibrium (under applied electric field) molecular dynamics (MD) simulations was used to establish the conductance of the open and blocked channel. It was found that the presence of the unstructured C-terminal tail of tubulin in the VDAC pore decreases its conductance by more than 40% and switches its selectivity from anionic to cationic. The subsequent 1D potential of mean force (PMF) computations for the VDAC-tubulin complex show that the state renders ATP transport virtually impossible. A number of residues pivotal for tubulin binding to the channel were identified that help to clarify the molecular details of VDAC-tubulin interaction and to provide

Functional and phenotypic effects of AhR activation in inflammatory dendritic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bankoti, Jaishree; Center for Environmental Health Sciences, University of Montana, Missoula, MT; Rase, Ben

2010-07-15

Aryl hydrocarbon receptor (AhR) activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces immune suppression. Dendritic cells (DCs) are key antigen presenting cells governing T cell activation and differentiation. However, the consequences of AhR activation in DCs are not fully defined. We hypothesized that AhR activation alters DC differentiation and generates dysfunctional DCs. To test this hypothesis, inflammatory bone marrow-derived DCs (BMDCs) from C57Bl/6 mice were generated in the presence of vehicle or TCDD. TCDD decreased CD11c expression but increased MHC class II, CD86 and CD25 expression on the BMDCs. The effects of TCDD were strictly AhR-dependent but not exclusively DRE-mediated. Similar effects weremore » observed with two natural AhR ligands, 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid (ITE). TCDD increased LPS- and CpG-induced IL-6 and TNF-{alpha} production by BMDCs but decreased their NO production. TCDD decreased CpG-induced IL-12p70 production by BMDCs but did not affect their secretion of IL-10. TCDD downregulated LPS- and CpG-induced NF-kB p65 levels and induced a trend towards upregulation of RelB levels in the BMDCs. AhR activation by TCDD modulated BMDC uptake of both soluble and particulate antigens. Induction of indoleamine-2,3-dioxygenase (IDO) and TGF-{beta}3 has been implicated in the generation of regulatory T cells following AhR activation. TCDD increased IDO1, IDO2 and TGF-{beta}3 mRNA levels in BMDCs as compared to vehicle. Despite the induction of regulatory mediators, TCDD-treated BMDCs failed to suppress antigen-specific T cell activation. Thus, AhR activation can directly alter the differentiation and innate functions of inflammatory DCs without affecting their ability to successfully interact with T cells.« less

Serotonergic regulation of distention-induced ATP release from the urothelium.

PubMed

Matsumoto-Miyai, Kazumasa; Yamada, Erika; Shinzawa, Eriko; Koyama, Yoshihisa; Shimada, Shoichi; Yoshizumi, Masaru; Kawatani, Masahito

2016-04-01

Serotonin [5-hydroxytryptamine (5-HT)] is involved in both motor and sensory functions in hollow organs, especially in the gastrointestinal tract. However, the involvement of 5-HT in visceral sensation of the urinary bladder remains unknown. Because distention-induced ATP release from the urothelium plays an essential role in visceral sensation of the urinary bladder, we investigated the regulation of urothelial ATP release by the 5-HT signaling system. RT-PCR and immunohistochemical analyses of the urothelium revealed specific expression of 5-HT 1D and 5-HT 4 receptors. The addition of 5-HT did not affect urothelial ATP release without bladder distention, but it significantly reduced distention-induced ATP release by physiological pressure during urine storage (5 cmH 2 O). The inhibitory effect of 5-HT on distention-elicited ATP release was blocked by preincubation with the 5-HT 1B/1D antagonist GR-127935 but not by the 5-HT 4 antagonist SB-204070. mRNA encoding tryptophan hydroxylase 1 was detected in the urinary bladder by nested RT-PCR amplification, and l-tryptophan or the selective serotonin reuptake inhibitor citalopram also inhibited ATP release, indicating that 5-HT is endogenously synthesized and released in the urinary bladder. The addition of GR-127935 significantly enhanced the distention-elicited ATP release 40 min after distention, whereas SB-204070 reduced the amount of ATP release 20 min after distention. These data suggest that 5-HT 4 facilitates the distention-induced ATP release at an earlier stage, whereas 5-HT 1D inhibits ATP release at a later stage. The net inhibitory effect of 5-HT indicates that the action of 5-HT on the urothelium is mediated predominantly by 5-HT 1D . Copyright © 2016 the American Physiological Society.

INTERACTION OF ESTRADIOL AND 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN AN OVULATION MODEL: EVIDENCE FOR SYSTEMIC POTENTIATION AND LOCAL OVARIAN EFFECTS. (R826132)

EPA Science Inventory

Immature rats were treated with estradiol cypionate, (ECP, 0, 0.1, 1, or 2 mg/kg s.c.) followed 24 h later by TCDD (0 or 10 g/kg orally). Follicular development was induced with eC...

Ca++-sensitizing mutations in troponin, Pi, and 2-deoxyATP alter the depressive effect of acidosis on regulated thin-filament velocity

PubMed Central

Longyear, Thomas J.; Turner, Matthew A.; Davis, Jonathan P.; Lopez, Joseph; Biesiadecki, Brandon

2014-01-01

Repeated, intense contractile activity compromises the ability of skeletal muscle to generate force and velocity, resulting in fatigue. The decrease in velocity is thought to be due, in part, to the intracellular build-up of acidosis inhibiting the function of the contractile proteins myosin and troponin; however, the underlying molecular basis of this process remains poorly understood. We sought to gain novel insight into the decrease in velocity by determining whether the depressive effect of acidosis could be altered by 1) introducing Ca++-sensitizing mutations into troponin (Tn) or 2) by agents that directly affect myosin function, including inorganic phosphate (Pi) and 2-deoxy-ATP (dATP) in an in vitro motility assay. Acidosis reduced regulated thin-filament velocity (VRTF) at both maximal and submaximal Ca++ levels in a pH-dependent manner. A truncated construct of the inhibitory subunit of Tn (TnI) and a Ca++-sensitizing mutation in the Ca++-binding subunit of Tn (TnC) increased VRTF at submaximal Ca++ under acidic conditions but had no effect on VRTF at maximal Ca++ levels. In contrast, both Pi and replacement of ATP with dATP reversed much of the acidosis-induced depression of VRTF at saturating Ca++. Interestingly, despite producing similar magnitude increases in VRTF, the combined effects of Pi and dATP were additive, suggesting different underlying mechanisms of action. These findings suggest that acidosis depresses velocity by slowing the detachment rate from actin but also by possibly slowing the attachment rate. PMID:24651988

Loss of the gene for the alpha subunit of ATP synthase (ATP5A1) from the W chromosome in the African grey parrot (Psittacus erithacus).

PubMed

de Kloet, S R

2001-08-01

This study describes the results of an analysis using Southern blotting, the polymerase chain reaction, and sequencing which shows that the African grey parrot (Psittacus erithacus) lacks the W-chromosomal gene for the alpha subunit of mitochondrial ATP synthase (ATP5A1W). Additional evidence shows that in other psittacines a fragment of the ATP5A1W gene contains five times as many nonsynonymous nucleotide replacements as the homologous fragment of the Z gene. Therefore, whereas in these other psittacines the corresponding ATP5A1Z protein fragment is highly conserved and varies by only a few, moderately conservative amino acid substitutions, the homologous ATP5A1W fragments contain a considerable number of, sometimes highly nonconservative, amino acid replacements. In one of these species, the ringneck parakeet (Psittacula krameri), the ATP5A1W gene is present in an inactive form because of the presence of a nonsense codon. Other changes, possibly leading to an inactive ATP5A1W gene product, involve the substitution of arginine residues by cysteine in the ATP5A1W protein of the mitred conure (Aratinga mitrata) and the blue and gold macaw (Ara ararauna). The data suggest also that although the divergence of the psittacine ATP5A1W and ATP5A1Z genes preceded the origin of the psittacidae, this divergence occurred independently of a similar process in the myna (Gracula religiosa), the outgroup used in this study.

The Chloroplast atpA Gene Cluster in Chlamydomonas reinhardtii1

PubMed Central

Drapier, Dominique; Suzuki, Hideki; Levy, Haim; Rimbault, Blandine; Kindle, Karen L.; Stern, David B.; Wollman, Francis-André

1998-01-01

Most chloroplast genes in vascular plants are organized into polycistronic transcription units, which generate a complex pattern of mono-, di-, and polycistronic transcripts. In contrast, most Chlamydomonas reinhardtii chloroplast transcripts characterized to date have been monocistronic. This paper describes the atpA gene cluster in the C. reinhardtii chloroplast genome, which includes the atpA, psbI, cemA, and atpH genes, encoding the α-subunit of the coupling-factor-1 (CF1) ATP synthase, a small photosystem II polypeptide, a chloroplast envelope membrane protein, and subunit III of the CF0 ATP synthase, respectively. We show that promoters precede the atpA, psbI, and atpH genes, but not the cemA gene, and that cemA mRNA is present only as part of di-, tri-, or tetracistronic transcripts. Deletions introduced into the gene cluster reveal, first, that CF1-α can be translated from di- or polycistronic transcripts, and, second, that substantial reductions in mRNA quantity have minimal effects on protein synthesis rates. We suggest that posttranscriptional mRNA processing is common in C. reinhardtii chloroplasts, permitting the expression of multiple genes from a single promoter. PMID:9625716

Monitoring ATP dynamics in electrically active white matter tracts

PubMed Central

Trevisiol, Andrea; Saab, Aiman S; Winkler, Ulrike; Marx, Grit; Imamura, Hiromi; Möbius, Wiebke; Kusch, Kathrin; Nave, Klaus-Armin; Hirrlinger, Johannes

2017-01-01

In several neurodegenerative diseases and myelin disorders, the degeneration profiles of myelinated axons are compatible with underlying energy deficits. However, it is presently impossible to measure selectively axonal ATP levels in the electrically active nervous system. We combined transgenic expression of an ATP-sensor in neurons of mice with confocal FRET imaging and electrophysiological recordings of acutely isolated optic nerves. This allowed us to monitor dynamic changes and activity-dependent axonal ATP homeostasis at the cellular level and in real time. We find that changes in ATP levels correlate well with compound action potentials. However, this correlation is disrupted when metabolism of lactate is inhibited, suggesting that axonal glycolysis products are not sufficient to maintain mitochondrial energy metabolism of electrically active axons. The combined monitoring of cellular ATP and electrical activity is a novel tool to study neuronal and glial energy metabolism in normal physiology and in models of neurodegenerative disorders. DOI: http://dx.doi.org/10.7554/eLife.24241.001 PMID:28414271

Ammonium tetrathiomolybdate treatment targets the copper transporter ATP7A and enhances sensitivity of breast cancer to cisplatin

PubMed Central

Wong, Ada Hang-Heng; Vazquez-Ortiz, Guelaguetza; Chen, Weiping; Xu, Xiaoling; Deng, Chu-Xia

2016-01-01

Cisplatin is an effective breast cancer drug but resistance often develops over prolonged chemotherapy. Therefore, we performed a candidate approach RNAi screen in combination with cisplatin treatment to identify molecular pathways conferring survival advantages. The screen identified ATP7A as a therapeutic target. ATP7A is a copper ATPase transporter responsible for intercellular movement and sequestering of cisplatin. Pharmaceutical replacement for ATP7A by ammonium tetrathiomolybdate (TM) enhanced cisplatin treatment in breast cancer cells. Allograft and xenograft models in athymic nude mice treated with cisplatin/TM exhibited retarded tumor growth, reduced accumulation of cancer stem cells and decreased cell proliferation as compared to mono-treatment with cisplatin or TM. Cisplatin/TM treatment of cisplatin-resistant tumors reduced ATP7A protein levels, attenuated cisplatin sequestering by ATP7A, increased nuclear availability of cisplatin, and subsequently enhanced DNA damage and apoptosis. Microarray analysis of gene ontology pathways that responded uniquely to cisplatin/TM double treatment depicted changes in cell cycle regulation, specifically in the G1/S transition. These findings offer the potential to combat platinum-resistant tumors and sensitize patients to conventional breast cancer treatment by identifying and targeting the resistant tumors' unique molecular adaptations. PMID:27806319