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Sample records for atp iii definition

  1. JIS definition identified more Malaysian adults with metabolic syndrome compared to the NCEP-ATP III and IDF criteria.

    PubMed

    Ramli, Anis Safura; Daher, Aqil Mohammad; Nor-Ashikin, Mohamed Noor Khan; Mat-Nasir, Nafiza; Ng, Kien Keat; Miskan, Maizatullifah; Ambigga, Krishnapillai S; Ariffin, Farnaza; Mazapuspavina, Md Yasin; Abdul-Razak, Suraya; Abdul-Hamid, Hasidah; Abd-Majid, Fadhlina; Abu-Bakar, Najmin; Nawawi, Hapizah; Yusoff, Khalid

    2013-01-01

    Metabolic syndrome (MetS) is a steering force for the cardiovascular diseases epidemic in Asia. This study aimed to compare the prevalence of MetS in Malaysian adults using NCEP-ATP III, IDF, and JIS definitions, identify the demographic factors associated with MetS, and determine the level of agreement between these definitions. The analytic sample consisted of 8,836 adults aged ≥30 years recruited at baseline in 2007-2011 from the Cardiovascular Risk Prevention Study (CRisPS), an ongoing, prospective cohort study involving 18 urban and 22 rural communities in Malaysia. JIS definition gave the highest overall prevalence (43.4%) compared to NCEP-ATP III (26.5%) and IDF (37.4%), P < 0.001. Indians had significantly higher age-adjusted prevalence compared to other ethnic groups across all MetS definitions (30.1% by NCEP-ATP III, 50.8% by IDF, and 56.5% by JIS). The likelihood of having MetS amongst the rural and urban populations was similar across all definitions. A high level of agreement between the IDF and JIS was observed (Kappa index = 0.867), while there was a lower level of agreement between the IDF and NCEP-ATP III (Kappa index = 0.580). JIS definition identified more Malaysian adults with MetS and therefore should be recommended as the preferred diagnostic criterion.

  2. JIS Definition Identified More Malaysian Adults with Metabolic Syndrome Compared to the NCEP-ATP III and IDF Criteria

    PubMed Central

    Daher, Aqil Mohammad; Noor Khan Nor-Ashikin, Mohamed; Mat-Nasir, Nafiza; Keat Ng, Kien; Ambigga, Krishnapillai S.; Ariffin, Farnaza; Yasin Mazapuspavina, Md; Abdul-Razak, Suraya; Abdul-Hamid, Hasidah; Abd-Majid, Fadhlina; Abu-Bakar, Najmin; Nawawi, Hapizah; Yusoff, Khalid

    2013-01-01

    Metabolic syndrome (MetS) is a steering force for the cardiovascular diseases epidemic in Asia. This study aimed to compare the prevalence of MetS in Malaysian adults using NCEP-ATP III, IDF, and JIS definitions, identify the demographic factors associated with MetS, and determine the level of agreement between these definitions. The analytic sample consisted of 8,836 adults aged ≥30 years recruited at baseline in 2007–2011 from the Cardiovascular Risk Prevention Study (CRisPS), an ongoing, prospective cohort study involving 18 urban and 22 rural communities in Malaysia. JIS definition gave the highest overall prevalence (43.4%) compared to NCEP-ATP III (26.5%) and IDF (37.4%), P < 0.001. Indians had significantly higher age-adjusted prevalence compared to other ethnic groups across all MetS definitions (30.1% by NCEP-ATP III, 50.8% by IDF, and 56.5% by JIS). The likelihood of having MetS amongst the rural and urban populations was similar across all definitions. A high level of agreement between the IDF and JIS was observed (Kappa index = 0.867), while there was a lower level of agreement between the IDF and NCEP-ATP III (Kappa index = 0.580). JIS definition identified more Malaysian adults with MetS and therefore should be recommended as the preferred diagnostic criterion. PMID:24175300

  3. The reliability of the National Cholesterol Education Program's Adult Treatment Panel III (NCEP/ATP III) and the International Diabetes Federation (IDF) definitions in diagnosing metabolic syndrome (MetS) among Gaza Strip Palestinians.

    PubMed

    Sirdah, Mahmoud M; Abu Ghali, Asmaa S; Al Laham, Nahed A

    2012-01-01

    Metabolic syndrome (MetS) which is a multifaceted syndrome, has been demonstrated as a common precursor for developing cardiovascular diseases and/or type 2 diabetes mellitus. Different diagnostic definitions for MetS have been proposed and recommended. We set up to evaluate the reliabilities of the National Cholesterol Education Program's Adult Treatment Panel III (NCEP/ATP III) and the International Diabetes Federation (IDF) definitions in diagnosing MetS among Gaza Strip Palestinians. This cross sectional study involved a randomly selected two hundred and thirty apparently healthy adults from the Gaza Strip. Anthropometric measurements, blood pressure, fasting plasma glucose, lipid profile, and questionnaire interviews were performed. The overall prevalence of MetS in our Gaza Strip cohort was 23.0% and 39.5% according to NCEP/ATP III and IDF definitions respectively (p<0.001). No significant differences were seen in the number of MetS components in individuals having MetS by either definition (mean 3.42 ± 0.63 vs 3.52 ± 0.69 respectively, p=0.865). Both IDF and NCEP/ATP III showed an increased prevalence of MetS with age, and body mass index (BMI), however they revealed different prevalence trends with sex. Except for BMI, there were no significant differences in the general and metabolic related characteristics between subjects with MetS of IDF and NCEP/ATP III definitions. Independently of the definition used, MetS is highly prevalent in Gaza Strip population, with a steady increase in MetS prevalence through age and BMI. The IDF definition tends to give higher values for MetS prevalence, and therefore could be more appropriate for diagnosing MetS in Gaza Strip cohort. Copyright © 2012 Diabetes India. Published by Elsevier Ltd. All rights reserved.

  4. A Comparison between Revised NCEP ATP III and IDF Definitions in Diagnosing Metabolic Syndrome in an Urban Sri Lankan Population: The Ragama Health Study

    PubMed Central

    Chackrewarthy, S.; Gunasekera, D.; Pathmeswaren, A.; Wijekoon, C. N.; Ranawaka, U. K.; Kato, N.; Takeuchi, F.; Wickremasinghe, A. R.

    2013-01-01

    Background. The prevalence of metabolic syndrome (MetS) within individual cohorts varies with the definition used. The aim of this study was to compare the prevalence of MetS between IDF and revised NCEP ATP III criteria in an urban Sri Lankan population and to investigate the characteristics of discrepant cases. Methods. 2985 individuals, aged 35–65 years, were recruited to the study. Anthropometric and blood pressure measurements and laboratory investigations were carried out following standard protocols. Results. Age and sex-adjusted prevalences of MetS were 46.1% and 38.9% by revised NCEP and IDF definitions, respectively. IDF criteria failed to identify 21% of men and 7% of women identified by the revised NCEP criteria. The discrepant group had more adverse metabolic profiles despite having a lower waist circumference than those diagnosed by both criteria. Conclusion. MetS is common in this urban Sri Lankan cohort regardless of the definition used. The revised NCEP definition was more appropriate in identifying the metabolically abnormal but nonobese individuals, especially among the males predisposed to type 2 diabetes or cardiovascular disease. Further research is needed to determine the suitability of the currently accepted Asian-specific cut-offs for waist circumference in Sri Lankan adults. PMID:23533799

  5. Coordination of substrate binding and ATP hydrolysis in Vps4-mediated ESCRT-III disassembly.

    PubMed

    Davies, Brian A; Azmi, Ishara F; Payne, Johanna; Shestakova, Anna; Horazdovsky, Bruce F; Babst, Markus; Katzmann, David J

    2010-10-01

    ESCRT-III undergoes dynamic assembly and disassembly to facilitate membrane exvagination processes including multivesicular body (MVB) formation, enveloped virus budding, and membrane abscission during cytokinesis. The AAA-ATPase Vps4 is required for ESCRT-III disassembly, however the coordination of Vps4 ATP hydrolysis with ESCRT-III binding and disassembly is not understood. Vps4 ATP hydrolysis has been proposed to execute ESCRT-III disassembly as either a stable oligomer or an unstable oligomer whose dissociation drives ESCRT-III disassembly. An in vitro ESCRT-III disassembly assay was developed to analyze Vps4 function during this process. The studies presented here support a model in which Vps4 acts as a stable oligomer during ATP hydrolysis and ESCRT-III disassembly. Moreover, Vps4 oligomer binding to ESCRT-III induces coordination of ATP hydrolysis at the level of individual Vps4 subunits. These results suggest that Vps4 functions as a stable oligomer that acts upon individual ESCRT-III subunits to facilitate ESCRT-III disassembly.

  6. Cloning, characterization and mapping of the human ATP5E gene, identification of pseudogene ATP5EP1, and definition of the ATP5E motif.

    PubMed

    Tu, Q; Yu, L; Zhang, P; Zhang, M; Zhang, H; Jiang, J; Chen, C; Zhao, S

    2000-04-01

    A cDNA encoding the epsilon subunit of human ATP synthase, ATP5E, was isolated from heart, skeletal muscle and spleen cDNA libraries respectively. Its genome structure was characterized as comprising three exons and two introns within a stretch of 5 kb, according to the genomic sequence AL109840. The gene was mapped to human chromosome 20q13.3 between marker D20S173 and 20qter using the radiation hybrid GB4 panel. Northern blot analysis showed that the ATP5E gene was expressed as a single 0.6 kb transcript in all 16 human tissues tested, with a high level present in heart and skeletal muscle. A new conserved motif composed of 24 residues, termed the ATP5E motif [W(R/K)X(5)YX(2)(Y/F)X(3)(C/A)X(4)RX(3)K], was defined on the basis of sequences of ATP synthase epsilon subunits from ten different organisms. In addition, a pseudogene ATP5EP1 was also identified on the basis of genomic sequence AC004066, localized on human chromosome 4q25. By analysing these results combined with the Southern blot patterns of human DNA hybridized with bovine ATP5E cDNA reported previously [Vinas, Powell, Runswick, Iacobazzi and Walker (1990) Biochem. J. 265, 321-326], we provide evidence of yet further homologous sequences (either gene or pseudogene) of ATP5E, in addition to ATP5E and ATP5EP1 in the human genome.

  7. Re-evaluating the kinetics of ATP hydrolysis during initiation of DNA sliding by Type III restriction enzymes

    PubMed Central

    Tóth, Júlia; Bollins, Jack; Szczelkun, Mark D.

    2015-01-01

    DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This ‘DNA sliding’ is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ∼10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding. PMID:26538601

  8. Re-evaluating the kinetics of ATP hydrolysis during initiation of DNA sliding by Type III restriction enzymes.

    PubMed

    Tóth, Júlia; Bollins, Jack; Szczelkun, Mark D

    2015-12-15

    DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This 'DNA sliding' is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ∼10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding.

  9. Prevalence of metabolic syndrome with International Diabetes Federation Criteria and ATP III Program in patients 65 years of age or older.

    PubMed

    De Luis, D A; Lopez Mongil, R; Gonzalez Sagrado, M; Lopez Trigo, J A; Mora, P F; Castrodeza Sanz, J

    2010-05-01

    The relationship between cardiovascular factors and death can vary with age, very few studies have examined metabolic syndrome in the elderly. The aim of this study is to assess the prevalence of the MS in a sample of elderly institutionalized patients (> 65 years) using ATPIII and IDF definitions. This was a cross-sectional survey covering a sample of representative of the institutionalized Spanish population aged above 65 years. The final sample study consisted of 862 patients, 556 females and 306 males. ATPIII and IDF definitions were used to classify the patients. Prevalence of MS was different according to the two definitions used. When the IDF definition was applied, total prevalence was 48.91% (CI 95%:43.47-50.25), while prevalence according to ATPIII criteria was 46.80% (CI = 43.47-50.25). a higher prevalence of MS was found in females as compared to males. Using IDF criteria, odds ratio was 1.9 (CI 95%:1.4-2.6) and 1.7 (CI 95%:1.2-2.2) according to ATPIII criteria. a steady decrease is seen in MS prevalence as the age of patients increases (the last two groups (85-94 ys and > 95 ys), both for the ATP III and the IDF definitions. A higher prevalence of MS in this elderly population as compared to general population was observed. A decrease of this prevalence above 95 years was detected.

  10. A Label-Free Luminescent Switch-On Assay for ATP Using a G-Quadruplex-Selective Iridium(III) Complex

    PubMed Central

    Wang, Modi; Mak, Tsun-Yin; Chan, Daniel Shiu-Hin; Tang, Fung-Kit; Leung, Chung-Hang; Kwan, Hiu-Yee; Yu, Zhiling; Ma, Dik-Lung

    2013-01-01

    We report herein the G-quadruplex-selective property of a luminescent cyclometallated iridium(III) complex for the detection of adenosine-5′-triphosphate (ATP) in aqueous solution. The ATP-binding aptamer was employed as the ATP recognition unit, while the iridium(III) complex was used to monitor the formation of the G-quadruplex structure induced by ATP. The sensitivity and fold enhancement of the assay were higher than those of the previously reported assay using the organic dye crystal violet as a fluorescent probe. This label-free luminescent switch-on assay exhibits high sensitivity and selectivity towards ATP with a limit of detection of 2.5 µM. PMID:24204723

  11. 2013 ACC/AHA versus 2004 NECP ATP III Guidelines in the Assignment of Statin Treatment in a Korean Population with Subclinical Coronary Atherosclerosis

    PubMed Central

    Kang, Yu Mi; Yang, Dong Hyun; Kang, Joon-Won; Kim, Eun Hee; Park, Duk-Woo; Park, Joong-Yeol; Kim, Hong-Kyu; Lee, Woo Je

    2015-01-01

    Background The usefulness of the 2013 ACC/AHA guidelines for the management of blood cholesterol in the Asian population remains controversial. In this study, we investigated whether eligibility for statin therapy determined by the 2013 ACC/AHA guidelines is better aligned with the presence of subclinical coronary atherosclerosis detected by CCTA (coronary computed tomography angiography) compared to the previously recommended 2004 NCEP ATP III guidelines. Methods We collected the data from 5,837 asymptomatic subjects who underwent CCTA using MDCT during routine health examinations. Based on risk factor assessment and lipid data, we determined guideline-based eligibility for statin therapy according to the 2013 ACC/AHA and 2004 NCEP ATP III guidelines. We defined the presence and severity of subclinical coronary atherosclerosis detected in CCTA according to the presence of significant coronary artery stenosis (defined as >50% stenosis), plaques, and the degree of coronary calcification. Results As compared to the 2004 ATP III guidelines, a significantly higher proportion of subjects with significant coronary stenosis (61.8% vs. 33.8%), plaques (52.3% vs. 24.7%), and higher CACS (CACS >100, 63.6% vs. 26.5%) was assigned to statin therapy using the 2013 ACC/AHA guidelines (P < .001 for all variables). The area under the curves of the pooled cohort equation of the new guidelines in detecting significant stenosis, plaques, and higher CACS were significantly higher than those of the Framingham risk calculator. Conclusions Compared to the previous ATP III guidelines, the 2013 ACC/AHA guidelines were more sensitive in identifying subjects with subclinical coronary atherosclerosis detected by CCTA in an Asian population. PMID:26372638

  12. Major CHD risk factors predominate among African-American women who are eligible for lipid-lowering drug therapy under the new ATP III guidelines.

    PubMed

    Koro, Carol E; L'italien, Gilbert J; Fedder, Donald O

    2004-10-01

    Coronary heart disease remains the leading cause of morbidity and death among African-Americans. We studied the cardiovascular risk factor distributions among African-American men and women deemed eligible for lipid-lowering treatment under the new Adult Treatment Panel Guidelines (ATPIII). A sub-sample of African-American NHANES III subjects aged 20-79 years, with known cardiovascular risk factors and LDL-C levels was identified (n=4,213). We assessed their eligibility for drug therapy using the new ATP III criteria and compared CHD risk factor distributions across gender. Both conservative and drug-optional LDL-C target levels were applied. An estimated 5.7 million African-Americans aged 20-79 are eligible for drug therapy under ATP III, and the overall eligibility prevalence is 24.3%; 47.8% are males and 52.2% are females (P<0.001). Of these, 1.87 million are eligible based on drug-optional LDL-C targets and 54.5% of these are female. Of treatment-eligible individuals, 61% of males versus 72% of females exhibited LDL-C > or =160 mg/dl (P=0.0001). The prevalence and levels of important CHD risk factors such as diabetes, hypertension, mean total and LDL-C cholesterol levels, and body mass index were all greater for eligible females compared to males despite lower absolute Framingham risk estimates for females. Among African-Americans, more women than men are eligible for treatment under the new ATP III guidelines. Eligibility in women is based primarily on diabetes and lipid levels rather than absolute Framingham risk, which seems to be underestimated in African-American women. As compensation for this underestimate, drug-optional (lower) targets should be applied to this population.

  13. In vitro study of accuracy of cervical pedicle screw insertion using an electronic conductivity device (ATPS part III).

    PubMed

    Koller, Heiko; Hitzl, Wolfgang; Acosta, Frank; Tauber, Mark; Zenner, Juliane; Resch, Herbert; Yukawa, Yasutsugu; Meier, Oliver; Schmidt, Rene; Mayer, Michael

    2009-09-01

    Reconstruction of the highly unstable, anteriorly decompressed cervical spine poses biomechanical challenges to current stabilization strategies, including circumferential instrumented fusion, to prevent failure. To avoid secondary posterior surgery, particularly in the elderly population, while increasing primary construct rigidity of anterior-only reconstructions, the authors introduced the concept of anterior transpedicular screw (ATPS) fixation and plating. We demonstrated its morphological feasibility, its superior biomechanical pull-out characteristics compared with vertebral body screws and the accuracy of inserting ATPS using a manual fluoroscopically assisted technique. Although accuracy was high, showing non-critical breaches in the axial and sagittal plane in 78 and 96%, further research was indicated refining technique and increasing accuracy. In light of first clinical case series, the authors analyzed the impact of using an electronic conductivity device (ECD, PediGuard) on the accuracy of ATPS insertion. As there exist only experiences in thoracolumbar surgery the versatility of the ECD was also assessed for posterior cervical pedicle screw fixation (pCPS). 30 ATPS and 30 pCPS were inserted alternately into the C3-T1 vertebra of five fresh-frozen specimen. Fluoroscopic assistance was only used for the entry point selection, pedicle tract preparation was done using the ECD. Preoperative CT scans were assessed for sclerosis at the pedicle entrance or core, and vertebrae with dense pedicles were excluded. Pre- and postoperative reconstructed CT scans were analyzed for pedicle screw positions according to a previously established grading system. Statistical analysis revealed an astonishingly high accuracy for the ATPS group with no critical screw position (0%) in axial or sagittal plane. In the pCPS group, 88.9% of screws inserted showed non-critical screw position, while 11.1% showed critical pedicle perforations. The usage of an ECD for posterior and

  14. National Cholesterol Education Program Adult Treatment Panel III Versus International Diabetic Federation Definition of Metabolic Syndrome, Which One is Associated with Diabetes Mellitus and Coronary Artery Disease?

    PubMed

    Rezaianzadeh, Abbas; Namayandeh, Seyedeh-Mahdieh; Sadr, Seyed-Mahmood

    2012-08-01

    A cluster of risk factors for cardiovascular diseases and type 2 diabetes mellitus, which occur together more often than by chance alone, have been known as the metabolic syndrome. Various definitions have been proposed by different organizations over the past decade. This study was designed to evaluate a new definition of the metabolic syndrome for the prediction of diabetes mellitus among the Iranian population. This study was carried out in an urban population, aged 20 to 74 years, from Yazd, a city in the center of Iran. The study is a part of the phase I of Yazd Healthy Heart Program, that is, a community-based intervention study for the prevention of cardiovascular disease. The significance level has been defined as P<0.05. Prevalence of the metabolic syndrome by the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) criteria was 21.3 ± .017%, and by International Diabetes Federation (IDF) criteria it was 30.16 ± .02%. The multivariate analysis showed that the most important relevant factors of diabetes mellitus were: Increased age and metabolic syndrome by both definitions of NCEP and IDF criteria, and also, the most important relevant factors of stable angina were: Increased age, male sex, and metabolic syndrome by only IDF definitions, but the NCEP definition of the metabolic syndrome cannot predict diabetes mellitus independent of age and sex. This study showed that increased age and metabolic syndrome are the most important relevant factors for diabetes mellitus, especially by using the IDF criteria for definition of the metabolic syndrome.

  15. Structural Features Reminiscent of ATP-Driven Protein Translocases Are Essential for the Function of a Type III Secretion-Associated ATPase

    PubMed Central

    Kato, Junya; Lefebre, Matthew

    2015-01-01

    ABSTRACT Many bacterial pathogens and symbionts utilize type III secretion systems to interact with their hosts. These machines have evolved to deliver bacterial effector proteins into eukaryotic target cells to modulate a variety of cellular functions. One of the most conserved components of these systems is an ATPase, which plays an essential role in the recognition and unfolding of proteins destined for secretion by the type III pathway. Here we show that structural features reminiscent of other ATP-driven protein translocases are essential for the function of InvC, the ATPase associated with a Salmonella enterica serovar Typhimurium type III secretion system. Mutational and functional analyses showed that a two-helix-finger motif and a conserved loop located at the entrance of and within the predicted pore formed by the hexameric ATPase are essential for InvC function. These findings provide mechanistic insight into the function of this highly conserved component of type III secretion machines. IMPORTANCE Type III secretion machines are essential for the virulence or symbiotic relationships of many bacteria. These machines have evolved to deliver bacterial effector proteins into host cells to modulate cellular functions, thus facilitating bacterial colonization and replication. An essential component of these machines is a highly conserved ATPase, which is necessary for the recognition and secretion of proteins destined to be delivered by the type III secretion pathway. Using modeling and structure and function analyses, we have identified structural features of one of these ATPases from Salmonella enterica serovar Typhimurium that help to explain important aspects of its function. PMID:26170413

  16. Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis.

    PubMed

    Meisel, A; Mackeldanz, P; Bickle, T A; Krüger, D H; Schroeder, C

    1995-06-15

    Type III restriction/modification systems recognize short non-palindromic sequences, only one strand of which can be methylated. Replication of type III-modified DNA produces completely unmethylated recognition sites which, according to classical mechanisms of restriction, should be signals for restriction. We have shown previously that suicidal restriction by the type III enzyme EcoP15I is prevented if all the unmodified sites are in the same orientation: restriction by EcoP15I requires a pair of unmethylated, inversely oriented recognition sites. We have now addressed the molecular mechanism of site orientation-specific DNA restriction. EcoP15I is demonstrated to possess an intrinsic ATPase activity, the potential driving force of DNA translocation. The ATPase activity is uniquely recognition site-specific, but EcoP15I-modified sites also support the reaction. EcoP15I DNA restriction patterns are shown to be predetermined by the enzyme-to-site ratio, in that site-saturating enzyme levels elicit cleavage exclusively between the closest pair of head-to-head oriented sites. DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15I sites. These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently tracking EcoP15I-DNA complexes.

  17. Complexation of bisphosphonates with ytterbium(III): application of phosphate and ATP detection assay based on Yb(3+)-pyrocatechol violet.

    PubMed

    Gaidamauskas, Ernestas; Parker, Helen; Kashemirov, Boris A; Holder, Alvin A; Saejueng, Kanokkarn; McKenna, Charles E; Crans, Debbie C

    2009-12-01

    The coordination chemistry of bisphosphonates with Yb(3+) was investigated to evaluate the potential of the UV-vis based detection method using the Yb(3+)-pyrocatechol complexation reaction as a sensor for bisphosphonates. The complexation chemistry of Yb(3+) with phosphate and ATP analogs was previously described (E. Gaidamauskas, K. Saejueng, A.A. Holder, S. Bharuah, B.A. Kashemirov, D.C. Crans, C.E. McKenna, J. Biol. Inorg. Chem. 13 (2008) 1291-1299), and we here studied the complexation chemistry of bisphosphonates in this system. The spectrophotometric assay yields direct evidence for formation of a 4:3 metal to ligand complex at neutral pH. Direct evidence for Yb(3+):methylenebis(phosphonate) complexes with 1:1 and 1:2 stoichiometry was also obtained by potentiometry at acidic and basic pH. Direct evidence for complex formation was obtained using (1)H NMR spectroscopy although the stoichiometry was not accessed at neutral pH. Our results suggest that the spectroscopic observation of the YbPV complex can be used to conveniently measure concentrations of bisphosphonates down to 2-3 microM.

  18. Utility of the modified ATP III defined metabolic syndrome and severe obesity as predictors of insulin resistance in overweight children and adolescents: a cross-sectional study

    PubMed Central

    Dhuper, Sarita; Cohen, Hillel W; Daniel, Josephine; Gumidyala, Padmasree; Agarwalla, Vipin; St Victor, Rosemarie; Dhuper, Sunil

    2007-01-01

    Background The rising prevalence of obesity and metabolic syndrome (MetS) has received increased attention since both place individuals at risk for Type II diabetes and cardiovascular disease. Insulin resistance (IR) has been implicated in the pathogenesis of obesity and MetS in both children and adults and is a known independent cardiovascular risk factor. However measures of IR are not routinely performed in children while MetS or severe obesity when present, are considered as clinical markers for IR. Objective The study was undertaken to assess the utility of ATPIII defined metabolic syndrome (MetS) and severe obesity as predictors of insulin resistance (IR) in a group of 576 overweight children and adolescents attending a pediatric obesity clinic in Brooklyn. Methods Inclusion criteria were children ages 3–19, and body mass index > 95th percentile for age. MetS was defined using ATP III criteria, modified for age. IR was defined as upper tertile of homeostasis model assessment (HOMA) within 3 age groups (3–8, n = 122; 9–11, n = 164; 12–19, n = 290). Sensitivity, specificity, positive predictive values and odds ratios (OR) with 95% confidence intervals (CI) were calculated within age groups for predicting IR using MetS and severe obesity respectively. Results MetS was present in 45%, 48% and 42% of the respective age groups and significantly predicted IR only in the oldest group (OR = 2.0, 95% CI 1.2, 3.4; p = .006). Sensitivities were <55%; specificities <63% and positive predictive values ≤ 42% in all groups. Severe obesity was significantly associated with IR in both the 9–11 (p = .002) and 12–18 (p = .01) groups but positive predictive values were nonetheless ≤ 51% for all groups. Conclusion The expression of IR in overweight children and adolescents is heterogeneous and MetS or severe obesity may not be sufficiently sensitive and specific indicators of insulin resistance. In addition to screening for MetS in overweight children markers for

  19. 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.

  20. Identification of critical amino acid residues of Saccharomyces cerevisiae carbamoyl-phosphate synthetase: definition of the ATP site involved in carboxy-phosphate formation.

    PubMed

    Zheng, W; Lim, A L; Powers-Lee, S G

    1997-08-15

    Carbamoyl-phosphate synthetases (CPSases) utilize two molecules of ATP at two homologous domains, B and C, with ATP(B) used to form the enzyme-bound intermediate carboxy-phosphate and ATP(C) used to phosphorylate the carbamate intermediate. To further define the role of one CPSase peptide suggested by affinity labeling studies to be near the ATP(B) site, we have carried out site-directed mutagenic analysis of peptide 234-242 of the Saccharomyces cerevisiae arginine-specific CPSase. Mutants E234A, E234D, E236A, E236D and E238A were unable to complement the CPSase-deficient yeast strain LPL26 whereas mutants Y237A, E238D, R241K, R241E and R241P supported LPL26 growth as well as wild-type CPSase. Kinetic analysis of E234A and Y237A indicated impaired utilization of ATP(B) but not of ATP(C). D242A, a temperature-sensitive mutant, retained no detectable activity when assayed in vitro. These findings, together with the affinity labeling data and primary sequence analysis, strongly suggest that the yeast CPSase peptide 234-242 is located at the ATP(B) site and that some of its residues are important for functioning of the enzyme. D242 appears to occupy a critical structural position and E234, E236 and E238 appear to be critical for function, with the spatial arrangement of the carboxyl side chain also critical for E234 and E236.

  1. Subunit III of the chloroplast ATP-synthase can form a Ca(2+)-binding site on the lumenal side of the thylakoid membrane.

    PubMed

    Zakharov, S D; Ewy, R G; Dilley, R A

    1993-12-20

    Subunit III, the 8 kDa component of the chloroplast CFo H+ channel, was isolated and purified from pea thylakoids for the purpose of studying its Ca(2+)-binding properties. After n-butanol extraction and ether precipitation, HPLC purification was accomplished using a poly(styrene-divinylbenzene) column which removes lipid and protein contaminations. The main components of protein contamination were two hydrophobic proteins of near 4 kDa molecular mass, the psaI and psbK gene products associated with PSI and PSII reaction centers, respectively. Purified subunit III as well as the unfractionated organic-solvent soluble preparation were used in a 45Ca(2+)-ligand blot assay known to detect high affinity Ca(2+)-binding sites in proteins. Polypeptides were separated with SDS-PAGE and were transferred onto PVDF membranes. Treatment of the membrane with 45CaCl2 in the presence of 10-fold excess of MgCl2 and 200-fold excess KCl led to the labeling of only the 8 kDa polypeptide. The Ca2+ binding was inhibited after derivatizing aqueously exposed carboxyl groups with a water soluble carbodiimide plus a nucleophile, after de-formylation of the N-terminal methionine, or with a subsequent treatment with La3+. Ca2+ binding was maximum at pH 7.5-8.5 and was greatly decreased at acidic pH. Dicyclohexylcarbodiimide treatment (no nucleophile was added) of thylakoid membranes, which derivatizes the hydrophobically located Glu-61, decreased the electrophoretical mobility of isolated subunit III but did not inhibit the Ca2+ binding. The data indicate that the carbonyl group of the formylated N-terminal Met-1 and probably the carboxyl group of the subunit III C-terminal Val-81 provide some of seven essential oxygen ligands normally required for defining a Ca(2+)-binding site in proteins. It is probable, but not yet established that an oligomeric form of subunit III polypeptides is essential for forming the Ca(2+)-binding site. Based on the accepted models for the hairpin conformation of

  2. Understanding radio polarimetry. III. Interpreting the IAU/IEEE definitions of the Stokes parameters.

    NASA Astrophysics Data System (ADS)

    Hamaker, J. P.; Bregman, J. D.

    1996-05-01

    In two companion papers (Paper I, Hamaker et al. 1996; Paper II, Sault et al. 1996), a new theory of radio-interferometric polarimetry and its application to the calibration of interferometer arrays are presented. To complete our study of radio polarimetry, we examine here the definition of the Stokes parameters adopted by Commission 40 of the IAU (1974) and the way this definition works out in the mathematical equations. Using the formalism of Paper I, we give a simplified derivation of the frequently-cited `black-box' formula originally derived by Morris et al. (1964). We show that their original version is in error in the sign of Stokes V, the correct sign being that given by Weiler (1973) and Thompson et al. (1986).

  3. AtMRP1 gene of Arabidopsis encodes a glutathione S-conjugate pump: isolation and functional definition of a plant ATP-binding cassette transporter gene.

    PubMed

    Lu, Y P; Li, Z S; Rea, P A

    1997-07-22

    Because plants produce cytotoxic compounds to which they, themselves, are susceptible and are exposed to exogenous toxins (microbial products, allelochemicals, and agrochemicals), cell survival is contingent on mechanisms for detoxifying these agents. One detoxification mechanism is the glutathione S-transferase-catalyzed glutathionation of the toxin, or an activated derivative, and transport of the conjugate out of the cytosol. We show here that a transporter responsible for the removal of glutathione S-conjugates from the cytosol, a specific Mg2+-ATPase, is encoded by the AtMRP1 gene of Arabidopsis thaliana. The sequence of AtMRP1 and the transport capabilities of membranes prepared from yeast cells transformed with plasmid-borne AtMRP1 demonstrate that this gene encodes an ATP-binding cassette transporter competent in the transport of glutathione S-conjugates of xenobiotics and endogenous substances, including herbicides and anthocyanins.

  4. [Impact of the new definitions in the prevalence of the metabolic syndrome in an adult population at Bucaramanga, Colombia].

    PubMed

    Pinzón, Juan Bernardo; Serrano, Norma Cecilia; Díaz, Luis Alfonso; Mantilla, Gerardo; Velasco, Harvey Mauricio; Martínez, Luz Ximena; Millán, Paula Andrea; Acevedo, Sandra Milena; Moreno, Daniel

    2007-06-01

    The prevalence of metabolic syndrome depends on the criteria used for its classification. Three criteria in common use are those from International Diabetes Federation (IDF), the Adult Treatment Panel (ATP-III) or its update (ATP-IIIa). The prevalence statistic for generated metabolic syndrome was compared on the basis of each of the three criteria in an adult population. The sample consisted of 155 teachers and employees in the school of medicine. The average age was 40.9; 54.2% were men. The three criteria were applied and the prevalences were compared with the Wilcoxon test and Cohen's kappa. Metabolic syndrome prevalence generated by each criterion was as follows: ATP-III was 12.3% (95%CI 7.5-18.5), ATP-IIIa was 34.8% (95%CI 27.4-42.9) and IDF 32.9% (95%CI 25.6--40.9). The prevalence indicated by ATP-III was lower than the ATP-IIIa or IDF prevalences (p < 0,001); however those of ATPIII-a and IDF were similar (p=0,083). Poor agreement was seen between ATP-III and ATP-IIIa (k=0.414, IC95% 0.409-0.420), and between ATP-III and IDF (k=0.374, IC95% 0.368-0.379); however, very good agreement was obtained between ATP-IIIa and IDF (k=0.957, IC95% 0.950-0.963). The new definitions for metabolic syndrome, ATP-IIIa and IDF, increase the prevalence statistic by three times. This occurred despite the inclusion in IDF of an obesity factor in the criteria set.

  5. Comparison of definitions for the metabolic syndrome in adolescents. The HELENA study.

    PubMed

    Vanlancker, Tine; Schaubroeck, Emmily; Vyncke, Krishna; Cadenas-Sanchez, Cristina; Breidenassel, Christina; González-Gross, Marcela; Gottrand, Frederic; Moreno, Luis A; Beghin, Laurent; Molnár, Denes; Manios, Yannis; Gunter, Marc J; Widhalm, Kurt; Leclercq, Catherine; Dallongeville, Jean; Ascensión, Marcos; Kafatos, Anthony; Castillo, Manuel J; De Henauw, Stefaan; Ortega, Francisco B; Huybrechts, Inge

    2017-02-01

    Various definitions are used to define metabolic syndrome in adolescents. This study aimed to compare, in terms of prevalence and differences, five frequently used definitions for this population: International Diabetes Federation, National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP) modified by Cook, pediatric American Heart Association (AHA), World Health Organization, and Jolliffe and Janssen. A sample of 1004 adolescents (12.5-17.0 years) from the Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) study was considered. The components of the definitions (waist circumference/BMI, plasma lipids, glycemia, and blood pressure) were applied, and definitions were compared by using crosstabs, sensitivity, specificity, and kappa coefficient. The prevalence of metabolic syndrome varied from 1.6 to 3.8% depending on the used definitions. Crosstabs comparing the definitions showed the fewest cases being misclassified (having metabolic syndrome or not) between NCEP-ATP and AHA. Analyses for kappa coefficient, sensitivity, and specificity confirmed this finding.

  6. Limited predictive value of the IDF definition of metabolic syndrome for the diagnosis of insulin resistance measured with the oral minimal model.

    PubMed

    Ghanassia, E; Raynaud de Mauverger, E; Brun, J-F; Fedou, C; Mercier, J

    2009-01-01

    To assess the agreement of the NCEP ATP-III and the IDF definitions of metabolic syndrome and to determine their predictive values for the diagnosis of insulin resistance. For this purpose, we recruited 150 subjects (94 women and 56 men) and determined the presence of metabolic syndrome using the NCEP-ATP III and IDF definitions. We evaluated their insulin sensitivity S(I) using Caumo's oral minimal model after a standardized hyperglucidic breakfast test. Subjects whose S(I) was in the lowest quartile were considered as insulin resistant. We then calculated sensitivity, specificity, positive and negative predictive values of both definitions for the diagnosis of insulin resistance. The prevalence of metabolic syndrome was 37.4% (NCEP-ATP III) and 40% (IDF). Agreement between the two definitions was 96%. Using NCEP-ATP III and IDF criteria for the identification of insulin resistant subjects, sensitivity was 55.3% and 63%, specificity was 68.8% and 67.8%, positive predictive value was 37.5% and 40%, negative predictive value was 81.9% and 84.5%, respectively. Positive predictive value increased with the number of criteria for both definitions. Whatever the definition, the scoring of metabolic syndrome is not a reliable tool for the individual diagnosis of insulin resistance, and is more useful for excluding this diagnosis.

  7. 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

  8. Impact of metabolic syndrome traits on cardiovascular function: should the Adult Treatment Panel III definition be further stratified?

    PubMed

    Antonini-Canterin, Francesco; Mateescu, Anca D; Vriz, Olga; La Carrubba, Salvatore; Di Bello, Vitantonio; Carerj, Scipione; Zito, Concetta; Sparacino, Lina; Uşurelu, Cătălin; Ticulescu, Răzvan; Ginghină, Carmen; Nicolosi, Gian L; Popescu, Bogdan A

    2014-10-01

    The aims of the study were to evaluate whether a further classification of metabolic syndrome according to the number of traits (based on the Adult Treatment Panel III definition) could better explain the impact on cardiovascular remodeling and function, and to assess the role of single metabolic syndrome components in this regard. We studied by echocardiography and carotid ultrasound 435 asymptomatic patients with metabolic syndrome. Patients with coronary artery disease or more than mild valvular heart disease were excluded. Carotid stiffness index (β) was measured using a high-resolution echo-tracking system. Patients with metabolic syndrome were divided into two groups: metabolic syndrome with three traits (Gr.1) and metabolic syndrome with four or five traits (Gr. 2). Patients in Gr. 2 had higher left ventricular mass index (P < 0.001), left ventricular end-diastolic volume index (P = 0.029), left atrial volume index (P = 0.002), E/e' ratio (P = 0.002), intima-media thickness (P = 0.031), and prevalence of plaques (P = 0.01) than patients in Gr. 1. Left ventricular ejection fraction was similar in both groups. The mean carotid β index tended to be higher in Gr. 2. Considering metabolic syndrome traits separately, in an age-corrected multivariate analysis, abdominal obesity was found to have the strongest association with cardiac structure and carotid artery atherosclerosis and stiffness. An increasing number of metabolic syndrome traits had a significantly worse impact on cardiac remodeling and function and carotid artery atherosclerosis. Abdominal obesity showed the strongest association with cardiac structure, carotid artery stiffness, and intima-media thickness. Prospective studies are needed to evaluate whether a new classification of metabolic syndrome using the number of traits could add prognostic information.

  9. Assessment of severe malaria in a multicenter, phase III, RTS, S/AS01 malaria candidate vaccine trial: case definition, standardization of data collection and patient care

    PubMed Central

    2011-01-01

    Background An effective malaria vaccine, deployed in conjunction with other malaria interventions, is likely to substantially reduce the malaria burden. Efficacy against severe malaria will be a key driver for decisions on implementation. An initial study of an RTS, S vaccine candidate showed promising efficacy against severe malaria in children in Mozambique. Further evidence of its protective efficacy will be gained in a pivotal, multi-centre, phase III study. This paper describes the case definitions of severe malaria used in this study and the programme for standardized assessment of severe malaria according to the case definition. Methods Case definitions of severe malaria were developed from a literature review and a consensus meeting of expert consultants and the RTS, S Clinical Trial Partnership Committee, in collaboration with the World Health Organization and the Malaria Clinical Trials Alliance. The same groups, with input from an Independent Data Monitoring Committee, developed and implemented a programme for standardized data collection. The case definitions developed reflect the typical presentations of severe malaria in African hospitals. Markers of disease severity were chosen on the basis of their association with poor outcome, occurrence in a significant proportion of cases and on an ability to standardize their measurement across research centres. For the primary case definition, one or more clinical and/or laboratory markers of disease severity have to be present, four major co-morbidities (pneumonia, meningitis, bacteraemia or gastroenteritis with severe dehydration) are excluded, and a Plasmodium falciparum parasite density threshold is introduced, in order to maximize the specificity of the case definition. Secondary case definitions allow inclusion of co-morbidities and/or allow for the presence of parasitaemia at any density. The programmatic implementation of standardized case assessment included a clinical algorithm for evaluating

  10. Assessment of severe malaria in a multicenter, phase III, RTS, S/AS01 malaria candidate vaccine trial: case definition, standardization of data collection and patient care.

    PubMed

    Vekemans, Johan; Marsh, Kevin; Greenwood, Brian; Leach, Amanda; Kabore, William; Soulanoudjingar, Solange; Asante, Kwaku Poku; Ansong, Daniel; Evans, Jennifer; Sacarlal, Jahit; Bejon, Philip; Kamthunzi, Portia; Salim, Nahya; Njuguna, Patricia; Hamel, Mary J; Otieno, Walter; Gesase, Samwel; Schellenberg, David

    2011-08-04

    An effective malaria vaccine, deployed in conjunction with other malaria interventions, is likely to substantially reduce the malaria burden. Efficacy against severe malaria will be a key driver for decisions on implementation. An initial study of an RTS, S vaccine candidate showed promising efficacy against severe malaria in children in Mozambique. Further evidence of its protective efficacy will be gained in a pivotal, multi-centre, phase III study. This paper describes the case definitions of severe malaria used in this study and the programme for standardized assessment of severe malaria according to the case definition. Case definitions of severe malaria were developed from a literature review and a consensus meeting of expert consultants and the RTS, S Clinical Trial Partnership Committee, in collaboration with the World Health Organization and the Malaria Clinical Trials Alliance. The same groups, with input from an Independent Data Monitoring Committee, developed and implemented a programme for standardized data collection.The case definitions developed reflect the typical presentations of severe malaria in African hospitals. Markers of disease severity were chosen on the basis of their association with poor outcome, occurrence in a significant proportion of cases and on an ability to standardize their measurement across research centres. For the primary case definition, one or more clinical and/or laboratory markers of disease severity have to be present, four major co-morbidities (pneumonia, meningitis, bacteraemia or gastroenteritis with severe dehydration) are excluded, and a Plasmodium falciparum parasite density threshold is introduced, in order to maximize the specificity of the case definition. Secondary case definitions allow inclusion of co-morbidities and/or allow for the presence of parasitaemia at any density. The programmatic implementation of standardized case assessment included a clinical algorithm for evaluating seriously sick children

  11. Customized ATP towpreg

    NASA Astrophysics Data System (ADS)

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

    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.

  12. 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

  13. Dose-Response Relationship between Radiation Dose and Loco-regional Control in Patients with Stage II-III Esophageal Cancer Treated with Definitive Chemoradiotherapy.

    PubMed

    Kim, Hyun Ju; Suh, Yang-Gun; Lee, Yong Chan; Lee, Sang Kil; Shin, Sung Kwan; Cho, Byung Chul; Lee, Chang Geol

    2017-07-01

    The correlation between radiation dose and loco-regional control (LRC) was evaluated in patients with stage II-III esophageal cancer treated with definitive concurrent chemoradiotherapy (CRT). Medical records of 236 stage II-III esophageal cancer patients treated with definitive CRT at Yonsei Cancer Center between 1994 and 2013 were retrospectively reviewed. Among these, 120 received a radiation dose of < 60 Gy (standard-dose group), while 116 received ≥ 60 Gy (high-dose group). The median doses of radiation in the standard- and high-dose groups were 50.4 and 63 Gy, respectively. Concurrent 5-fluorouracil/cisplatin chemotherapy was administered to most patients. There were no differences in patient characteristics between the two groups except for high Karnofsky performance status and lower-thoracic lesions being more prevalent in the standard-dose group. The median progression-free survival (PFS) and overall survival (OS) times were 13.2 months and 26.2 months, respectively. Patients in the high-dose group had significantly better 2-year LRC (69.1% vs. 50.3%, p=0.002), median PFS (16.7 months vs. 11.7 months, p=0.029), and median OS (35.1 months vs. 22.3 months, p=0.043). Additionally, LRC exhibited a dose-response relationship and the complete response rate was significantly higher in the high-dose group (p=0.006). There were no significant differences in treatment-related toxicities between the groups. A higher radiation dose (> 60 Gy) is associated with increased LRC, PFS, and OS in patients with stage II-III esophageal cancer treated with definitive CRT.

  14. SCOPE1: a randomised phase II/III multicentre clinical trial of definitive chemoradiation, with or without cetuximab, in carcinoma of the oesophagus

    PubMed Central

    2011-01-01

    Background Chemoradiotherapy is the standard of care for patients with oesophageal cancer unsuitable for surgery due to the presence of co-morbidity or extent of disease, and is a standard treatment option for patients with squamous cell carcinoma of the oesophagus. Modern regimens of chemoradiotherapy can lead to significant long-term survival. However the majority of patients will die of their disease, most commonly with local progression/recurrence of their tumours. Cetuximab may overcome one of the principal mechanisms of tumour radio-resistance, namely tumour repopulation, in patients treated with chemoradiotherapy. The purpose of this research is first to determine whether the addition of cetuximab to definitive chemoradiotherapy for treatment of patients with non-metastatic carcinoma of the oesophagus is active (in terms of failure-free rate), safe, and feasible within the context of a multi-centre randomised controlled trial in the UK. If the first stage is successful then the trial will continue to accrue sufficient patients to establish whether the addition of cetuximab to the standard treatment improves overall survival. Methods/Design SCOPE1 is a two arm, open, randomised multicentre Phase II/III trial. Eligible patients will have histologically confirmed carcinoma of the oesophagus and have been chosen to receive definitive chemoradiotherapy by an accredited multidisciplinary team including a specialist Upper GI surgeon. 420 patients will be randomised to receive definitive chemoradiotherapy with or without cetuximab using a 1:1 allocation ratio. During Phase II of the study, the trial will assess safety (toxicity), activity (failure-free rate) and feasibility (recruitment rate and protocol dose modifications/delays) in 90 patients in the experimental arm. If the experimental arm is found to be active, safe, and feasible by the Independent Data Monitoring Committee then recruitment will continue into Phase III. This second stage will recruit a further

  15. Prognostic factors for survival in stage III non-small-cell lung cancer treated with definitive radiation therapy: Impact of tumor volume

    SciTech Connect

    Basaki, Kiyoshi . E-mail: basaki-rad@umin.ac.jp; Abe, Yoshinao; Aoki, Masahiko; Kondo, Hidehiro; Hatayama, Yoshiomi; Nakaji, Shigeyuki

    2006-02-01

    Purpose: To investigate the impact of tumor volume on overall survival in patients with Stage III non-small-cell lung cancer (NSCLC) treated with definitive radiation therapy (RT). Methods and Materials: Between May 1997 and February 2003, 71 patients with Stage III NSCLC were treated with radiation therapy of 60 Gy or more. The total target dose was between 60 and 77 Gy (average, 66.3 Gy). Chemotherapy was used in 45 cases. The primary tumor and nodal volume were identified in pretreatment computed tomography scans. Univariate and multivariate analyses were used to evaluate the impact of tumor volume on survival after RT. Results: The overall 2-year survival rate was 23%, with a median survival time of 14 months. The median survival times were 10 months and 19 months with large primary tumor volume more than median volume and smaller primary tumor volume, respectively. At a univariate analysis, the total tumor volume (TTV) (p < 0.0003) and the primary tumor volume (p < 0.00008) were significant and the nodal volume was not. At multivariate analyses, both the TTV and the primary tumor volume were significant prognostic factors. Conclusion: The primary tumor volume as well as TTV is a significant prognostic factor on survival in patients with Stage III NSCLC treated with RT and should be recorded in clinical results when the survivals are compared among clinical studies.

  16. Apyrases, extracellular ATP and the regulation of growth.

    PubMed

    Clark, Greg; Roux, Stanley J

    2011-12-01

    Although no definitive receptor for extracellular ATP (eATP) has been identified in plants, there is now stronger physiological evidence that the effects of eATP on plant growth are mediated by a receptor, or, as in animals, by multiple receptors. Recent papers clarify how extracellular nucleotides induce changes in [Ca(2+)](cyt), and the production of nitric oxide (NO) and reactive oxygen species. They document links between eATP signaling and the synthesis or transport of hormones, and they reveal that applied nucleotides can regulate the aperture of stomates, which release ATP when stimulated by light and hormones. Ectoapyrases (ecto-nucleoside triphosphate-diphosphohydrolase) help control both the diverse signaling changes and downstream growth changes induced by extracellular nucleotides by limiting their concentration in the extracellular matrix (ECM). Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Curtains for ATP?

    NASA Astrophysics Data System (ADS)

    The administration's efforts to keep various technology-transfer programs afloat in the budget process appear to be stalled. House Science Committee chair Robert Walker (R-Pa.) advised in early April that the Republican agenda for the pending budget process entails zeroing out the Commerce Department's Advanced Technology Program (ATP), which was funded at 431 million in fiscal year 1995. The ATP would lose about 90 million from its FY 95 budget. Although Walker says that the Republican leadership has no intention to dictate to the subcommittees how cuts should be made, they will be held to the "fairly severe caps" established by the House Budget Committee. In other words, Walker says, if ATP stays, something else will have to go in its place. In addition, a bill to rescind about 223 million from the FY 1995 budget of the Technology Reinvestment Project and another 77 million from TRP's FY 1994 budget, which has not been spent, is heading for the president's signature. Yet Walker says while he supports the merits of technology transfer, "the question is do you have to create government programs to get the technology out?"

  18. Strategies for Primary Prevention of Coronary Heart Disease Based on Risk Stratification by the ACC/AHA Lipid Guidelines, ATP III Guidelines, Coronary Calcium Scoring, and C-Reactive Protein, and a Global Treat-All Strategy: A Comparative--Effectiveness Modeling Study

    PubMed Central

    Galper, Benjamin Z.; Wang, Y. Claire; Einstein, Andrew J.

    2015-01-01

    Background Several approaches have been proposed for risk-stratification and primary prevention of coronary heart disease (CHD), but their comparative and cost-effectiveness is unknown. Methods We constructed a state-transition microsimulation model to compare multiple approaches to the primary prevention of CHD in a simulated cohort of men aged 45–75 and women 55–75. Risk-stratification strategies included the 2013 American College of Cardiology/American Heart Association (ACC/AHA) guidelines on the treatment of blood cholesterol, the Adult Treatment Panel (ATP) III guidelines, and approaches based on coronary artery calcium (CAC) scoring and C-reactive protein (CRP). Additionally we assessed a treat-all strategy in which all individuals were prescribed either moderate-dose or high-dose statins and all males received low-dose aspirin. Outcome measures included CHD events, costs, medication-related side effects, radiation-attributable cancers, and quality-adjusted-life-years (QALYs) over a 30-year timeframe. Results Treat-all with high-dose statins dominated all other strategies for both men and women, gaining 15.7 million QALYs, preventing 7.3 million myocardial infarctions, and saving over $238 billion, compared to the status quo, far outweighing its associated adverse events including bleeding, hepatitis, myopathy, and new-onset diabetes. ACC/AHA guidelines were more cost-effective than ATP III guidelines for both men and women despite placing 8.7 million more people on statins. For women at low CHD risk, treat-all with high-dose statins was more likely to cause a statin-related adverse event than to prevent a CHD event. Conclusions Despite leading to a greater proportion of the population placed on statin therapy, the ACC/AHA guidelines are more cost-effective than ATP III. Even so, at generic prices, treating all men and women with statins and all men with low-dose aspirin appears to be more cost-effective than all risk-stratification approaches for the

  19. Is a unified definition of metabolic syndrome needed? Comparison of three definitions of metabolic syndrome in 60-year-old men and women.

    PubMed

    Carlsson, Axel C; Wändell, Per E; Halldin, Mats; de Faire, Ulf; Hellénius, Mai-Lis

    2009-06-01

    There are three commonly used definitions of the metabolic syndrome, making scientific studies hard to compare. The aim of this study was to investigate agreement in the prevalence of the metabolic syndrome defined by three different definitions and to analyze definition and gender differences. A population-based, cross-sectional study of a total of 4232 participants--2039 men and 2193 women, aged 60 years--was employed. Three different metabolic syndrome definitions were compared: European Group for the Study of Insulin Resistance (EGIR), International Diabetes Federation (IDF), and National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III). Medical history, socioeconomic information, and lifestyle data were collected by a questionnaire. A medical examination including laboratory tests was performed. Significant factors for the metabolic syndrome were calculated by multivariate logistic regression. Forty five percent of men and 30% of women met the criteria for the metabolic syndrome by any definition, but only 17% of men and 9% of women met the criteria of all three definitions. The highest agreement was found between IDF and NCEP ATP III definition. Two significant associations were identified in both men and women by the three metabolic syndrome definitions; former smokers were highly associated with the metabolic syndrome (odds ratio [OR] congruent with 1.5), and regular physical activity (OR congruent with 0.6) was inversely associated with the metabolic syndrome. Depending on the definition used, different individuals were identified as having the metabolic syndrome, which affects the reliability of interpretations to be made from scientific studies of the metabolic syndrome. Unified criteria are warranted. Physicians facing a physically inactive former smoker may consider diagnosing metabolic syndrome.

  20. Influence of Surveillance PET/CT on Detection of Early Recurrence After Definitive Radiation in Stage III Non-small-cell Lung Cancer.

    PubMed

    Reddy, Jay P; Tang, Chad; Shih, Tina; Kim, Bumyang; Kim, Charissa; Nguyen, Quynh-Nhu; Welsh, James; Benveniste, Marcelo; Zhang, Jianjun; Liao, Zhongxing; Gomez, Daniel R

    2017-03-01

    There are few data to support the use of varying imaging modalities in evaluating recurrence in non-small-cell lung cancer (NSCLC). We compared the efficacy of surveillance positron emission tomography (PET)/computed tomography (CT) versus CT scans of the chest in detecting recurrences after definitive radiation for NSCLC. We retrospectively analyzed 200 patients treated between 2000 and 2011 who met the inclusion criteria of stage III NSCLC, completion of definitive radiation treatment, and absence of recurrence within the initial 6 months. These patients were then grouped on the basis of the use of PET/CT imaging during postradiation surveillance. Patients who received ≥ 1 PET/CT scans within 6 months of the end of radiation treatment were placed in the PET group whereas all others were placed in the CT group. We compared survival times from the end of treatment to the date of death or last follow-up using log rank tests. Multivariate analysis was conducted to identify factors associated with decreased survival. In the entire cohort, median event-free survival (EFS) was 26.7 months, and median overall survival (OS) was 41.2 months. The CT group had a median EFS of 21.4 months versus 29.4 months for the PET group (P = .59). There was no difference in OS between the CT and PET groups (median OS of 41.2 and 41.3 months, respectively; P = .59). There was also no difference in local recurrence-free survival or distant metastases-free survival between the CT-only and PET/CT groups (P = .92 and P = .30, respectively). Similarly, in multivariate analysis, stratification into the PET group was not associated with improved EFS (hazard ratio [HR], 0.90; 95% confidence interval [CI], 0.61-1.34; P = .60) or OS (HR, 1.2; 95% CI, 0.83-1.7; P = .34). In stage III NSCLC patients treated with definitive radiation and without early recurrence, PET/CT scan surveillance did not result in decreased time to detection of locoregional or distant recurrence or improved survival

  1. Does the new International Diabetes Federation definition of metabolic syndrome improve prediction of coronary artery disease and carotid intima-media thickening?

    PubMed

    Timóteo, Ana; Santos, Rui; Lima, Sandra; Mamede, Andreia; Fernandes, Rita; Ferreira, Rui

    2009-02-01

    Metabolic syndrome (MS) is associated with increased incidence of diabetes and atherosclerotic complications. The new definition of the International Diabetes Federation (IDF) increases the population with this entity, compared to the NCEP ATP III definition. To study the prevalence of coronary artery disease (CAD) and carotid intima-media thickness (IMT) in patients with and without MS, according to the NCEP ATP III and IDF definitions, and the predictive ability of carotid IMT for CAD. We studied 270 consecutive patients admitted for elective coronary angiography due to suspicion of CAD. All patients underwent ultrasound study of the carotid arteries to measure IMT (the highest value between the right and left common carotid arteries was used in the analysis). Coronary stenosis of > or =70% (or 50% for the left main coronary artery) was considered significant. By the ATP III definition, 14% of the patients had MS, and these patients had a higher prevalence of CAD (87% vs. 63%, p = 0.004), but no significant difference was found for carotid IMT (1.03 +/- 0.36 mm vs. 0.95 +/- 0.35 mm, p=NS). With the IDF definition, 61% of the patients had MS; this group was slightly older and included more women. There were no differences in terms of CAD (68% vs. 63%) or carotid IMT (0.97 +/- 0.34 vs. 0.96 +/- 0.39 mm). On multivariate analysis, the ATP III definition of MS predicts CAD (OR 4.76, 95% CI 1.71-13.25, p = 0.003), but the IDF definition does not (OR 1.29, 95% CI 0.74-2.27, p = 0.37). On ROC curve analysis, an IMT of > or = 0.95 mm predicts CAD (AUC 0.66, p < 0.001), with a sensitivity of 52% and specificity of 75%. The new IDF definition increases the population with MS, decreasing the capacity to predict the presence of CAD. In our population, neither the ATP III nor the IDF definition showed differences in terms of carotid IMT. Carotid IMT can predict CAD, but with only modest sensitivity.

  2. 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.

  3. ATP release through pannexon channels

    PubMed Central

    Dahl, Gerhard

    2015-01-01

    Extracellular adenosine triphosphate (ATP) serves as a signal for diverse physiological functions, including spread of calcium waves between astrocytes, control of vascular oxygen supply and control of ciliary beat in the airways. ATP can be released from cells by various mechanisms. This review focuses on channel-mediated ATP release and its main enabler, Pannexin1 (Panx1). Six subunits of Panx1 form a plasma membrane channel termed ‘pannexon’. Depending on the mode of stimulation, the pannexon has large conductance (500 pS) and unselective permeability to molecules less than 1.5 kD or is a small (50 pS), chloride-selective channel. Most physiological and pathological stimuli induce the large channel conformation, whereas the small conformation so far has only been observed with exclusive voltage activation of the channel. The interaction between pannexons and ATP is intimate. The pannexon is not only the conduit for ATP, permitting ATP efflux from cells down its concentration gradient, but the pannexon is also modulated by ATP. The channel can be activated by ATP through both ionotropic P2X as well as metabotropic P2Y purinergic receptors. In the absence of a control mechanism, this positive feedback loop would lead to cell death owing to the linkage of purinergic receptors with apoptotic processes. A control mechanism preventing excessive activation of the purinergic receptors is provided by ATP binding (with low affinity) to the Panx1 protein and gating the channel shut. PMID:26009770

  4. Incidental receipt of cardiac medications and survival outcomes among patients with stage III non-small-cell lung cancer after definitive radiotherapy.

    PubMed

    Wang, Hongmei; Liao, Zhongxing; Zhuang, Yan; Liu, Ying; Levy, Lawrence B; Xu, Ting; Yusuf, Syed Wamique; Gomez, Daniel R

    2015-03-01

    Preclinical and epidemiologic studies suggest that receipt of some cardiac medications such as angiotensin-converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs), β-blockers, or aspirin may have antiproliferative effects in several types of cancer. The aim of this study was to estimate survival outcomes in patients receiving incidental cardiac medications during treatment for lung cancer, and to compare outcomes with those patients not receiving these medications. We retrospectively reviewed 673 patients who had received definitive radiotherapy for stage III non-small-cell lung cancer (NSCLC). Cox proportional hazard models were used to assess associations between receipt of ACEIs, ARBs, β-blockers, or aspirin and locoregional progression-free survival (LRPFS), distant metastasis-free survival (DMFS), disease-free survival (DFS), and overall survival (OS). Multivariate analyses showed that ACEI receipt was associated with poorer LRPFS but had no effect on DMFS, DFS, or OS. Aspirin receipt was associated only with improved DMFS, and β-blocker receipt was associated with improved DMFS, DFS, and OS. Incidental receipt of ACEIs was associated with a higher prevalence of local failure, whereas receipt of either β-blockers or aspirin had protective effects on survival outcomes in this large group of patients with lung cancer. This finding warrants further clinical and preclinical exploration, as it may have important implications for treating patients with lung cancer who are also receiving cardiac medications. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. [New definition of metabolic syndrome: does it have the same cardiovascular risk?].

    PubMed

    Rodilla, E; González, C; Costa, J A; Pascual, J M

    2007-02-01

    The International Diabetes Federation (IDF) has recently published the new criteria for the diagnosis of metabolic syndrome. The aim of this study was to compare the clinical characteristics and cardiovascular risk of the new patients with MS compared to the previous National Cholesterol Education Program ATP III definition, its differential characteristics and cardiovascular risk. Cross sectional study in a hypertension clinic. Coronary risk was calculated (Framingham function NCEP-ATP III) and other cardiovascular markers, urinary albumin excretion (UAE in mg/24 hours) and high sensitivity C-reactive protein (CRP) were assessed. A total 2,404 patients were evaluated, 1,901 non-diabetic and 503 diabetic hypertensive subjects. The non-diabetics 726 (38.2%) had MS with the previous NCEP ATP-III definition, the number increasing sharply to 1,091 (57.4%) with the new IDF definition. The proportion did not increase in diabetics (93% vs. 92%). Concordance in the diagnosis was 78% in non-diabetics and 91% in diabetics. The new patients had a similar coronary risk (Framingham) but lower values of other cardiovascular markers: logUAE 1.00 (0.49) mg/24 hours vs. 1.06 (0.55) mg/24 hours (p = 0.003), and CRP 1.9 (2.7) mg/L vs. 2.5 (3.2) mg/L (median, interquartile range; p < 0.001). The new IDF definition of MS increases the number of patients with MS. The new patients have a similar coronary risk (Framingham) but the new parameters used to assess cardiovascular risk (UAE and CRP) were lower. The relationship of the new definition of MS and cardiovascular risk remains to be defined.

  6. Structure of ATP-Bound Human ATP:Cobalamin Adenosyltransferase

    SciTech Connect

    Schubert,H.; Hill, C.

    2006-01-01

    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, thereby 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.

  7. Empirical derivation to improve the definition of the metabolic syndrome in the evaluation of cardiovascular disease risk.

    PubMed

    Wildman, Rachel P; McGinn, Aileen P; Kim, Mimi; Muntner, Paul; Wang, Dan; Cohen, Hillel W; Ogorodnikova, Alexandra D; Reynolds, Kristi; Fonseca, Vivian

    2011-03-01

    To examine whether a quantitatively derived metabolic syndrome definition predicts incident cardiovascular disease (CVD) events better than do existing definitions. Data were pooled from the Atherosclerosis Risk in Communities, Cardiovascular Health, and Framingham Offspring studies (n = 20,581). Incident coronary heart disease and stroke events were ascertained over 9 years. The sensitivity for incident CVD events was higher and the specificity lower for the empirically derived versus the Adult Treatment Panel (ATP) III, International Diabetes Federation (IDF), or Harmonized metabolic syndrome definitions (sensitivity/specificity 0.65/0.53 vs. 0.53/0.63, 0.51/0.66, and 0.64/0.56, respectively), resulting in no overall improvement in discrimination. Multivariable-adjusted hazard ratios for incident CVD events were similar across definitions and were 1.7 (95% CI 1.6-1.9) for ATP III, 1.8 (1.6-2.0) for IDF, 1.9 (1.7-2.0) for Harmonized, and 1.7 (1.6-1.9) for the empirically derived definition. Empirical derivation of the metabolic syndrome definition did not improve CVD discrimination or risk prediction.

  8. The ATP-dependent ClpXP and Lon proteases regulate expression of the Yersinia pestis type III secretion system via regulated proteolysis of YmoA, a small histone-like protein.

    PubMed

    Jackson, Michael W; Silva-Herzog, Eugenia; Plano, Gregory V

    2004-12-01

    The Yersinia pestis plasmid pCD1-encoded type III secretion system (T3SS) is essential for the pathogenicity of Y. pestis in mammalian hosts. T3SS-associated genes are maximally expressed at 37 degrees C in the absence of extracellular calcium. Expression of T3SS genes requires LcrF, an AraC-like transcriptional activator, and is repressed by YmoA, a small histone-like protein. The mechanism by which temperature regulates T3SS gene expression has not been determined; however, changes in DNA topology have been implicated in this process. We report here that a Y. pestis strain deficient in production of the ClpXP and Lon proteases does not express a functional T3SS partly because of high cytosolic levels of YmoA. YmoA is rapidly degraded at 37 degrees C in wild-type Y. pestis, but remains stable in a clpXPlon deletion mutant. The stability of YmoA in wild-type Y. pestis increased as the growth temperature of the culture decreased; in contrast, YmoA was stable at all temperatures examined in the clpXPlon deletion mutant. These results indicate that the ClpXP and Lon proteases contribute to the environmental regulation of the Y. pestis T3SS system through regulated proteolysis of YmoA.

  9. Comparison of the prevalence of metabolic syndrome and its association with diabetes and cardiovascular disease in the rural population of Bangladesh using the modified National Cholesterol Education Program Expert Panel Adult Treatment Panel III and International Diabetes Federation definitions

    PubMed Central

    Bhowmik, Bishwajit; Afsana, Faria; Siddiquee, Tasnima; Munir, Sanjida B; Sheikh, Fareeha; Wright, Erica; Bhuiyan, Farjana R; Ashrafuzzaman, Sheikh Mohammad; Mahtab, Hajera; Azad Khan, Abul Kalam; Hussain, Akhtar

    2015-01-01

    Aims/Introduction To compare the prevalence of metabolic syndrome (MS) using the modified National Cholesterol Education Program Adult Treatment Plan III (NCEP) and the International Diabetes Federation (IDF) definitions and, using both definitions, determine and compare the association of MS, prediabetes, type 2 diabetes, hypertension (HTN) and cardiovascular disease risk (CVD). Materials and Methods A total of 2,293 randomly selected participants (aged ≥20 years) in a rural community in Bangladesh were investigated in a population-based cross-sectional study. Sociodemographic and anthropometric characteristics, blood pressure, blood glucose, and lipid profiles were studied. Age-adjusted data for MS and cardiometabolic risk factors were assessed, and their relationships were examined. Results The age-adjusted prevalence of MS was 30.7% (males 30.5%; females 30.5%) using the NCEP definition, and 24.5% (males 19.2%, females 27.5%) using the IDF definition. The prevalence of MS using the NCEP definition was also higher in study participants with prediabetes, type 2 diabetes, HTN and CVD risk. The agreement rate between both definitions was 92% (k = 0.80). The NCEP definition had a stronger association with type 2 diabetes and HTN (odds ratio 12.4 vs 5.2; odds ratio 7.0 vs 4.7, respectively) than the IDF definition. However, the odds ratios for prediabetes and CVD risk were not significantly different. Conclusions The prevalence of MS was higher using the NCEP definition, and was more strongly associated with prediabetes, type 2 diabetes, HTN and CVD in this Bangladeshi population. PMID:25969712

  10. Stages III and IV Squamous Cell Carcinoma of the Mouth: Three-Year Experience with Superselective Intraarterial Chemotherapy Using Cisplatin Prior to Definitive Treatment

    SciTech Connect

    Hirai, Toshinori; Korogi, Yukunori; Hamatake, Satoshi; Nishimura, Ryuichi; Baba, Yuji; Takahashi, Mutsumasa; Uji, Yasuyoshi; Taen, Akira

    1999-05-15

    Purpose: This study was designed to assess the 3-year experience with superselective intraarterial chemotherapy prior to definitive treatment for stages III and IV squamous cell carcinomas of the mouth. Methods: Twenty-two patients prospectively received superselective intraarterial chemotherapy using relatively low-dose cisplatin via a transfemoral approach. The locations of the tumors were the tongue (n= 12), gingiva (n= 5), buccal mucosa (n= 2), hard palate (n= 1), floor of the mouth (n= 1), and lip (n= 1). After intraarterial chemotherapy, 21 patients underwent surgery (n= 14), radiation therapy (n= 6), or both (n= 1). The survival rate of 25 patients who underwent surgery with/without radiation therapy until 1992 at Kumamoto University Hospital was also evaluated as a historical control. The survival curve was calculated with the Kaplan-Meier method, and the statistical difference between survival curves was determined with the generalized Wilcoxon test. Results: The overall response rate was 95% [complete response (tumor completely resolved), 24%; partial response (tumor reduction {>=}50%), 71%]. Fifty-two intraarterial infusions were performed without any catheter-related complications. Mild and transient local toxicity such as edema or mucositis of the infused area was relatively common. One patient died of renal failure from cisplatin. After a median follow-up of 20 months (range 2-41 months), the estimated 3-year survival rate for patients who underwent intraarterial chemotherapy plus surgery was 91%. The survival of the patients who underwent intraarterial chemotherapy plus surgery tended to be longer than that of the historical control. Conclusions: Early tumor reduction without delay of subsequent treatments can be obtained by intraarterial chemotherapy while minimizing complications and possibly improving survival. Further investigations of long-term survival with larger series need to be performed.

  11. Treatment outcomes of and prognostic factors for definitive radiotherapy with and without chemotherapy for Stage I/II nasal extranodal NK/T-cell lymphoma

    PubMed Central

    Yang, Claire Wen-Chi; Wang, Chun-Wei; Hong, Ruey-Long; Tsai, Chiao-Ling; Yao, Ming; Tang, Jih-Luh; Lin, Chung-Wu; Cheng, Ann-Lii; Kuo, Sung-Hsin

    2017-01-01

    Treatment strategies for nasal extranodal NK/T-cell lymphoma (ENKTL), including sequential chemotherapy followed by radiotherapy (SCRT), concurrent chemoradiotherapy (CCRT), or radiotherapy alone (RT), remain varied. The purpose of this study was to assess the treatment outcome, the toxicity, and the potential prognostic factors for patients with early-stage nasal ENKTL treated using definitive RT (minimum of 50 Gy) with or without chemotherapy. From 1998 to 2014, 37 patients were included in the study. Eight patients were treated with RT alone, 1 with CCRT, and 28 with SCRT. Local regional control (LRC), progression-free survival (PFS), and overall survival (OS) were calculated using the Kaplan–Meier method. RT resulted in an overall response rate of 91.2%, with a complete response rate of 78.4%. After a median follow-up time of 36.8 months, the 3-year LRC, PFS and OS were 87.4%, 64.0% and 76.3%, respectively. Acute severe toxicity (Grade 3) of mucositis was observed in 6 (16.2%) of the 37 patients. In univariate analyses, extensive disease (Stage I/II with local invasiveness) and the presence of B symptoms were significantly associated with a poor PFS, whereas extensive disease was significantly associated with a poor OS. Multivariate analysis identified the presence of extensive disease as an independent predictor of PFS (P < 0.001) and OS (P = 0.015). High-dose RT with or without chemotherapy reported promising locoregional control and a favorable outcome for patients with early-stage nasal ENKTL without local invasiveness. Further investigation of new treatment strategies for patients with local invasiveness is warranted. PMID:27534792

  12. Comparison and correlation of binding mode of ATP in the kinase domains of Hexokinase family

    PubMed Central

    Kumar, Yellapu Nanda; Kumar, Pasupuleti Santhosh; Sowjenya, Gopal; Rao, Valasani Koteswara; Yeswanth, Sthanikam; Prasad, Uppu Venkateswara; Pradeepkiran, Jangampalli Adi; Sarma, PVGK; Bhaskar, Matcha

    2012-01-01

    Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family. PMID:22829728

  13. Evaluation of ATP measurements to detect microbial ingress by wastewater and surface water in drinking water.

    PubMed

    Vang, Óluva K; Corfitzen, Charlotte B; Smith, Christian; Albrechtsen, Hans-Jørgen

    2014-11-01

    Fast and reliable methods are required for monitoring of microbial drinking water quality in order to protect public health. Adenosine triphosphate (ATP) was investigated as a potential real-time parameter for detecting microbial ingress in drinking water contaminated with wastewater or surface water. To investigate the ability of the ATP assay in detecting different contamination types, the contaminant was diluted with non-chlorinated drinking water. Wastewater, diluted at 10(4) in drinking water, was detected with the ATP assay, as well as 10(2) to 10(3) times diluted surface water. To improve the performance of the ATP assay in detecting microbial ingress in drinking water, different approaches were investigated, i.e. quantifying microbial ATP or applying reagents of different sensitivities to reduce measurement variations; however, none of these approaches contributed significantly in this respect. Compared to traditional microbiological methods, the ATP assay could detect wastewater and surface water in drinking water to a higher degree than total direct counts (TDCs), while both heterotrophic plate counts (HPC 22 °C and HPC 37 °C) and Colilert-18 (Escherichia coli and coliforms) were more sensitive than the ATP measurements, though with much longer response times. Continuous sampling combined with ATP measurements displays definite monitoring potential for microbial drinking water quality, since microbial ingress in drinking water can be detected in real-time with ATP measurements. The ability of the ATP assay to detect microbial ingress is influenced by both the ATP load from the contaminant itself and the ATP concentration in the specific drinking water. Consequently, a low ATP concentration of the specific drinking water facilitates a better detection of a potential contamination of the water supply with the ATP assay.

  14. Energy transduction in ATP synthase

    NASA Astrophysics Data System (ADS)

    Elston, Timothy; Wang, Hongyun; Oster, George

    1998-01-01

    Mitochondria, bacteria and chloroplasts use the free energy stored in transmembrane ion gradients to manufacture ATP by the action of ATP synthase. This enzyme consists of two principal domains. The asymmetric membrane-spanning Fo portion contains the proton channel, and the soluble F1 portion contains three catalytic sites which cooperate in the synthetic reactions. The flow of protons through Fo is thought to generate a torque which is transmitted to F1 by an asymmetric shaft, the coiled-coil γ-subunit. This acts as a rotating `cam' within F1, sequentially releasing ATPs from the three active sites. The free-energy difference across the inner membrane of mitochondria and bacteria is sufficient to produce three ATPs per twelve protons passing through the motor. It has been suggested that this protonmotive force biases the rotor's diffusion so that Fo constitutes a rotary motor turning the γ shaft. Here we show that biased diffusion, augmented by electrostatic forces, does indeed generate sufficient torque to account for ATP production. Moreover, the motor's reversibility - supplying torque from ATP hydrolysis in F1 converts the motor into an efficient proton pump - can also be explained by our model.

  15. Association of metabolic risk factors with uncontrolled hypertension: comparison of the several definitions of metabolic syndrome.

    PubMed

    Cortez-Dias, Nuno; Martins, Susana R; Belo, Adriana; Fiuza, Manuela

    2013-10-01

    To evaluate the influence of metabolic syndrome in the effectiveness of antihypertensive treatment and to compare it using the National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) (2001 and 2004), International Diabetes Federation (IDF) and American Heart Association/National Heart, Lung and Blood Institute (AHA-NHLBI) definitions. The VALSIM (Estudo de Prevalência da Síndrome Metabólica) survey was designed as an observational cross-sectional study performed in a primary healthcare setting in Portugal. The first two adult patients scheduled for an appointment on a given day were invited to participate. The treatment effectiveness was evaluated by the occurrence of uncontrolled hypertension (≥140/90 mmHg) in patients taking antihypertensive drugs. Logistic regression analysis was used to determine the association between uncontrolled hypertension and metabolic risk factors, with adjustments for age, sex, and pattern of antihypertensive treatment. Among the 16,856 individuals evaluated, 8925-treated hypertensive patients were identified. Only 35.8% of them had controlled hypertension. The risk of poor blood pressure control increased with age, waist circumference, serum levels of triglycerides and HDL-cholesterol. Among treatable risk factors, metabolic syndrome as defined by NCEP-ATP III 2001 diagnostic criteria was the strongest independent predictor of uncontrolled hypertension (odds ratio: 1.23; 95% CI: 1.08-1.41; P=0.002). In opposition, the IDF or AHA-NHLBI definitions of metabolic syndrome failed to identify patients at risk of poor blood pressure control. Metabolic syndrome is associated with lower effectiveness of antihypertensive therapy and the NCEP-ATP III 2001 definition of metabolic syndrome is the one that better identifies patients at risk of poor blood pressure control.

  16. The 3'-5' exonuclease site of DNA polymerase III from gram-positive bacteria: definition of a novel motif structure.

    PubMed

    Barnes, M H; Spacciapoli, P; Li, D H; Brown, N C

    1995-11-07

    The primary structure of the 3'-5' exonuclease (Exo) site of the Gram+ bacterial DNA polymerase III (Pol III) was examined by site-directed mutagenesis of Bacillus subtilis Pol III (BsPol III). It was found to differ significantly from the conventional three-motif substructure established for the Exo site of DNA polymerase I of Escherichia coli (EcPol I) and the majority of other DNA polymerase-exonucleases. Motifs I and II were conventionally organized and anchored functionally by the predicted carboxylate residues. However, the conventional downstream motif, motif III, was replaced by motif III epsilon, a novel 55-amino-acid (aa) segment incorporating three essential aa (His565, Asp533 and Asp570) which are strictly conserved in three Gram+ Pol III and in the Ec Exo epsilon (epsilon). Despite its unique substructure, the Gram+ Pol III-specific Exo site was conventionally independent of Pol, the site of 2'-deoxyribonucleoside 5-triphosphate (dNTP) binding and polymerization. The entire Exo site, including motif III epsilon, could be deleted without profoundly affecting the enzyme's capacity to polymerize dNTPs. Conversely, Pol and all other sequences downstream of the Exo site could be deleted with little apparent effect on Exo activity. Whether the three essential aa within the unique motif III epsilon substructure participate in the conventional two-metal-ion mechanism elucidated for the model Exo site of EcPol I, remains to be established.

  17. Automated Proof Compression by Invention of New Definitions

    NASA Astrophysics Data System (ADS)

    Vyskočil, Jiří; Stanovský, David; Urban, Josef

    State-of-the-art automated theorem provers (ATPs) are today able to solve relatively complicated mathematical problems. But as ATPs become stronger and more used by mathematicians, the length and human unreadability of the automatically found proofs become a serious problem for the ATP users. One remedy is automated proof compression by invention of new definitions.

  18. Long-term results and recurrence patterns from SCOPE-1: a phase II/III randomised trial of definitive chemoradiotherapy +/− cetuximab in oesophageal cancer

    PubMed Central

    Crosby, T; Hurt, C N; Falk, S; Gollins, S; Staffurth, J; Ray, R; Bridgewater, J A; Geh, J I; Cunningham, D; Blazeby, J; Roy, R; Maughan, T; Griffiths, G; Mukherjee, S

    2017-01-01

    Background: The SCOPE-1 study tested the role of adding cetuximab to conventional definitive chemoradiotherapy (dCRT), and demonstrated greater toxicity and worse survival outcomes. We present the long-term outcomes and patterns of recurrence. Methods: SCOPE-1 was a phase II/III trial in which patients were randomised to cisplatin 60 mg m−2 (day 1) and capecitabine 625 mg m−2 bd (days 1–21) for four cycles +/− cetuximab 400 mg m−2 day 1 then by 250 mg m−2 weekly. Radiotherapy consisted of 50 Gy/25# given concurrently with cycles 3 and 4. Recruitment was between February 2008 and February 2012, when the IDMC recommended closure on the basis of futility. Results: About 258 patients (dCRT=129; dCRT+cetuximab (dCRT+C)=129) were recruited from 36 centres. About 72.9% (n=188) had squamous cell histology. The median follow-up (IQR) was 46.2 (35.9–48.3) months for surviving patients. The median overall survival (OS; months; 95% CI) was 34.5 (24.7–42.3) in dCRT and 24.7 (18.6–31.3) in dCRT+C (hazard ratio (HR)=1.25, 95% CIs: 0.93–1.69, P=0.137). Median progression-free survival (PFS; months; 95% CI) was 24.1 (15.3–29.9) and 15.9 (10.7–20.8) months, respectively (HR=1.28, 95% CIs: 0.94–1.75; P=0.114). On multivariable analysis only earlier stage, full-dose RT, and higher cisplatin dose intensity were associated with improved OS. Conclusions: The mature analysis demonstrates that the dCRT regimen used in the study provided useful survival outcomes despite its use in patients who were largely unfit for surgery or who had inoperable disease. Given the competing risk of systemic and local failure, future studies should continue to focus on enhancing local control as well as optimising systemic therapy. PMID:28196063

  19. Extracellular ATP signaling in plants

    PubMed Central

    Tanaka, Kiwamu; Gilroy, Simon; Jones, Alan M.; Stacey, Gary

    2016-01-01

    Extracellular adenosine-5′-triphosphate (ATP) induces a number of cellular responses in plants and animals. Some of the molecular components for purinergic signaling in animal cells appear to be lacking in plant cells, although some cellular responses are similar in both systems [e.g. increased levels of cytosolic free calcium, nitric oxide (NO), and reactive oxygen species (ROS)]. The purpose of this review is to compare and contrast purinergic signaling mechanisms in animal and plant cells. This comparison will aid our overall understanding of plant physiology and also provide details of the general fundamentals of extracellular ATP signaling in eukaryotes. PMID:20817461

  20. Two-step ATP-driven opening of cohesin head.

    PubMed

    Marcos-Alcalde, Íñigo; Mendieta-Moreno, Jesús I; Puisac, Beatriz; Gil-Rodríguez, María Concepción; Hernández-Marcos, María; Soler-Polo, Diego; Ramos, Feliciano J; Ortega, José; Pié, Juan; Mendieta, Jesús; Gómez-Puertas, Paulino

    2017-06-12

    The cohesin ring is a protein complex composed of four core subunits: Smc1A, Smc3, Rad21 and Stag1/2. It is involved in chromosome segregation, DNA repair, chromatin organization and transcription regulation. Opening of the ring occurs at the "head" structure, formed of the ATPase domains of Smc1A and Smc3 and Rad21. We investigate the mechanisms of the cohesin ring opening using techniques of free molecular dynamics (MD), steered MD and quantum mechanics/molecular mechanics MD (QM/MM MD). The study allows the thorough analysis of the opening events at the atomic scale: i) ATP hydrolysis at the Smc1A site, evaluating the role of the carboxy-terminal domain of Rad21 in the process; ii) the activation of the Smc3 site potentially mediated by the movement of specific amino acids; and iii) opening of the head domains after the two ATP hydrolysis events. Our study suggests that the cohesin ring opening is triggered by a sequential activation of the ATP sites in which ATP hydrolysis at the Smc1A site induces ATPase activity at the Smc3 site. Our analysis also provides an explanation for the effect of pathogenic variants related to cohesinopathies and cancer.

  1. 'Domino' systems biology and the 'A' of ATP.

    PubMed

    Verma, Malkhey; Zakhartsev, Maksim; Reuss, Matthias; Westerhoff, Hans V

    2013-01-01

    We develop a strategic 'domino' approach that starts with one key feature of cell function and the main process providing for it, and then adds additional processes and components only as necessary to explain provoked experimental observations. The approach is here applied to the energy metabolism of yeast in a glucose limited chemostat, subjected to a sudden increase in glucose. The puzzles addressed include (i) the lack of increase in adenosine triphosphate (ATP) upon glucose addition, (ii) the lack of increase in adenosine diphosphate (ADP) when ATP is hydrolyzed, and (iii) the rapid disappearance of the 'A' (adenine) moiety of ATP. Neither the incorporation of nucleotides into new biomass, nor steady de novo synthesis of adenosine monophosphate (AMP) explains. Cycling of the 'A' moiety accelerates when the cell's energy state is endangered, another essential domino among the seven required for understanding of the experimental observations. This new domino analysis shows how strategic experimental design and observations in tandem with theory and modeling may identify and resolve important paradoxes. It also highlights the hitherto unexpected role of the 'A' component of ATP. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

  2. Homeostasis of Extracellular ATP in Human Erythrocytes*

    PubMed Central

    Montalbetti, Nicolas; Leal Denis, Maria F.; Pignataro, Omar P.; Kobatake, Eiry; Lazarowski, Eduardo R.; Schwarzbaum, Pablo J.

    2011-01-01

    We explored the intra- and extracellular processes governing the kinetics of extracellular ATP (ATPe) in human erythrocytes stimulated with agents that increase cAMP. Using the luciferin-luciferase reaction in off-line luminometry we found both direct adenylyl cyclase activation by forskolin and indirect activation through β-adrenergic stimulation with isoproterenol-enhanced [ATP]e in a concentration-dependent manner. A mixture (3V) containing a combination of these agents and the phosphodiesterase inhibitor papaverine activated ATP release, leading to a 3-fold increase in [ATP]e, and caused increases in cAMP concentration (3-fold for forskolin + papaverine, and 10-fold for 3V). The pannexin 1 inhibitor carbenoxolone and a pannexin 1 blocking peptide (10Panx1) decreased [ATP]e by 75–84%. The residual efflux of ATP resulted from unavoidable mechanical perturbations stimulating a novel, carbenoxolone-insensitive pathway. In real-time luminometry experiments using soluble luciferase, addition of 3V led to an acute increase in [ATP]e to a constant value of ∼1 pmol × (106 cells)−1. A similar treatment using a surface attached luciferase (proA-luc) triggered a rapid accumulation of surface ATP levels to a peak concentration of 2.4 pmol × (106 cells)−1, followed by a slower exponential decay (t½ = 3.7 min) to a constant value of 1.3 pmol × (106 cells)−1. Both for soluble luciferase and proA-luc, ATP efflux was fully blocked by carbenoxolone, pointing to a 3V-induced mechanism of ATP release mediated by pannexin 1. Ecto-ATPase activity was extremely low (∼28 fmol × (106 cells min)−1), but nevertheless physiologically relevant considering the high density of erythrocytes in human blood. PMID:21921036

  3. A Review of the Definition and Measurement of Poverty: Volume I, Summary Review Paper; Volume II, Annotated Bibliography. The Measure of Poverty, Technical Paper III.

    ERIC Educational Resources Information Center

    Oster, Sharon; And Others

    This study reviews the existing literature on a series of issues associated with the defintion and measurement of poverty, and it consists of a summary report covering this research (Volume I), and an annotated bibliography (Volume II). Eleven specific issues were identified and reviewed in this study: (1) the historical definitions of poverty,…

  4. 75 FR 25294 - Notice Pursuant to the National Cooperative Research and Production Act of 1993-High Definition...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-07

    ... Definition Metrology and Process-2 Micron Manufacturing Under ATP Award No. 70NANB77041 Notice is hereby... Manufacturing under ATP Award No. 70NANB7H7041 has filed written notifications simultaneously with the...

  5. Comparison of coronary heart disease risk among four diagnostic definitions of metabolic syndrome.

    PubMed

    Suzuki, T; Zeng, Z; Zhao, B; Wei, Z; Tanabe, M; Shimbo, T; Kajio, H; Kato, N; Naruse, M

    2016-11-01

    Metabolic syndrome (MetS) is now well known as one of the major risk factors for coronary heart disease (CHD). Currently, there are several methods used to define MetS. The aim of this study was to determine to what extent current MetS definition reflects CHD risk using the probability of CHD in 10 years based on Framingham risk score algorithms. A total of 7575 adults, aged 16-93 years (2532 men and 5043 women), were recruited. We conducted a cross-sectional health survey in China using MetS criteria from four different definitions: modified National Cholesterol Education Program-Adult Treatment Panel III (NCEP-ATP III), International Diabetes Federation (IDF), Chinese and Japanese. Differences in the prevalence of MetS by each definition were small in males (22.9-25.9 %), whereas in females, MetS was three times more prevalent using the IDF definition (29.1 %) versus the Japanese definition (9.7 %). Framingham risk scores in participants with MetS were significantly higher than in those without MetS by all definition criteria (p < 0.001). The CHD risk scores for participants with MetS by each definition showed similar values in males (range 11.5-12.1 %) with no significant differences among definitions. Conversely, in females with MetS the risk score for CHD was low (range 3.5-4.3 %) by each MetS definition. These findings suggest that further studies are required to establish appropriate criteria of MetS in females.

  6. Is there role of additional chemotherapy after definitive local treatment for stage I/II marginal zone lymphoma?: Consortium for Improving Survival of Lymphoma (CISL) study.

    PubMed

    Koh, Myeong Seok; Kim, Won Seog; Kim, Seok Jin; Oh, Sung Yong; Yoon, Dok Hyun; Lee, Soon Il; Hong, Junshik; Song, Moo Kon; Shin, Ho-Jin; Kwon, Jung Hye; Kim, Hyo Jung; Do, Yong Rok; Suh, Cheolwon; Kim, Hyo Jin

    2015-10-01

    Even though local stage (Ann Arbor stage I/II) marginal zone lymphoma (MZL) is well controlled with local treatment-based therapy, no data exist on the role of additional chemotherapy after local treatment for stage I/II MZL. Patients with biopsy-confirmed Ann Arbor stage I/II MZL (n = 210) were included for analysis in this study. Of these, 180 patients (85.7 %) were stage I and 30 (14.3 %) were stage II. Most patients (n = 182, 86.7 %) were treated with a local modality including radiation therapy or surgery and 28 (13.3 %) received additional systemic chemotherapy after local treatment. The overall response rate was 98.3 % (95 % CI 96-100 %), with 187 complete responses and 20 partial responses. In the local treatment group, the mean progression-free survival (PFS) was 147.4 months (95 % CI 126.7-168.1 months) and the overall survival (OS) was 188.2 months (95 % CI 178.8-197.7 months). In the additional chemotherapy group, the mean PFS was 103.4 months (95 % CI 84.9-121.9 months) and the OS was 137.3 months (95 % CI 127.9-146.7 months). There was no difference between the two groups in OS (p = 0.836) and PFS (p = 0.695). Local stage MZL has a good clinical course and is well controlled with a local treatment modality without additional chemotherapy.

  7. Autocrine and paracrine roles for ATP and serotonin in mouse taste buds.

    PubMed

    Huang, Yijen A; Dando, Robin; Roper, Stephen D

    2009-11-04

    Receptor (type II) taste bud cells secrete ATP during taste stimulation. In turn, ATP activates adjacent presynaptic (type III) cells to release serotonin (5-hydroxytryptamine, or 5-HT) and norepinephrine (NE). The roles of these neurotransmitters in taste buds have not been fully elucidated. Here we tested whether ATP or 5-HT exert feedback onto receptor (type II) cells during taste stimulation. Our previous studies showed NE does not appear to act on adjacent taste bud cells, or at least on receptor cells. Our data show that 5-HT released from presynaptic (type III) cells provides negative paracrine feedback onto receptor cells by activating 5-HT(1A) receptors, inhibiting taste-evoked Ca(2+) mobilization in receptor cells, and reducing ATP secretion. The findings also demonstrate that ATP exerts positive autocrine feedback onto receptor (type II) cells by activating P2Y1 receptors and enhancing ATP secretion. These results begin to sort out how purinergic and aminergic transmitters function within the taste bud to modulate gustatory signaling in these peripheral sensory organs.

  8. ATP-Dependent Interactions between Escherichia coli Min Proteins and the Phospholipid Membrane In Vitro

    PubMed Central

    Lackner, Laura L.; Raskin, David M.; de Boer, Piet A. J.

    2003-01-01

    Proper placement of the division apparatus in Escherichia coli requires pole-to-pole oscillation of the MinC division inhibitor. MinC dynamics involves a membrane association-dissociation cycle that is driven by the activities of the MinD ATPase and the MinE topological specificity factor, which themselves undergo coupled oscillatory localization cycles. To understand the biochemical mechanisms underlying Min protein dynamics, we studied the interactions of purified Min proteins with phospholipid vesicles and the role of ATP in these interactions. We show that (i) the ATP-bound form of MinD (MinD.ATP) readily associates with phospholipid vesicles in the presence of Mg2+, whereas the ADP-bound form (MinD.ADP) does not; (ii) MinD.ATP binds membrane in a self-enhancing fashion; (iii) both MinC and MinE can be recruited to MinD.ATP-decorated vesicles; (iv) MinE stimulates dissociation of MinD.ATP from the membrane in a process requiring hydrolysis of the nucleotide; and (v) MinE stimulates dissociation of MinC from MinD.ATP-membrane complexes, even when ATP hydrolysis is blocked. The results support and extend recent work by Z. Hu et al. (Z. Hu, E. P. Gogol, and J. Lutkenhaus, Proc. Natl. Acad. Sci. USA 99:6761-6766, 2002) and support models of protein oscillation wherein MinE induces Min protein dynamics by stimulating the conversion of the membrane-bound form of MinD (MinD.ATP) to the cytoplasmic form (MinD.ADP). The results also indicate that MinE-stimulated dissociation of MinC from the MinC-MinD.ATP-membrane complex can, and may, occur prior to hydrolysis of the nucleotide. PMID:12533449

  9. Novel and recurrent ATP2A2 mutations in Japanese patients with Darier’s disease

    PubMed Central

    Noda, Kana; Takeichi, Takuya; Okuno, Yusuke; Takama, Hiromichi; Miura, Shunsuke; Kagami, Shinji; Hino, Haruko; Nakamura, Yuki; Fujio, Yumi; Konohana, Izumi; Otani, Ayako; Mukai, Hideki; Sugiura, Kazumitsu; Akiyama, Masashi

    2016-01-01

    ABSTRACT Darier’s disease (DD, keratosis follicularis: OMIM#124200) is an autosomal dominant skin disorder characterized by multiple dark brown keratotic plaques and warty papules covered by thick crusts. Most cases of DD are caused by mutations in ATP2A2, which is expressed in both the skin and the brain. ATP2A2 encodes the cardiac muscle SERCA2a protein and the ubiquitously expressed SERCA2b. SERCA2 plays an important role as a calcium pump. It is thought that a mutation in ATP2A2 causes dyskeratosis and abnormality of cell-cell adhesion. Here, we report five DD patients from five independent families who presented or were referred to the Nagoya University Hospital in the past five years. We detected five mutations in ATP2A2, including a previously unreported mutation. We observed no apparent genotype/phenotype correlation between types and sites of the ATP2A2 mutations and DD phenotypes in the present series of DD patients. Genetic diagnosis from ATP2A2 mutation search is useful for the definite diagnosis of DD, although it is difficult to predict the severity and prognosis of skin symptoms from the results of the ATP2A2 mutation analysis in DD patients. PMID:28008204

  10. Initiation of Purinergic Signaling by Exocytosis of ATP-containing Vesicles in Liver Epithelium*

    PubMed Central

    Feranchak, Andrew P.; Lewis, Matthew A.; Kresge, Charles; Sathe, Meghana; Bugde, Abhijit; Luby-Phelps, Kate; Antich, Peter P.; Fitz, J. Gregory

    2010-01-01

    Extracellular ATP represents an important autocrine/paracrine signaling molecule within the liver. The mechanisms responsible for ATP release are unknown, and alternative pathways have been proposed, including either conductive ATP movement through channels or exocytosis of ATP-enriched vesicles, although direct evidence from liver cells has been lacking. Utilizing dynamic imaging modalities (confocal and total internal reflection fluorescence microscopy and luminescence detection utilizing a high sensitivity CCD camera) at different scales, including confluent cell populations, single cells, and the intracellular submembrane space, we have demonstrated in a model liver cell line that (i) ATP release is not uniform but reflects point source release by a defined subset of cells; (ii) ATP within cells is localized to discrete zones of high intensity that are ∼1 μm in diameter, suggesting a vesicular localization; (iii) these vesicles originate from a bafilomycin A1-sensitive pool, are depleted by hypotonic exposure, and are not rapidly replenished from recycling of endocytic vesicles; and (iv) exocytosis of vesicles in response to cell volume changes depends upon a complex series of signaling events that requires intact microtubules as well as phosphoinositide 3-kinase and protein kinase C. Collectively, these findings are most consistent with an essential role for exocytosis in regulated release of ATP and initiation of purinergic signaling in liver cells. PMID:20071341

  11. Epidermal Growth Factor Receptor Mutation Is Associated With Longer Local Control After Definitive Chemoradiotherapy in Patients With Stage III Nonsquamous Non–Small-Cell Lung Cancer

    SciTech Connect

    Yagishita, Shigehiro; Horinouchi, Hidehito; Katsui Taniyama, Tomoko; Nakamichi, Shinji; Kitazono, Satoru; Mizugaki, Hidenori; Kanda, Shintaro; Fujiwara, Yutaka; Nokihara, Hiroshi; Yamamoto, Noboru; Sumi, Minako; Shiraishi, Kouya; Kohno, Takashi; Furuta, Koh; Tsuta, Koji; Tamura, Tomohide

    2015-01-01

    Purpose: To determine the frequency and clinical significance of epidermal growth factor receptor (EGFR) mutations in patients with potentially curable stage III non–small-cell lung cancer (NSCLC) who are eligible for definitive chemoradiotherapy (CRT). Patients and Methods: Between January 2001 and December 2010, we analyzed the EGFR mutational status in consecutive NSCLC patients who were treated by CRT. The response rate, relapse-free survival, 2-year relapse-free rate, initial relapse sites, and overall survival of the patients were investigated. Results: A total of 528 patients received CRT at our hospital during the study period. Of these, 274 were diagnosed as having nonsquamous NSCLC. Sufficient specimens for mutational analyses could be obtained from 198 of these patients. The proportion of patients with EGFR activating mutations was 17%. In addition to the well-known characteristics of patients carrying EGFR mutations (female, adenocarcinoma, and never/light smoker), the proportion of cases with smaller primary lesions (T1/2) was found to be higher in patients with EGFR mutations than in those with wild-type EGFR. Patients with EGFR mutations showed similar response rate, relapse-free survival, and 2-year relapse-free rates as compared to patients with wild-type EGFR. Local relapses as the site of initial relapse occurred significantly less frequently in patients with EGFR mutation (4% vs 21%; P=.045). Patients with EGFR mutations showed longer local control (adjusted hazard ratio 0.49; P=.043). After disease progression, a majority of the patients with EGFR mutations received EGFR tyrosine kinase inhibitors (62%), and these patients showed longer postprogression survival than those with wild-type EGFR. Conclusions: Our study is the first to show radiosensitive biology of EGFR-mutated tumors in definitive CRT with curative intent. This finding could serve as a credible baseline estimate of EGFR-mutated population in stage III nonsquamous NSCLC.

  12. 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.

  13. Detecting ATP release by a biosensor method.

    PubMed

    Hayashi, Seiji; Hazama, Akihiro; Dutta, Amal K; Sabirov, Ravshan Z; Okada, Yasunobu

    2004-11-09

    Cells release adenosine 5'-triphosphate (ATP) into the extracellular space in response to various stimuli. This released ATP plays an important physiological role in cell-to-cell signal transduction. The bulk ATP concentration can be detected using a conventional luciferin-luciferase assay. However, the ATP concentration in the vicinity of the cell surface is often different from the bulk concentration because of its rapid degradation by ecto-ATPases and because of delayed diffusion due to unstirred layer effects. Here, we describe a simple biosensor method to measure the local ATP concentration on the cell surface in real time. The method is based on the ATP-dependent opening of ligand-gated cation channels of purinergic P2X receptors expressed in undifferentiated pheochromocytoma (PC12) cells or in human embryonic kidney 293 (HEK293) cells stably transfected with recombinant P2X2 purinergic receptors. Under the whole-cell configuration of patch-clamp, a sensor PC12 cell or HEK293 is positioned within the proximity of a target cell, and the P2X-mediated currents induced by ATP released from a given site on the target cell surface is measured. The ATP release is quantified by a calibration procedure utilizing local puff applications of ATP at preset concentrations.

  14. Structure of complex III with bound cytochrome c in reduced state and definition of a minimal core interface for electron transfer.

    PubMed

    Solmaz, Sozanne R N; Hunte, Carola

    2008-06-20

    In cellular respiration, cytochrome c transfers electrons from cytochrome bc(1) complex (complex III) to cytochrome c oxidase by transiently binding to the membrane proteins. Here, we report the structure of isoform-1 cytochrome c bound to cytochrome bc(1) complex at 1.9 A resolution in reduced state. The dimer structure is asymmetric. Monovalent cytochrome c binding is correlated with conformational changes of the Rieske head domain and subunit QCR6p and with a higher number of interfacial water molecules bound to cytochrome c(1). Pronounced hydration and a "mobility mismatch" at the interface with disordered charged residues on the cytochrome c side are favorable for transient binding. Within the hydrophobic interface, a minimal core was identified by comparison with the novel structure of the complex with bound isoform-2 cytochrome c. Four core interactions encircle the heme cofactors surrounded by variable interactions. The core interface may be a feature to gain specificity for formation of the reactive complex.

  15. 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

  16. Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*

    PubMed Central

    Villamor, Joji Grace; Kaschani, Farnusch; Colby, Tom; Oeljeklaus, Julian; Zhao, David; Kaiser, Markus; Patricelli, Matthew P.; van der Hoorn, Renier A. L.

    2013-01-01

    Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188. PMID:23722185

  17. Pyrazinoic acid decreases the proton motive force, respiratory ATP synthesis activity, and cellular ATP levels.

    PubMed

    Lu, Ping; Haagsma, Anna C; Pham, Hoang; Maaskant, Janneke J; Mol, Selena; Lill, Holger; Bald, Dirk

    2011-11-01

    Pyrazinoic acid, the active form of the first-line antituberculosis drug pyrazinamide, decreased the proton motive force and respiratory ATP synthesis rates in subcellular mycobacterial membrane assays. Pyrazinoic acid also significantly lowered cellular ATP levels in Mycobacterium bovis BCG. These results indicate that the predominant mechanism of killing by this drug may operate by depletion of cellular ATP reserves.

  18. The Prevalence of Metabolic Syndrome According to Various Definitions and Hypertriglyceridemic-Waist in Malaysian Adults

    PubMed Central

    Zainuddin, Laila Ruwaida Mohd; Isa, NurFirdaus; Muda, Wan Manan Wan; Mohamed, Hamid Jan

    2011-01-01

    Objectives: Metabolic syndrome can be diagnosed according to several different criteria such as the latest International Diabetes Federation (IDF), National Cholesterol Education Program Adult Treatment Program III (NCEP ATPIII), and World Health Organization (WHO). The objectives of this study were to determine the prevalence of metabolic syndrome and the concordance between the above mentioned definition, and hypertriglyceridemic-waist criteria. Methods: This cross sectional study was done in Bachok, Malaysia and involved 298 respondents aged between 18 to 70 years. Multistage random sampling method was used to identify study locations while convenient random sampling method was applied to select individuals. Hypertriglyceridemic waist was defined from an internationally acceptable cut-off criterion. Kappa statistic (κ test) was used to determine the concordance between various definitions and hypertriglyceridemic-waist. Results: The prevalence of metabolic syndrome based on different definitions was 32.2% (IDF), 28.5% (NCEP ATP III) and 12.4% (modified WHO). The prevalence of hypertriglyceridemic-waist was 19.7% and based on the IDF criteria a total of 97.5% participants with hypertriglyceridemic-waist had metabolic syndrome. The IDF criteria showed the highest concordance with NCEP ATPIII criteria (κ = 0.63), followed by hypertriglyceridemic-waist criteria (κ = 0.62) and WHO criteria (κ = 0.26). Conclusions: The prevalence of metabolic syndrome was highest using the IDF criteria compared to NCEP ATPIII, modified WHO and hypertriglyceridemic-waist. There was a good concordance of IDF criteria with NCEP ATP III and hypertriglyceridemic-waist criteria. PMID:22174962

  19. Catalytic strategy used by the myosin motor to hydrolyze ATP.

    PubMed

    Kiani, Farooq Ahmad; Fischer, Stefan

    2014-07-22

    Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (Pi) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantum-classical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose γ-phosphate is not in the previously reported HPγO4(2-) state, but in the H2PγO4(-) state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the γ-phosphate of ATP in a dissociated metaphosphate (PγO3(-)) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes.

  20. ATP-triggered anticancer drug delivery

    NASA Astrophysics Data System (ADS)

    Mo, Ran; Jiang, Tianyue; Disanto, Rocco; Tai, Wanyi; Gu, Zhen

    2014-03-01

    Stimuli-triggered drug delivery systems have been increasingly used to promote physiological specificity and on-demand therapeutic efficacy of anticancer drugs. Here we utilize adenosine-5'-triphosphate (ATP) as a trigger for the controlled release of anticancer drugs. We demonstrate that polymeric nanocarriers functionalized with an ATP-binding aptamer-incorporated DNA motif can selectively release the intercalating doxorubicin via a conformational switch when in an ATP-rich environment. The half-maximal inhibitory concentration of ATP-responsive nanovehicles is 0.24 μM in MDA-MB-231 cells, a 3.6-fold increase in the cytotoxicity compared with that of non-ATP-responsive nanovehicles. Equipped with an outer shell crosslinked by hyaluronic acid, a specific tumour-targeting ligand, the ATP-responsive nanocarriers present an improvement in the chemotherapeutic inhibition of tumour growth using xenograft MDA-MB-231 tumour-bearing mice. This ATP-triggered drug release system provides a more sophisticated drug delivery system, which can differentiate ATP levels to facilitate the selective release of drugs.

  1. ATP in the pathogenesis of lung emphysema.

    PubMed

    Mortaz, Esmaeil; Braber, Saskia; Nazary, Maiwand; Givi, Masoumh Ezzati; Nijkamp, Frans P; Folkerts, Gert

    2009-10-01

    Extracellular ATP is a signaling molecule that often serves as a danger signal to alert the immune system of tissue damage. This molecule activates P2 nucleotide receptors, that include the ionotropic P2X receptors and metabotropic P2Y receptors. Recently, it has been reported that ATP accumulates in the airways of both asthmatic patients and sensitized mice after allergen challenge. The role and function of ATP in the pathogenesis of chronic obstructive pulmonary diseases (COPD) are not well understood. In this study we investigated the effect of cigarette smoke on purinergic receptors and ATP release by neutrophils. Neutrophils and their mediators are key players in the pathogenesis of lung emphysema. Here we demonstrated that in an in vivo model of cigarette smoke-induced lung emphysema, the amount of ATP was increased in the bronchoalveolar lavage fluid. Moreover, activation of neutrophils with cigarette smoke extract induced ATP release. Treatment of neutrophils with apyrase (catalyses the hydrolysis of ATP to yield AMP) and suramin (P2-receptor antagonist) abrogated the release of CXCL8 and elastase induced by cigarette smoke extract and exogenous ATP. These observations indicate that activation of purinergic signaling by cigarette smoke may take part in the pathogenesis of lung emphysema.

  2. ATP synthase: two motors, two fuels.

    PubMed

    Oster, G; Wang, H

    1999-04-15

    FoF1 ATPase is the universal protein responsible for ATP synthesis. The enzyme comprises two reversible rotary motors: Fo is either an ion 'turbine' or an ion pump, and F1 is either a hydrolysis motor or an ATP synthesizer. Recent biophysical and biochemical studies have helped to elucidate the operating principles for both motors.

  3. [ATP in the metabolism of ruminants].

    PubMed

    Bergner, H

    1991-10-01

    The ATP yield from the carbohydrates of anaerobically living microorganisms in the rumen amounts to only 5-10% of the ATP yield of the intermediary metabolism in the presence of oxygen. Vital functions and thus microbial protein synthesis are due to protein degradation in the rumen. The ATP yield in the intermediary metabolism of ruminants is mainly achieved from propionate and microbial protein by means of gluconeogenesis because the absorption of glucose from digested starch is very low. The relationships between ATP yield in the rumen and the processes of glucose provision for the production of lactose as well as the protein content of the milk are shown. As important processes of ATP production in microorganisms from easily soluble carbohydrates take place in silage preparations before feed intake, the corresponding consequences for the metabolism of high-performance cows fed with silage are shown.

  4. Analysis of the influence of nucleotidases on the apparent activity of exogenous ATP and ADP at P2Y1 receptors

    PubMed Central

    Vigne, Paul; Philippe Breittmayer, Jean; Frelin, Christian

    1998-01-01

    ADP is a potent agonist of rat and human P2Y1 purinoceptors. ATP is a weak competitive antagonist. This study analyses the situation in which P2Y1 receptors are exposed to ATP in the presence of exogenous ecto-nucleotidases (apyrases) that have high or low ATPase/ADPase activity ratio.Rat brain capillary endothelial cells of the B10 clone express P2Y1 receptors that couple to intracellular Ca2+ mobilization. They have low endogenous ecto-ATPase and ecto-ADPase activities.ATP did not raise intracellular Ca2+ in B10 cells. Addition of apyrases III or VII (1 u ml−1) to ATP treated cells induced large intracellular Ca2+ transients. Apyrases had no action in the absence of ATP.A 1 u ml−1 apyrase III solution generated 20 μM ADP from 0.1 mM ATP within 15 s. This concentration of ADP was sufficient to produce maximal activation of P2Y1 receptors.ATP was a full agonist of P2Y1 receptors in the presence of 1 u ml−1 apyrase III. Dose response curves for the apparent actions of ATP were bell shaped in the presence of 0.1 u ml−1 apyrase III. Apyrase III did not alter ADP dose response curves when coincubated with ADP for 15 s.Apyrase VII (1 u ml−1) shifted dose response curves for the actions of ADP to larger concentrations. It induced a bell shaped ATP dose response curve.Results suggest that ATPDases prevent P2Y1 receptor activation by degrading ADP but may contribute to P2Y1 receptor activation by generating ADP from ATP. PMID:9831901

  5. Metabolic syndrome in Mexican children: Low effectiveness of diagnostic definitions.

    PubMed

    Peña-Espinoza, Barbara Itzel; Granados-Silvestre, María de Los Ángeles; Sánchez-Pozos, Katy; Ortiz-López, María Guadalupe; Menjivar, Marta

    Early identification of children with metabolic syndrome (MS) is essential to decrease the risk of developing diabetes and cardiovascular disease in adulthood. Detection of MS is however challenging because of the different definitions for diagnosis; as a result, preventive actions are not taken in some children at risk. The study objective was therefore to compare prevalence of MS in children according to the IDF, NCEP-ATP-III, Cook, de Ferranti and Weiss definitions, considering insulin resistance (IR) markers such as HOMA-IR and/or metabolic index (MI). A total of 508 Mexican children (aged 9 to 13 years) from seven schools were enrolled in a cross-sectional study. Somatometric, biochemical, and hormonal measurements were evaluated. Frequency of MS was 2.4-45.9% depending on the definition used. Frequency of IR in children not diagnosed with MS was 12.4-25.2% using HOMA-IR and 4.0-16.3% using MI. When HOMA-IR or MI was included in each of the definitions, frequency of MS was 8.5-50.2% and 7.7-46.9% respectively. The kappa value including HOMA-IR and/or MI was greater than 0.8. This study demonstrated the poor effectiveness of the current criteria used to diagnose MS in Mexican children, as shown by the variability in the definitions and by the presence of IR in children who not diagnosed with MS. Inclusion of HOMA-IR and/or MI in definitions of MS (thus increasing agreement between them) decreases the chance of excluding children at risk and allows for MS prevalence between populations. Copyright © 2017 SEEN y SED. Publicado por Elsevier España, S.L.U. All rights reserved.

  6. Phase I/II trial of definitive carbon ion radiotherapy for prostate cancer: evaluation of shortening of treatment period to 3 weeks

    PubMed Central

    Nomiya, T; Tsuji, H; Maruyama, K; Toyama, S; Suzuki, H; Akakura, K; Shimazaki, J; Nemoto, K; Kamada, T; Tsujii, H

    2014-01-01

    Background: The purpose of this study was to evaluate the feasibility of a new shortened 3-week treatment schedule of carbon ion radiotherapy (CIRT) for prostate cancer. Methods: Beginning in May 2010, patients with T1b–T3bN0M0, histologically proven prostate adenocarcinoma were enrolled in the phase II trial of CIRT. Patients received 51.6 GyE in 12 fractions over 3 weeks (protocol 1002). The primary end point was defined as the incidence of late adverse events that were evaluated based on the Common Terminology Criteria for Adverse Events version 4.0. Biochemical failure was determined using the Phoenix definition (nadir +2.0 ng ml−1). Results: Forty-six patients were enrolled, and all patients were included in the analysis. The number of low-, intermediate-, and high-risk patients was 12 (26%), 9 (20%), and 25 (54%), respectively. The median follow-up period of surviving patients was 32.3 months. Two patients had intercurrent death without recurrence, and the remaining 44 patients were alive at the time of this analysis. In the analysis of late toxicities, grade 1 (G1) rectal haemorrhage was observed in 3 (7%) patients. The incidence of G1 haematuria was observed in 6 (13%) patients, and G1 urinary frequency was observed in 17 (37%) patients. No ⩾G2 late toxicities were observed. In the analysis of acute toxicities, 2 (4%) patients showed G2 urinary frequency, and no other G2 acute toxicities were observed. Conclusions: The new shortened CIRT schedule over 3 weeks was considered as feasible. The analysis of long-term outcome is warranted. PMID:24722181

  7. EP2 receptors mediate airway relaxation to substance P, ATP, and PGE2.

    PubMed

    Fortner, C N; Breyer, R M; Paul, R J

    2001-08-01

    Substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of constricted mouse tracheal smooth muscle. Relaxation to either SP or ATP is blocked by indomethacin, but the specific eicosanoid(s) involved have not been definitively identified. SP and ATP are reported to release PGE2 from airway epithelium in other species, suggesting PGE2 as a likely mediator in epithelium-dependent airway relaxation. Using mice homozygous for a gene-targeted deletion of the EP2 receptor [EP2(-/-)], one of the PGE2 receptors, we tested the hypothesis that PGE2 is the primary mediator of relaxation to SP or ATP. Relaxation in response to SP or ATP was significantly reduced in tracheas from EP2(-/-) mice. There were no differences between EP2(-/-) and wild-type tracheas in their physical dimensions, contraction to ACh, or relaxation to isoproterenol, thus ruling out any general alterations of smooth muscle function. There were also no differences between EP2(-/-) and wild-type tracheas in basal or stimulated PGE2 production. Exogenous PGE2 produced significantly less relaxation in EP2(-/-) tracheas compared with the wild type. Taken together, this experimental evidence supports the following two conclusions: EP2 receptors are of primary importance in airway relaxation to PGE2 and relaxation to SP or ATP is mediated through PGE2 acting on EP2 receptors.

  8. 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

  9. Metal-Dependent Regulation of ATP7A and ATP7B in Fibroblast Cultures

    PubMed Central

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz; Jensen, Thomas G.; Møller, Lisbeth B.

    2016-01-01

    Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured the expression level of the two genes at various concentrations of iron, copper, and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and 10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency obtained by treatment with the copper chelator led to a downregulation of ATP7A in the control fibroblasts, but surprisingly not in the WD fibroblasts. In contrast, the addition of copper led to an increased expression of ATP7A, but a decreased expression of ATP7B. Thus, whereas similar regulation patterns for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6–8 weeks led to clonal selection. Cells that express the normal ATP7A allele had a selective growth advantage at high copper concentrations, whereas more surprisingly, cells that express the mutant ATP7A allele had a selective growth advantage at low copper concentrations. Thus, although the transcription of ATP7A is regulated by copper, clonal growth selection in mosaic cell cultures is affected by the level of copper. Female carriers of MD are rarely affected probably due to a skewed inactivation of the X-chromosome bearing the ATP7A mutation. PMID:27587995

  10. 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.

  11. 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.

  12. 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.

  13. Cleanup MAC and MBA code ATP

    SciTech Connect

    Russell, V.K.

    1994-10-17

    The K Basins Materials Accounting (MAC) and Material Balance (MBA) database system had some minor code cleanup performed to its code. This ATP describes how the code was to be tested to verify its correctness.

  14. The Rotary Mechanism of the ATP Synthase

    PubMed Central

    Nakamoto, Robert K.; Scanlon, Joanne A. Baylis; Al-Shawi, Marwan K.

    2008-01-01

    The FOF1 ATP synthase is a large complex of at least 22 subunits, more than half of which are in the membranous FO sector. This nearly ubiquitous transporter is responsible for the majority of ATP synthesis in oxidative and photo-phosphorylation, and its overall structure and mechanism have remained conserved throughout evolution. Most examples utilize the proton motive force to drive ATP synthesis except for a few bacteria, which use a sodium motive force. A remarkable feature of the complex is the rotary movement of an assembly of subunits that plays essential roles in both transport and catalytic mechanisms. This review addresses the role of rotation in catalysis of ATP synthesis/hydrolysis and the transport of protons or sodium. PMID:18515057

  15. Electrophysiology of autonomic neuromuscular transmission involving ATP.

    PubMed

    Sneddon, P

    2000-07-03

    Electrophysiological investigations of autonomic neuromuscular transmission have provided great insights into the role of ATP as a neurotransmitter. Burnstock and Holman made the first recordings of excitatory junction potentials (e.j.p.s) produced by sympathetic nerves innervating the smooth muscle of the guinea-pig vas deferens. This led to the identification of ATP as the mediator of e.j.p.s in this tissue, where ATP acts as a cotransmitter with noradrenaline. The e.j.p.s are mediated solely by ATP acting on P2X(1) receptors leading to action potentials and a rapid phasic contraction, whilst noradrenaline mediates a slower, tonic contraction which is not dependent on membrane depolarisation. Subsequent electrophysiological studies of the autonomic innervation of smooth muscles of the urogenital, gastrointestinal and cardiovascular systems have revealed a similar pattern of response, where ATP mediates a fast electrical and mechanical response, whilst another transmitter such as noradrenaline, acetylcholine, nitric oxide or a peptide mediates a slower response. The modulation of junction potentials by a variety of pre-junctional receptors and the mechanism of inactivation of ATP as a neurotransmitter will also be described.

  16. A tenth atp gene and the conserved atpI gene of a Bacillus atp operon have a role in Mg2+ uptake

    PubMed Central

    Hicks, David B.; Wang, ZhenXiong; Wei, Yi; Kent, Rebecca; Guffanti, Arthur A.; Banciu, Horia; Bechhofer, David H.; Krulwich, Terry A.

    2003-01-01

    The atp operon of alkaliphilic Bacillus pseudofirmus OF4, as in most prokaryotes, contains the eight structural genes for the F-ATPase (ATP synthase), which are preceded by an atpI gene that encodes a membrane protein of unknown function. A tenth gene, atpZ, has been found in this operon, which is upstream of and overlapping with atpI. Most Bacillus species, and some other bacteria, possess atpZ homologues. AtpZ is predicted to be a membrane protein with a hairpin topology, and was detected by Western analyses. Deletion of atpZ, atpI, or atpZI from B. pseudofirmus OF4 led to a requirement for a greatly increased concentration of Mg2+ for growth at pH 7.5. Either atpZ, atpI, or atpZI complemented the similar phenotype of a triple mutant of Salmonella typhimurium (MM281), which is deficient in Mg2+ uptake. atpZ and atpI, separately and together, increased the Mg2+-sensitive 45Ca2+ uptake by vesicles of an Escherichia coli mutant that is defective in Ca2+ and Na+ efflux. We hypothesize that AtpZ and AtpI, as homooligomers, and perhaps as heterooligomers, are Mg2+ transporter, Ca2+ transporter, or channel proteins. Such proteins could provide Mg2+, which is required by ATP synthase, and also support charge compensation, when the enzyme is functioning in the hydrolytic direction; e.g., during cytoplasmic pH regulation. PMID:12917488

  17. SAGE III

    Atmospheric Science Data Center

    2017-01-13

    SAGE III Data and Information The Stratospheric Aerosol and Gas ... on the spacecraft. SAGE III produced L1 and L2 scientific data from 5/07/2002 until 12/31/2005. The flight of the second instrument is as ... Additional Info:  Data Format: HDF-EOS or Big Endian/IEEE Binary SCAR-B Block:  ...

  18. 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

  19. 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

  20. Cyclic AMP regulates bicarbonate secretion in cholangiocytes through release of ATP into bile

    PubMed Central

    Minagawa, Noritaka; Nagata, Jun; Shibao, Kazunori; Masyuk, Anatoliy I.; Gomes, Dawidson A.; Rodrigues, Michele A.; LeSage, Gene; Akiba, Yasutada; Kaunitz, Jonathan D.; Ehrlich, Barbara E.; LaRusso, Nicholas F.; Nathanson, Michael H.

    2007-01-01

    Background & Aims Bicarbonate secretion is a primary function of cholangiocytes. Either cAMP or cytosolic Ca2+ can mediate bicarbonate secretion, but these are thought to act through separate pathways. We examined the role of the inositol 1,4,5-trisphosphate receptor (InsP3R) in mediating bicarbonate secretion, because this is the only intracellular Ca2+ release channel in cholangiocytes. Methods Intrahepatic bile duct units (IBDUs) were microdissected from rat liver, then luminal pH was examined by confocal microscopy during IBDU microperfusion. Cyclic AMP was increased using forskolin or secretin, and Ca2+ was increased using acetylcholine (ACh) or ATP. Apyrase was used to hydrolyze extracellular ATP, and suramin was used to block apical P2Y ATP receptors. In selected experiments IBDU were pre-treated with siRNA to silence expression of specific InsP3R isoforms. Results Both cAMP and Ca2+ agonists increased luminal pH. The effect of ACh on luminal pH was reduced by siRNA for basolateral (types I and II) but not apical (type III) InsP3R isoforms. The effect of forskolin on luminal pH was reduced by a CFTR inhibitor and by siRNA for the type III InsP3R. Luminal apyrase or suramin blocked the effects of forskolin but not ACh on luminal pH. Conclusions Cyclic AMP-induced ductular bicarbonate secretion depends upon an autocrine signaling pathway that involves CFTR, apical release of ATP, stimulation of apical nucleotide receptors, and then activation of apical, type III InsP3Rs. The primary role of CFTR in bile duct secretion may be to regulate secretion of ATP rather than to secrete chloride and/or bicarbonate. PMID:17916355

  1. Comparing the predictive abilities of different metabolic syndrome definitions for acute coronary syndrome: a case-control study in Chinese adults.

    PubMed

    Wang, Qun; Chair, Sek Ying; Wong, Eliza Mi Ling; Li, Xiaomei; Liu, Meili; Zhang, Yulian

    2014-09-01

    Different institutions have proposed various definitions for metabolic syndrome, which is a combination of risk factors for cardiovascular diseases (CVD). This study aimed to compare the feasibilities and abilities of different metabolic syndrome definitions in predicting acute coronary syndrome (ACS) in Chinese adults. A case-control study was designed. This study recruited 162 newly diagnosed ACS patients (the case group) and 162 non-ACS patients (the control group) according to the study criteria. Metabolic syndrome definitions proposed by the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III), International Diabetes Federation (IDF), American Heart Association/National Heart, Lung, and Blood Institute (AHA/NHLBI), Chinese Diabetes Society (CDS), and Joint Committee for Developing Chinese Guidelines on Dyslipidemia in Adults (JCDCG) were studied. After collecting demographic and clinical data, sensitivity, specificity, positive and negative predictive values (PPV, NPV), the likelihood ratio of a positive test and a negative test (LR+, LR-), odds ratios (OR), diagnostic accuracy, and the Youden index (YI) were compared. Of the 324 participants, the mean age was 59.1 ± 10.5 years, and 56.8% were males. The AHA/NHLBI and IDF definitions had better sensitivity (53.09%, 48.77%). The CDS definition was more specific (76.54%), but less sensitive (25.93%). The IDF definition performed better in PPV (53.74%), NPV (53.11%), LR+ (1.15) and LR- (0.89), OR (1.32), and diagnostic accuracy (53.4%). The IDF definition also provided optimal cutoff points with the biggest YI. The IDF definition performed better in detecting the onsets of nonfatal ACS in the northwestern Chinese population. All studied definitions were feasible in Chinese clinical settings.

  2. The chloroplast atpA gene cluster in Chlamydomonas reinhardtii. Functional analysis of a polycistronic transcription unit.

    PubMed

    Drapier, D; Suzuki, H; Levy, H; Rimbault, B; Kindle, K L; Stern, D B; Wollman, F A

    1998-06-01

    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 alpha-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-alpha 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.

  3. Blockade of Extracellular ATP Effect by Oxidized ATP Effectively Mitigated Induced Mouse Experimental Autoimmune Uveitis (EAU)

    PubMed Central

    Zhao, Ronglan; Liang, Dongchun; Sun, Deming

    2016-01-01

    Various pathological conditions are accompanied by ATP release from the intracellular to the extracellular compartment. Extracellular ATP (eATP) functions as a signaling molecule by activating purinergic P2 purine receptors. The key P2 receptor involved in inflammation was identified as P2X7R. Recent studies have shown that P2X7R signaling is required to trigger the Th1/Th17 immune response, and oxidized ATP (oxATP) effectively blocks P2X7R activation. In this study we investigated the effect of oxATP on mouse experimental autoimmune uveitis (EAU). Our results demonstrated that induced EAU in B6 mice was almost completely abolished by the administration of small doses of oxATP, and the Th17 response, but not the Th1 response, was significantly weakened in the treated mice. Mechanistic studies showed that the therapeutic effects involve the functional change of a number of immune cells, including dendritic cells (DCs), T cells, and regulatory T cells. OxATP not only directly inhibits the T cell response; it also suppresses T cell activation by altering the function of DCs and Foxp3+ T cell. Our results demonstrated that inhibition of P2X7R activation effectively exempts excessive autoimmune inflammation, which may indicate a possible therapeutic use in the treatment of autoimmune diseases. PMID:27196432

  4. Blockade of Extracellular ATP Effect by Oxidized ATP Effectively Mitigated Induced Mouse Experimental Autoimmune Uveitis (EAU).

    PubMed

    Zhao, Ronglan; Liang, Dongchun; Sun, Deming

    2016-01-01

    Various pathological conditions are accompanied by ATP release from the intracellular to the extracellular compartment. Extracellular ATP (eATP) functions as a signaling molecule by activating purinergic P2 purine receptors. The key P2 receptor involved in inflammation was identified as P2X7R. Recent studies have shown that P2X7R signaling is required to trigger the Th1/Th17 immune response, and oxidized ATP (oxATP) effectively blocks P2X7R activation. In this study we investigated the effect of oxATP on mouse experimental autoimmune uveitis (EAU). Our results demonstrated that induced EAU in B6 mice was almost completely abolished by the administration of small doses of oxATP, and the Th17 response, but not the Th1 response, was significantly weakened in the treated mice. Mechanistic studies showed that the therapeutic effects involve the functional change of a number of immune cells, including dendritic cells (DCs), T cells, and regulatory T cells. OxATP not only directly inhibits the T cell response; it also suppresses T cell activation by altering the function of DCs and Foxp3+ T cell. Our results demonstrated that inhibition of P2X7R activation effectively exempts excessive autoimmune inflammation, which may indicate a possible therapeutic use in the treatment of autoimmune diseases.

  5. Synthetic peptides target ATP translocase of ‘Candidatus Liberibacter asiaticus’ to block ATP uptake

    USDA-ARS?s Scientific Manuscript database

    As an obligate intracellular pathogen, ‘Candidatus Liberibacter asiaticus’ (Las) may act as an “energy parasite” by importing ATP from its host’s cells. We previously demonstrated that the Las translocase NttA (gb|ACX71867.1) is functional in Escherichia coli and enables the direct import of ATP/ADP...

  6. ATP: The crucial component of secretory vesicles.

    PubMed

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R; González-Santana, Ayoze; Westhead, Edward W; Borges, Ricardo; Machado, José David

    2016-07-12

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.

  7. ATP: The crucial component of secretory vesicles

    PubMed Central

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R.; González-Santana, Ayoze; Westhead, Edward W.; Borges, Ricardo; Machado, José David

    2016-01-01

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission. PMID:27342860

  8. 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.

  9. Voltage dependence of ATP secretion in mammalian taste cells.

    PubMed

    Romanov, Roman A; Rogachevskaja, Olga A; Khokhlov, Alexander A; Kolesnikov, Stanislav S

    2008-12-01

    Mammalian type II taste cells release the afferent neurotransmitter adenosine triphosphate (ATP) through ATP-permeable ion channels, most likely to be connexin (Cx) and/or pannexin hemichannels. Here, we show that ion channels responsible for voltage-gated (VG) outward currents in type II cells are ATP permeable and demonstrate a strong correlation between the magnitude of the VG current and the intensity of ATP release. These findings suggest that slowly deactivating ion channels transporting the VG outward currents can also mediate ATP secretion in type II cells. In line with this inference, we studied a dependence of ATP secretion on membrane voltage with a cellular ATP sensor using different pulse protocols. These were designed on the basis of predictions of a model of voltage-dependent transient ATP efflux. Consistently with curves that were simulated for ATP release mediated by ATP-permeable channels deactivating slowly, the bell-like and Langmuir isotherm-like potential dependencies were characteristic of ATP secretion obtained for prolonged and short electrical stimulations of taste cells, respectively. These observations strongly support the idea that ATP is primarily released via slowly deactivating channels. Depolarizing voltage pulses produced negligible Ca(2+) transients in the cytoplasm of cells releasing ATP, suggesting that ATP secretion is mainly governed by membrane voltage under our recording conditions. With the proviso that natural connexons and pannexons are kinetically similar to exogenously expressed hemichannels, our findings suggest that VG ATP release in type II cells is primarily mediated by Cx hemichannels.

  10. 25 CFR 291.2 - Definitions

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 25 Indians 1 2011-04-01 2011-04-01 false Definitions 291.2 Section 291.2 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR ECONOMIC ENTERPRISES CLASS III GAMING PROCEDURES § 291.2 Definitions (a) All terms have the same meaning as set forth in the definitional section of IGRA, 25 U.S.C...

  11. 20 CFR 631.2 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 20 Employees' Benefits 3 2011-04-01 2011-04-01 false Definitions. 631.2 Section 631.2 Employees' Benefits EMPLOYMENT AND TRAINING ADMINISTRATION, DEPARTMENT OF LABOR PROGRAMS UNDER TITLE III OF THE JOB TRAINING PARTNERSHIP ACT General Provisions § 631.2 Definitions. In addition to the definitions contained...

  12. 20 CFR 631.2 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 20 Employees' Benefits 3 2012-04-01 2012-04-01 false Definitions. 631.2 Section 631.2 Employees' Benefits EMPLOYMENT AND TRAINING ADMINISTRATION, DEPARTMENT OF LABOR PROGRAMS UNDER TITLE III OF THE JOB TRAINING PARTNERSHIP ACT General Provisions § 631.2 Definitions. In addition to the definitions contained...

  13. 25 CFR 291.2 - Definitions

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 25 Indians 1 2012-04-01 2011-04-01 true Definitions 291.2 Section 291.2 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR ECONOMIC ENTERPRISES CLASS III GAMING PROCEDURES § 291.2 Definitions (a) All terms have the same meaning as set forth in the definitional section of IGRA, 25...

  14. 5 CFR 630.201 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Definitions. 630.201 Section 630.201 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS ABSENCE AND LEAVE Definitions and General Provisions for Annual and Sick Leave § 630.201 Definitions. (a) In section 6301(2)(iii) of...

  15. 25 CFR 291.2 - Definitions

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Definitions 291.2 Section 291.2 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR ECONOMIC ENTERPRISES CLASS III GAMING PROCEDURES § 291.2 Definitions (a) All terms have the same meaning as set forth in the definitional section of IGRA, 25...

  16. BIOPLUME III

    EPA Pesticide Factsheets

    BIOPLUME III is a two-dimensional finite difference model for simulating the natural attenuation of organic contaminants in groundwater due to the processes of advection, dispersion, sorption, and biodegradation.

  17. Dynamic regulation of extracellular ATP in Escherichia coli.

    PubMed

    Alvarez, Cora Lilia; Corradi, Gerardo; Lauri, Natalia; Marginedas-Freixa, Irene; Leal Denis, María Florencia; Enrique, Nicolás; Mate, Sabina María; Milesi, Verónica; Ostuni, Mariano Anibal; Herlax, Vanesa; Schwarzbaum, Pablo Julio

    2017-04-04

    We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [(32)P]Pi released from [γ-(32)P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-(32)P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1-1 µM). Exposure to MST7 and MEL enhanced ATP release by 3-7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6-7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

  18. 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

  19. Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant

    PubMed Central

    Boominathan, Amutha; Vanhoozer, Shon; Basisty, Nathan; Powers, Kathleen; Crampton, Alexandra L.; Wang, Xiaobin; Friedricks, Natalie; Schilling, Birgit; Brand, Martin D.; O'Connor, Matthew S.

    2016-01-01

    We explore the possibility of re-engineering mitochondrial genes and expressing them from the nucleus as an approach to rescue defects arising from mitochondrial DNA mutations. We have used a patient cybrid cell line with a single point mutation in the overlap region of the ATP8 and ATP6 genes of the human mitochondrial genome. These cells are null for the ATP8 protein, have significantly lowered ATP6 protein levels and no Complex V function. Nuclear expression of only the ATP8 gene with the ATP5G1 mitochondrial targeting sequence appended restored viability on Krebs cycle substrates and ATP synthesis capabilities but, failed to restore ATP hydrolysis and was insensitive to various inhibitors of oxidative phosphorylation. Co-expressing both ATP8 and ATP6 genes under similar conditions resulted in stable protein expression leading to successful integration into Complex V of the oxidative phosphorylation machinery. Tests for ATP hydrolysis / synthesis, oxygen consumption, glycolytic metabolism and viability all indicate a significant functional rescue of the mutant phenotype (including re-assembly of Complex V) following stable co-expression of ATP8 and ATP6. Thus, we report the stable allotopic expression, import and function of two mitochondria encoded genes, ATP8 and ATP6, resulting in simultaneous rescue of the loss of both mitochondrial proteins. PMID:27596602

  20. Continuous intravenous infusion of ATP in humans yields large expansions of erythrocyte ATP pools but extracellular ATP pools are elevated only at the start followed by rapid declines.

    PubMed

    Rapaport, Eliezer; Salikhova, Anna; Abraham, Edward H

    2015-06-01

    The pharmacokinetics of adenosine 5'-triphosphate (ATP) was investigated in a clinical trial that included 15 patients with advanced malignancies (solid tumors). ATP was administered by continuous intravenous infusions of 8 h once weekly for 8 weeks. Three values of blood ATP levels were determined. These were total blood (erythrocyte) and blood plasma (extracellular) ATP pools along with the initial rate of release of ATP into the blood plasma. We found that values related to erythrocyte ATP pools showed great variability (diversity) among individuals (standard deviation of about 30-40% of mean at baseline). It was discovered that erythrocyte baseline ATP pool sizes are unique to each individual and that they fall within a narrow range in each individual. At the end of an 8 h continuous intravenous infusion of ATP, intracellular erythrocyte ATP pools were increased in the range of 40-60% and extracellular ATP declined from elevated levels achieved at the beginning and middle of the infusion, to baseline levels. The ability of erythrocytes to sequester exogenously administered ATP to this degree, after its initial conversion to adenosine in the blood plasma is unexpected, considering that some of the adenosine is likely to have been degraded by in vivo catabolic activities or taken up by organs. The data suggest that administration of ATP by short-term intravenous infusions, of up to 4 h, may be a favorable way for elevating extracellular ATP pools. A large fraction of the total exogenously administered ATP is sequestered into the intracellular compartments of the erythrocytes after an 8 h intravenous infusion. Erythrocytes loaded with ATP are known to release their ATP pools by the application of previously established agents or conditions applied locally or globally to circulating erythrocytes. Rapid degradation of intravenously administered ATP to adenosine and subsequent accumulation of ATP inside erythrocytes indicate the existence of very effective mechanisms

  1. Local detection of mechanically induced ATP release from bone cells with ATP microbiosensors.

    PubMed

    Hecht, Elena; Liedert, Astrid; Ignatius, Anita; Mizaikoff, Boris; Kranz, Christine

    2013-06-15

    The mechanically induced release of adenosine-5'-triphosphate (ATP) from osteoblastic cells (MC3T3-E1) was measured in real time. A stretching device integrated into scanning electrochemical microscopy was developed to apply controlled mechanical strain to MC3T3-E1 cells. For ATP secretion, a stepwise yet uniform mechanical stress was imposed onto MC3T3-E1 cells. The ATP biosensors were positioned at a distance of approximately 30-40 μm above the cell surface. Calibration functions were recorded prior to the cell measurements and revealed a linear response up to 40 μM with a sensitivity of 1-5pA/μM ATP. Stretching MC3T3-E1 cells up to 21% resulted in a concentration of 30.57±4.82 μM of extracellular ATP (N=12) detected above the cell surface. As a control experiment, nifedipine, a L-type voltage sensitive calcium channel (L-VSCC) inhibitor was applied, which blocks Ca(2+)entry from the outer medium into the cell. Inhibition resulted in a significantly smaller amount of released ATP, i.e., 7.08±1.93 μM ATP (N=10). Further control experiments with glucose microbiosensors did not yield significant changes of the baseline current (N=8). Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Sulfide-based ATP production in Urechis unicinctus

    NASA Astrophysics Data System (ADS)

    Ma, Zhuojun; Bao, Zhenmin; Wang, Sifeng; Zhang, Zhifeng

    2010-05-01

    We measured sulfide-based ATP production by isolated mitochondria from four tissues of Urechis unicinctus and the effects of inhibitors of respiratory complexes on ATP production were evaluated. The results show that these mitochondria could oxidize sulfide to produce ATP. The yield of sulfide-stimulated ATP varied from 5 nmol ATP/min/mg to 90 nmol ATP/min/mg according to the sulfide concentration and the source of the mitochondria. The maximum ATP synthesis occurred in hindgut mitochondria using 5 μmol/L sulfide as a substrate. The effects of inhibitors (Rotenone, Antimycin A, Cyanide, and Salicylhydroxamic acid) on mitochondrial ATP production varied with the source of the mitochondria. Our results indicate that sulfide-based ATP production and the associated electron transport pathway are tissue-specific in U. unicinctus.

  3. Dynein ATPase pathway: ATP analogs and regulation by phosphorylation

    SciTech Connect

    Chilcote, T.J.

    1988-01-01

    Three biochemical aspects of 22S dynein from Tetrahymena cilia have been investigated: its ATP binding polypeptides and the manner in which they bind ATP, its AMPPNP-induced dissociation from microtubules, and its phosphorylation. We have attempted to identify the polypeptides of dynein that bind ATP, i.e., the active site polypeptides, with the photoaffinity ATP analog 8-N{sub 3}ATP. The 8-N{sub 3}ATP has been shown to bind to dyneins active sites and in a manner similar to that of ATP. Upon irradiation, (2-{sup 3}H)8-N{sub 3}ATP covalently labels the three heavy chains, i.e., heads, which is detected by autoradiography of SDS PAG's. Thus, the three heads are considered to be the three active sites of dynein. AMPPNP is a nonhydrolyzable ATP analog which we have assayed for the ability to induce dynein dissociation from microtubules.

  4. Firefly bioluminescent assay of ATP in the presence of ATP extractant by using liposomes.

    PubMed

    Kamidate, Tamio; Yanashita, Kenji; Tani, Hirofumi; Ishida, Akihiko; Notani, Mizuyo

    2006-01-01

    Liposomes containing phosphatidylcholine (PC) and cholesterol (Chol) were applied to the enhancer for firefly bioluminescence (BL) assay for ATP in the presence of cationic surfactants using as an extractant for the release of ATP from living cells. Benzalkonium chloride (BAC) was used as an ATP extractant. However, BAC seriously inhibited the activity of luciferase, thus resulting in the remarkable decrease in the sensitivity of the BL assay for ATP. On the other hand, we found that BAC was associated with liposomes to form cationic liposomes containing BAC. The association rate of BAC with liposomes was faster than that of BAC with luciferase. As a result, the inhibitory effect of BAC on luciferase was eliminated in the presence of liposomes. In addition, cationic liposomes thus formed enhanced BL emission. BL measurement conditions were optimized in terms of liposome charge type, liposome size, and total concentration of PC and Chol. ATP can be sensitively determined without dilution of analytical samples by using liposomes. The detection limit of ATP with and without liposomes was 100 amol and 25 fmol in aqueous ATP standard solutions containing 0.06% BAC, respectively. The method was applied to the determination of ATP in Escherichia coli extracts. The BL intensity was linear from 4 x 10(4) to 1 x 10(7) cells mL(-1) in the absence of liposomes. On the other hand, the BL intensity was linear from 4 x 10(3) to 4 x 10(6) cells mL(-1) in the presence of liposomes. The detection limit of ATP in E. coli extracts was improved by a factor of 10 via use of liposomes.

  5. Mechanisms that match ATP supply to demand in cardiac pacemaker cells during high ATP demand

    PubMed Central

    Yaniv, Yael; Spurgeon, Harold A.; Ziman, Bruce D.; Lyashkov, Alexey E.

    2013-01-01

    The spontaneous action potential (AP) firing rate of sinoatrial node cells (SANCs) involves high-throughput signaling via Ca2+-calmodulin activated adenylyl cyclases (AC), cAMP-mediated protein kinase A (PKA), and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of SR Ca2+ cycling and surface membrane ion channel proteins. When the throughput of this signaling increases, e.g., in response to β-adrenergic receptor activation, the resultant increase in spontaneous AP firing rate increases the demand for ATP. We hypothesized that an increase of ATP production to match the increased ATP demand is achieved via a direct effect of increased mitochondrial Ca2+ (Ca2+m) and an indirect effect via enhanced Ca2+-cAMP/PKA-CaMKII signaling to mitochondria. To increase ATP demand, single isolated rabbit SANCs were superfused by physiological saline at 35 ± 0.5°C with isoproterenol, or by phosphodiesterase or protein phosphatase inhibition. We measured cytosolic and mitochondrial Ca2+ and flavoprotein fluorescence in single SANC, and we measured cAMP, ATP, and O2 consumption in SANC suspensions. Although the increase in spontaneous AP firing rate was accompanied by an increase in O2 consumption, the ATP level and flavoprotein fluorescence remained constant, indicating that ATP production had increased. Both Ca2+m and cAMP increased concurrently with the increase in AP firing rate. When Ca2+m was reduced by Ru360, the increase in spontaneous AP firing rate in response to isoproterenol was reduced by 25%. Thus, both an increase in Ca2+m and an increase in Ca2+ activated cAMP-PKA-CaMKII signaling regulate the increase in ATP supply to meet ATP demand above the basal level. PMID:23604710

  6. Mechanisms that match ATP supply to demand in cardiac pacemaker cells during high ATP demand.

    PubMed

    Yaniv, Yael; Spurgeon, Harold A; Ziman, Bruce D; Lyashkov, Alexey E; Lakatta, Edward G

    2013-06-01

    The spontaneous action potential (AP) firing rate of sinoatrial node cells (SANCs) involves high-throughput signaling via Ca(2+)-calmodulin activated adenylyl cyclases (AC), cAMP-mediated protein kinase A (PKA), and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of SR Ca(2+) cycling and surface membrane ion channel proteins. When the throughput of this signaling increases, e.g., in response to β-adrenergic receptor activation, the resultant increase in spontaneous AP firing rate increases the demand for ATP. We hypothesized that an increase of ATP production to match the increased ATP demand is achieved via a direct effect of increased mitochondrial Ca(2+) (Ca(2+)m) and an indirect effect via enhanced Ca(2+)-cAMP/PKA-CaMKII signaling to mitochondria. To increase ATP demand, single isolated rabbit SANCs were superfused by physiological saline at 35 ± 0.5°C with isoproterenol, or by phosphodiesterase or protein phosphatase inhibition. We measured cytosolic and mitochondrial Ca(2+) and flavoprotein fluorescence in single SANC, and we measured cAMP, ATP, and O₂ consumption in SANC suspensions. Although the increase in spontaneous AP firing rate was accompanied by an increase in O₂ consumption, the ATP level and flavoprotein fluorescence remained constant, indicating that ATP production had increased. Both Ca(2+)m and cAMP increased concurrently with the increase in AP firing rate. When Ca(2+)m was reduced by Ru360, the increase in spontaneous AP firing rate in response to isoproterenol was reduced by 25%. Thus, both an increase in Ca(2+)m and an increase in Ca(2+) activated cAMP-PKA-CaMKII signaling regulate the increase in ATP supply to meet ATP demand above the basal level.

  7. Metabolic Syndrome in Military Aircrew Using a Candidate Definition.

    PubMed

    Sharma, Sanjiv; Chandrashekar, A M; Singh, Vishal

    2016-09-01

    Prevalence of metabolic syndrome (MetS) in the Indian population varies from 31.6 to 41.1%. Indians, without being conventionally obese, but inherently insulin resistant, have higher risk of developing cardiovascular diseases and diabetes. Since military aircrew, belonging to the same ethnic pool, may reflect similar prevalence of MetS as the general Indian populace, this study was undertaken to find the prevalence of MetS among Indian military aircrew using one candidate definition. In this cross sectional descriptive study, 210 military aircrew voluntarily participated. Besides demographic and lifestyle related details, their anthropometric measurements, including height, weight, waist circumference, hip circumference, and skin fold thickness were recorded. Body mass index and waist-to-hip ratio were deduced from the recorded measurements. Resting heart rate and blood pressure were recorded and appropriate laboratory investigations were undertaken. Prevalence of MetS, as per chosen definition, was 33.3% (N = 70), which had moderate, fair, and slight agreement with NCEP ATP III (k = 0.43), IDF (k = 0.27), and WHO (k = 0.15) definitions, respectively. Decadal prevalence of MetS was found to be highest in the fourth decade (46.8%), followed by the third decade (41.3%). Reported prevalence of MetS highlights an urgent need to define preventive strategies to minimize loss of trained manpower among military aircrew. Flight surgeons have an important role to play to educate aircrew about modifying their lifestyle to reduce morbidity and mortality among themselves in the future. Sharma S, Chandrashekar AM, Singh V. Metabolic syndrome in military aircrew using a candidate definition. Aerosp Med Hum Perform. 2016; 87(9):790-794.

  8. Complex processing patterns of mRNAs of the large ATP synthase operon in Arabidopsis chloroplasts.

    PubMed

    Malik Ghulam, Mustafa; Ghulam, Mustafa Malik; Courtois, Florence; Lerbs-Mache, Silva; Merendino, Livia

    2013-01-01

    Chloroplasts are photosynthetic cell organelles which have evolved from endosymbiosis of the cyanobacterial ancestor. In chloroplasts, genes are still organized into transcriptional units as in bacteria but the corresponding poly-cistronic mRNAs undergo complex processing events, including inter-genic cleavage and 5' and 3' end-definition. The current model for processing proposes that the 3' end of the upstream cistron transcripts and the 5' end of the downstream cistron transcripts are defined by the same RNA-binding protein and overlap at the level of the protein-binding site. We have investigated the processing mechanisms that operate within the large ATP synthase (atp) operon, in Arabidopsis thaliana chloroplasts. This operon is transcribed by the plastid-encoded RNA polymerase starting from two promoters, which are upstream and within the operon, respectively, and harbors four potential sites for RNA-binding proteins. In order to study the functional significance of the promoters and the protein-binding sites for the maturation processes, we have performed a detailed mapping of the atp transcript ends. Our data indicate that in contrast to maize, atpI and atpH transcripts with overlapping ends are very rare in Arabidopsis. In addition, atpA mRNAs, which overlap with atpF mRNAs, are even truncated at the 3' end, thus representing degradation products. We observe, instead, that the 5' ends of nascent poly-cistronic atp transcripts are defined at the first protein-binding site which follows either one of the two transcription initiation sites, while the 3' ends are defined at the subsequent protein-binding sites or at hairpin structures that are encountered by the progressing RNA polymerase. We conclude that the overlapping mechanisms of mRNA protection have only a limited role in obtaining stable processed atp mRNAs in Arabidopsis. Our findings suggest that during evolution of different plant species as maize and Arabidopsis, chloroplasts have evolved multiple

  9. Complex Processing Patterns of mRNAs of the Large ATP Synthase Operon in Arabidopsis Chloroplasts

    PubMed Central

    Ghulam, Mustafa Malik; Courtois, Florence; Lerbs-Mache, Silva; Merendino, Livia

    2013-01-01

    Chloroplasts are photosynthetic cell organelles which have evolved from endosymbiosis of the cyanobacterial ancestor. In chloroplasts, genes are still organized into transcriptional units as in bacteria but the corresponding poly-cistronic mRNAs undergo complex processing events, including inter-genic cleavage and 5′ and 3′ end-definition. The current model for processing proposes that the 3′ end of the upstream cistron transcripts and the 5′ end of the downstream cistron transcripts are defined by the same RNA-binding protein and overlap at the level of the protein-binding site. We have investigated the processing mechanisms that operate within the large ATP synthase (atp) operon, in Arabidopsis thaliana chloroplasts. This operon is transcribed by the plastid-encoded RNA polymerase starting from two promoters, which are upstream and within the operon, respectively, and harbors four potential sites for RNA-binding proteins. In order to study the functional significance of the promoters and the protein-binding sites for the maturation processes, we have performed a detailed mapping of the atp transcript ends. Our data indicate that in contrast to maize, atpI and atpH transcripts with overlapping ends are very rare in Arabidopsis. In addition, atpA mRNAs, which overlap with atpF mRNAs, are even truncated at the 3′ end, thus representing degradation products. We observe, instead, that the 5′ ends of nascent poly-cistronic atp transcripts are defined at the first protein-binding site which follows either one of the two transcription initiation sites, while the 3′ ends are defined at the subsequent protein-binding sites or at hairpin structures that are encountered by the progressing RNA polymerase. We conclude that the overlapping mechanisms of mRNA protection have only a limited role in obtaining stable processed atp mRNAs in Arabidopsis. Our findings suggest that during evolution of different plant species as maize and Arabidopsis, chloroplasts have

  10. A reusable prepositioned ATP reaction chamber

    NASA Technical Reports Server (NTRS)

    Hoffman, D. G.

    1972-01-01

    Luminescence biometer detects presence of life by means of light-emitting chemical reaction of luciferin and luciferase with adenosine triphosphate (ATP) that occurs in all living cells. Amount of light in reaction chamber is measured to determine presence and extent of life.

  11. Monitoring enzymatic ATP hydrolysis by EPR spectroscopy.

    PubMed

    Hacker, Stephan M; Hintze, Christian; Marx, Andreas; Drescher, Malte

    2014-07-14

    An adenosine triphosphate (ATP) analogue modified with two nitroxide radicals is developed and employed to study its enzymatic hydrolysis by electron paramagnetic resonance spectroscopy. For this application, we demonstrate that EPR holds the potential to complement fluorogenic substrate analogues in monitoring enzymatic activity.

  12. Calcium and ATP control multiple vital functions.

    PubMed

    Petersen, Ole H; Verkhratsky, Alexei

    2016-08-05

    Life on Planet Earth, as we know it, revolves around adenosine triphosphate (ATP) as a universal energy storing molecule. The metabolism of ATP requires a low cytosolic Ca(2+) concentration, and hence tethers these two molecules together. The exceedingly low cytosolic Ca(2+) concentration (which in all life forms is kept around 50-100 nM) forms the basis for a universal intracellular signalling system in which Ca(2+) acts as a second messenger. Maintenance of transmembrane Ca(2+) gradients, in turn, requires ATP-dependent Ca(2+) transport, thus further emphasizing the inseparable links between these two substances. Ca(2+) signalling controls the most fundamental processes in the living organism, from heartbeat and neurotransmission to cell energetics and secretion. The versatility and plasticity of Ca(2+) signalling relies on cell specific Ca(2+) signalling toolkits, remodelling of which underlies adaptive cellular responses. Alterations of these Ca(2+) signalling toolkits lead to aberrant Ca(2+) signalling which is fundamental for the pathophysiology of numerous diseases from acute pancreatitis to neurodegeneration. This paper introduces a theme issue on this topic, which arose from a Royal Society Theo Murphy scientific meeting held in March 2016.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'.

  13. Electric Field Driven Torque in ATP Synthase

    PubMed Central

    Miller, John H.; Rajapakshe, Kimal I.; Infante, Hans L.; Claycomb, James R.

    2013-01-01

    FO-ATP synthase (FO) is a rotary motor that converts potential energy from ions, usually protons, moving from high- to low-potential sides of a membrane into torque and rotary motion. Here we propose a mechanism whereby electric fields emanating from the proton entry and exit channels act on asymmetric charge distributions in the c-ring, due to protonated and deprotonated sites, and drive it to rotate. The model predicts a scaling between time-averaged torque and proton motive force, which can be hindered by mutations that adversely affect the channels. The torque created by the c-ring of FO drives the γ-subunit to rotate within the ATP-producing complex (F1) overcoming, with the aid of thermal fluctuations, an opposing torque that rises and falls with angular position. Using the analogy with thermal Brownian motion of a particle in a tilted washboard potential, we compute ATP production rates vs. proton motive force. The latter shows a minimum, needed to drive ATP production, which scales inversely with the number of proton binding sites on the c-ring. PMID:24040370

  14. Electric field driven torque in ATP synthase.

    PubMed

    Miller, John H; Rajapakshe, Kimal I; Infante, Hans L; Claycomb, James R

    2013-01-01

    FO-ATP synthase (FO) is a rotary motor that converts potential energy from ions, usually protons, moving from high- to low-potential sides of a membrane into torque and rotary motion. Here we propose a mechanism whereby electric fields emanating from the proton entry and exit channels act on asymmetric charge distributions in the c-ring, due to protonated and deprotonated sites, and drive it to rotate. The model predicts a scaling between time-averaged torque and proton motive force, which can be hindered by mutations that adversely affect the channels. The torque created by the c-ring of FO drives the γ-subunit to rotate within the ATP-producing complex (F1) overcoming, with the aid of thermal fluctuations, an opposing torque that rises and falls with angular position. Using the analogy with thermal Brownian motion of a particle in a tilted washboard potential, we compute ATP production rates vs. proton motive force. The latter shows a minimum, needed to drive ATP production, which scales inversely with the number of proton binding sites on the c-ring.

  15. Electrophysiological effects of ATP on brain neurones.

    PubMed

    Illes, P; Nieber, K; Nörenberg, W

    1996-12-01

    1. The electrophysiological effects of ATP on brain neurones are either due to the direct activation of P2 purinoceptors by the unmetabolized nucleotide or to the indirect activation of P1. purinoceptors by the degradation product adenosine. 2. Two subtypes of P2 purinoceptors are involved, a ligand-activated ion channel (P2X) and a G protein-coupled receptor (P2Y). Hence, the stimulation of P2X purinoceptors leads to a cationic conductance increase, while the stimulation of P2Y purinoceptors leads to a G protein-mediated opening or closure of potassium channels. 3. ATP may induce a calcium-dependent potassium current by increasing the intracellular Ca2+ concentration. This is due either to the entry of Ca2+ via P2X purinoceptors or to the activation of metabotropic P2Y purinoceptors followed by signaling via the G protein/phospholipase C/inositol 1,4,5-trisphosphate (IP3) cascade. Eventually, IP3 releases Ca2+ from its intracellular pools. 4. There is no convincing evidence for the presence of P2U purinoceptors sensitive to both ATP and UTP, or pyrimidinoceptors sensitive to UTP only, in the central nervous system (CNS). 5. ATP-sensitive P2X and P2Y purinoceptors show a wide distribution in the CNS and appear to regulate important neuronal functions.

  16. Mitochondrial ATP 6 and 8 polymorphisms in irritable bowel syndrome with diarrhea.

    PubMed

    Wang, Wei-Feng; Li, Xin; Guo, Ming-Zhou; Chen, Jian-De; Yang, Yun-Sheng; Peng, Li-Hua; Wang, Yong-Hua; Zhang, Chun-Yan; Li, Hui-Hui

    2013-06-28

    To investigate mitochondrial ATP 6 and 8 polymorphisms in the colon and ileum of patients with irritable bowel syndrome with diarrhea (IBS-D). Twenty-eight patients fulfilling the Rome III criteria for IBS-D and 28 healthy subjects were investigated. All study participants underwent screening colonoscopy and mucosal biopsies were obtained from the colon and/or terminal ileum. Genomic DNA was extracted from specimens based on standard protocols. Mitochondrial ATP (MT-ATP) 6 and 8 genes in specimens were polymerase chain reaction amplified and sequenced. Sequencing data were analyzed via Variant Reporter™ Software and compared with the reference sequence from Genbank (accession No. NC_012920) to indicate possible polymorphisms. The protocol was registered at www.clinicaltrials.gov as NCT01028898. Twenty-five polymorphic sites of MT-ATP 6 and 8 genes were detected and 12 of them were missense mutations. A median of two polymorphic sites in MT-ATP genes was found in colon specimens of controls while a median of three polymorphic sites was noted in patients with IBS-D (Mann-Whitney test, P = 0.012). The variants of the colon and ileum specimens from the same subjects were identical in all but one case. Symptom duration in IBS was not found to be a significant factor associated with the mtDNA polymorphism (Spearman correlation, P = 0.592). The mitochondrial DNA change at 8860 was present in all cases of both groups. The frequency of the 8701 polymorphism was found to be the second most frequent; however, no statistical difference was noted between the groups (χ(2) test, P = 0.584). Patients with IBS-D have a higher incidence of MT-ATP 6 and 8 polymorphisms than healthy subjects, implying that the mtDNA polymorphism may play a role in IBS-D.

  17. Torque Generation and Utilization in Motor Enzyme F0F1-ATP Synthase

    PubMed Central

    Usukura, Eiji; Suzuki, Toshiharu; Furuike, Shou; Soga, Naoki; Saita, Ei-ichiro; Hisabori, Toru; Kinosita, Kazuhiko; Yoshida, Masasuke

    2012-01-01

    ATP synthase (F0F1) is made of two motors, a proton-driven motor (F0) and an ATP-driven motor (F1), connected by a common rotary shaft, and catalyzes proton flow-driven ATP synthesis and ATP-driven proton pumping. In F1, the central γ subunit rotates inside the α3β3 ring. Here we report structural features of F1 responsible for torque generation and the catalytic ability of the low-torque F0F1. (i) Deletion of one or two turns in the α-helix in the C-terminal domain of catalytic β subunit at the rotor/stator contact region generates mutant F1s, termed F1(1/2)s, that rotate with about half of the normal torque. This helix would support the helix-loop-helix structure acting as a solid “pushrod” to push the rotor γ subunit, but the short helix in F1(1/2)s would fail to accomplish this task. (ii) Three different half-torque F0F1(1/2)s were purified and reconstituted into proteoliposomes. They carry out ATP-driven proton pumping and build up the same small transmembrane ΔpH, indicating that the final ΔpH is directly related to the amount of torque. (iii) The half-torque F0F1(1/2)s can catalyze ATP synthesis, although slowly. The rate of synthesis varies widely among the three F0F1(1/2)s, which suggests that the rate reflects subtle conformational variations of individual mutants. PMID:22128167

  18. Global Positioning System III (GPS III)

    DTIC Science & Technology

    2013-12-01

    Global Positioning System III ( GPS III) As of FY 2015 President’s Budget...00-00-2013 to 00-00-2013 4. TITLE AND SUBTITLE Global Positioning System III ( GPS III) 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT...Responsible Office References Program Name Global Positioning System III ( GPS III) DoD Component Air Force

  19. Sequential (gemcitabine/vinorelbine) and concurrent (gemcitabine) radiochemotherapy with FDG-PET-based target volume definition in locally advanced non-small cell lung cancer: first results of a phase I/II study

    PubMed Central

    Gagel, Bernd; Piroth, Marc; Pinkawa, Michael; Reinartz, Patrick; Krohn, Thomas; Kaiser, Hans J; Stanzel, Sven; Breuer, Christian; Asadpour, Branka; Schmachtenberg, Axel; Eble, Michael J

    2007-01-01

    Background The aim of the study was to determine the maximal tolerated dose (MTD) of gemcitabine every two weeks concurrent to radiotherapy, administered during an aggressive program of sequential and simultaneous radiochemotherapy for locally advanced, unresectable non-small cell lung cancer (NSCLC) and to evaluate the efficacy of this regime in a phase II study. Methods 33 patients with histologically confirmed NSCLC were enrolled in a combined radiochemotherapy protocol. 29 patients were assessable for evaluation of toxicity and tumor response. Treatment included two cycles of induction chemotherapy with gemcitabine (1200 mg/m2) and vinorelbine (30 mg/m2) at day 1, 8 and 22, 29 followed by concurrent radiotherapy (2.0 Gy/d; total dose 66.0 Gy) and chemotherapy with gemcitabine every two weeks at day 43, 57 and 71. Radiotherapy planning included [18F] fluorodeoxyglucose positron emission tomography (FDG PET) based target volume definition. 10 patients were included in the phase I study with an initial gemcitabine dose of 300 mg/m2. The dose of gemcitabine was increased in steps of 100 mg/m2 until the MTD was realized. Results MTD was defined for the patient group receiving gemcitabine 500 mg/m2 due to grade 2 (next to grade 3) esophagitis in all patients resulting in a mean body weight loss of 5 kg (SD = 1.4 kg), representing 8% of the initial weight. These patients showed persisting dysphagia 3 to 4 weeks after completing radiotherapy. In accordance with expected complications as esophagitis, dysphagia and odynophagia, we defined the MTD at this dose level, although no dose limiting toxicity (DLT) grade 3 was reached. In the phase I/II median follow-up was 15.7 months (4.1 to 42.6 months). The overall response rate after completion of therapy was 64%. The median overall survival was 19.9 (95% CI: [10.1; 29.7]) months for all eligible patients. The median disease-free survival for all patients was 8.7 (95% CI: [2.7; 14.6]) months. Conclusion After induction

  20. A cell wall-bound adenosine nucleosidase is involved in the salvage of extracellular ATP in Solanum tuberosum.

    PubMed

    Riewe, David; Grosman, Lukasz; Fernie, Alisdair R; Zauber, Henrik; Wucke, Cornelia; Geigenberger, Peter

    2008-10-01

    Extracellular ATP (eATP) has recently been demonstrated to play a crucial role in plant development and growth. To investigate the fate of eATP within the apoplast, we used intact potato (Solanum tuberosum) tuber slices as an experimental system enabling access to the apoplast without interference of cytosolic contamination. (i) Incubation of intact tuber slices with ATP led to the formation of ADP, AMP, adenosine, adenine and ribose, indicating operation of apyrase, 5'-nucleotidase and nucleosidase. (ii) Measurement of apyrase, 5'-nucleotidase and nucleosidase activities in fractionated tuber tissue confirmed the apoplastic localization for apyrase and phosphatase in potato and led to the identification of a novel cell wall-bound adenosine nucleosidase activity. (iii) When intact tuber slices were incubated with saturating concentrations of adenosine, the conversion of adenosine into adenine was much higher than adenosine import into the cell, suggesting a potential bypass of adenosine import. Consistent with this, import of radiolabeled adenine into tuber slices was inhibited when ATP, ADP or AMP were added to the slices. (iv) In wild-type plants, apyrase and adenosine nucleosidase activities were found to be co-regulated, indicating functional linkage of these enzymes in a shared pathway. (v) Moreover, adenosine nucleosidase activity was reduced in transgenic lines with strongly reduced apoplastic apyrase activity. When taken together, these results suggest that a complete ATP salvage pathway is present in the apoplast of plant cells.

  1. The endogenous mitochondrial complex II inhibitor malonate regulates mitochondrial ATP-sensitive potassium channels: implications for ischemic preconditioning.

    PubMed

    Wojtovich, Andrew P; Brookes, Paul S

    2008-01-01

    Ischemic preconditioning (IPC) affords cardioprotection against ischemia-reperfusion (IR) injury, and while the molecular mechanisms of IPC are debated, the mitochondrial ATP-sensitive K(+) channel (mK(ATP)) has emerged as a candidate effector for several IPC signaling pathways. The molecular identity of this channel is unknown, but significant pharmacologic overlap exists between mK(ATP) and mitochondrial respiratory complex II (succinate dehydrogenase). In this investigation, we utilized isolated cardiac mitochondria, Langendorff perfused hearts, and a variety of biochemical methods, to make the following observations: (i) The competitive complex II inhibitor malonate is formed in mitochondria under conditions resembling IPC. (ii) IPC leads to a reversible inhibition of complex II that has likely been missed in previous investigations due to the use of saturating concentrations of succinate. (iii) Malonate opens mK(ATP) channels even when mitochondria are respiring on complex I-linked substrates, suggesting an effect of this inhibitor on the mK(ATP) channel independent of complex II inhibition. Together, these observations suggest that complex II inhibition by endogenously formed malonate may represent an important activation pathway for mK(ATP) channels during IPC.

  2. Welding III.

    ERIC Educational Resources Information Center

    Allegheny County Community Coll., Pittsburgh, PA.

    Instructional objectives and performance requirements are outlined in this course guide for Welding III, an advanced course in arc welding offered at the Community College of Allegheny County to provide students with the proficiency necessary for industrial certification. The course objectives, which are outlined first, specify that students will…

  3. Welding III.

    ERIC Educational Resources Information Center

    Allegheny County Community Coll., Pittsburgh, PA.

    Instructional objectives and performance requirements are outlined in this course guide for Welding III, an advanced course in arc welding offered at the Community College of Allegheny County to provide students with the proficiency necessary for industrial certification. The course objectives, which are outlined first, specify that students will…

  4. Prevalence of Metabolic Syndrome in Type 2 Diabetes Mellitus Using NCEP-ATPIII, IDF and WHO Definition and Its Agreement in Gwalior Chambal Region of Central India

    PubMed Central

    Yadav, Dhananjay; Mahajan, Sunil; Subramanian, Senthil K.; Bisen, Prakash Singh; Chung, Choon Hee; Prasad, GBKS

    2013-01-01

    The aim of study was to determine the prevalence of metabolic syndrome (MetS) in people with type 2 diabetes mellitus (T2DM). National Cholesterol Education Program (NCEP) ATPIII Criteria, International Diabetes Federation and the World Health Organization (WHO) definitions were used in quantifying the metabolic syndrome and also the concordance between these three criteria’s used for identifying metabolic syndrome. Methods: This cross-sectional study involved 700 type 2 diabetic subjects from the urban areas of Gwalior Chambal region (Central India). Subjects in the age group of 28-87 yrs were included in the study. Type I diabetics, pregnant ladies and those with chronic viral and bacterial infections and serious metabolic disorders were excluded from the study. Fasting blood glucose, Blood lipids (T-cholesterol, triglyceride, HDL-cholesterol) were assessed and anthropometry blood pressure were measured from all the subjects. Results: The Prevalence of metabolic syndrome was found to be 45.8%, 57.7% and 28% following NCEP-ATPIII Criteria, IDF and WHO definitions, respectively. Using all the three definitions the prevalence was higher in women in all age groups. ATP III and IDF criteria showed good agreement (κ 0.68) compared to ATP III with WHO (κ 0.54) and IDF with WHO (κ 0.34) criteria. Highest prevalence was observed following IDF definition. Conclusions: A good agreement was observed between ATPIII and IDF criteria. Maximum prevalence of Metabolic syndrome was recorded when IDF criteria was followed. NCEP-ATPIII criteria for the diagnosis of MetS and this criterion reflected equal importance to the every variable and showed a good agreement between the different criteria used. PMID:24171882

  5. Prevalence of metabolic syndrome in type 2 diabetes mellitus using NCEP-ATPIII, IDF and WHO definition and its agreement in Gwalior Chambal region of Central India.

    PubMed

    Yadav, Dhananjay; Mahajan, Sunil; Subramanian, Senthil Kumar; Bisen, Prakash Singh; Chung, Choon Hee; Prasad, G B K S

    2013-09-17

    The aim of study was to determine the prevalence of metabolic syndrome (MetS) in people with type 2 diabetes mellitus (T2DM). National Cholesterol Education Program (NCEP) ATPIII Criteria, International Diabetes Federation and the World Health Organization (WHO) definitions were used in quantifying the metabolic syndrome and also the concordance between these three criteria's used for identifying metabolic syndrome. This cross-sectional study involved 700 type 2 diabetic subjects from the urban areas of Gwalior Chambal region (Central India). Subjects in the age group of 28- 87 yrs were included in the study. Type I diabetics, pregnant ladies and those with chronic viral and bacterial infections and serious metabolic disorders were excluded from the study. Fasting blood glucose, Blood lipids (T-cholesterol, triglyceride, HDL-cholesterol) were assessed and anthropometry blood pressure were measured from all the subjects. The Prevalence of metabolic syndrome was found to be 45.8%, 57.7% and 28% following NCEP-ATPIII Criteria, IDF and WHO definitions, respectively. Using all the three definitions the prevalence was higher in women in all age groups. ATP III and IDF criteria showed good agreement (k 0.68) compared to ATP III with WHO (k 0.54) and IDF with WHO (k 0.34) criteria. Highest prevalence was observed following IDF definition. A good agreement was observed between ATPIII and IDF criteria. Maximum prevalence of Metabolic syndrome was recorded when IDF criteria was followed. NCEP-ATPIII criteria for the diagnosis of MetS and this criterion reflected equal importance to the every variable and showed a good agreement between the different criteria used.

  6. Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes

    PubMed Central

    Denis, María Florencia Leal; Incicco, J. Jeremías; Espelt, María Victoria; Verstraeten, Sandra V.; Pignataro, Omar P.; Lazarowski, Eduardo R.; Schwarzbaum, Pablo J.

    2014-01-01

    SUMMARY Background The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). Methods Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry. Results Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10 μM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. Conclusions MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. General Significance Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation. PMID:23742824

  7. The c-Ring of the F1FO-ATP Synthase: Facts and Perspectives.

    PubMed

    Nesci, Salvatore; Trombetti, Fabiana; Ventrella, Vittoria; Pagliarani, Alessandra

    2016-04-01

    The F1FO-ATP synthase is the only enzyme in nature endowed with bi-functional catalytic mechanism of synthesis and hydrolysis of ATP. The enzyme functions, not only confined to energy transduction, are tied to three intrinsic features of the annular arrangement of c subunits which constitutes the so-called c-ring, the core of the membrane-embedded FO domain: (i) the c-ring constitution is linked to the number of ions (H(+) or Na(+)) channeled across the membrane during the dissipation of the transmembrane electrochemical gradient, which in turn determines the species-specific bioenergetic cost of ATP, the "molecular currency unit" of energy transfer in all living beings; (ii) the c-ring is increasingly involved in the mitochondrial permeability transition, an event linked to cell death and to most mitochondrial dysfunctions; (iii) the c subunit species-specific amino acid sequence and susceptibility to post-translational modifications can address antibacterial drug design according to the model of enzyme inhibitors which target the c subunits. Therefore, the simple c-ring structure not only allows the F1FO-ATP synthase to perform the two opposite tasks of molecular machine of cell life and death, but it also amplifies the enzyme's potential role as a drug target.

  8. Heat stress prevents mitochondrial injury in ATP-depleted renal epithelial cells.

    PubMed

    Li, F; Mao, H P; Ruchalski, K L; Wang, Y H; Choy, W; Schwartz, J H; Borkan, S C

    2002-09-01

    The events that precipitate cell death and the stress proteins responsible for cytoprotection during ATP depletion remain elusive. We hypothesize that exposure to metabolic inhibitors damages mitochondria, allowing proapoptotic proteins to leak into the cytosol, and suggest that heat stress-induced hsp72 accumulation prevents mitochondrial membrane injury. To test these hypotheses, renal epithelial cells were transiently ATP depleted with sodium cyanide and 2-deoxy-D-glucose in the absence of medium dextrose. Recovery from ATP depletion was associated with the release into the cytosol of cytochrome c and apoptosis-inducing factor (AIF), proapoptotic proteins that localize to the intermitochondrial membrane space. Concomitant with mitochondrial cytochrome c leak, a seven- to eightfold increase in caspase 3 activity was observed. In controls, state III mitochondrial respiration was reduced by 30% after transient exposure to metabolic inhibitors. Prior heat stress preserved mitochondrial ATP production and significantly reduced both cytochrome c release and caspase 3 activation. Despite less cytochrome c release, prior heat stress increased binding between cytochrome c and hsp72. The present study demonstrates that mitochondrial injury accompanies exposure to metabolic inhibitors. By reducing outer mitochondrial membrane injury and by complexing with cytochrome c, hsp72 could inhibit caspase activation and subsequent apoptosis.

  9. Yeast mitochondria import ATP through the calcium-dependent ATP-Mg/Pi carrier Sal1p, and are ATP consumers during aerobic growth in glucose.

    PubMed

    Traba, Javier; Froschauer, Elisabeth Maria; Wiesenberger, Gerlinde; Satrústegui, Jorgina; Del Arco, Araceli

    2008-08-01

    Sal1p, a novel Ca2+-dependent ATP-Mg/Pi carrier, is essential in yeast lacking all adenine nucleotide translocases. By targeting luciferase to the mitochondrial matrix to monitor mitochondrial ATP levels, we show in isolated mitochondria that both ATP-Mg and free ADP are taken up by Sal1p with a K(m) of 0.20 +/- 0.03 mM and 0.28 +/- 0.06 mM respectively. Nucleotide transport along Sal1p is strictly Ca2+ dependent. Ca2+ increases the V(max) with a S(0.5) of 15 muM, and no changes in the K(m) for ATP-Mg. Glucose sensing in yeast generates Ca2+ transients involving Ca2+ influx from the external medium. We find that carbon-deprived cells respond to glucose with an immediate increase in mitochondrial ATP levels which is not observed in the presence of EGTA or in Sal1p-deficient cells. Moreover, we now report that during normal aerobic growth on glucose, yeast mitochondria import ATP from the cytosol and hydrolyse it through H+-ATP synthase. We identify two pathways for ATP uptake in mitochondria, the ADP/ATP carriers and Sal1p. Thus, during exponential growth on glucose, mitochondria are ATP consumers, as those from cells growing in anaerobic conditions or deprived of mitochondrial DNA which depend on cytosolic ATP and mitochondrial ATPase working in reverse to generate a mitochondrial membrane potential. In conclusion, the results show that growth on glucose requires ATP hydrolysis in mitochondria and recruits Sal1p as a Ca2+-dependent mechanism to import ATP-Mg from the cytosol. Whether this mechanism is used under similar settings in higher eukaryotes is an open question.

  10. External Dentin Stimulation Induces ATP Release in Human Teeth.

    PubMed

    Liu, X; Wang, C; Fujita, T; Malmstrom, H S; Nedergaard, M; Ren, Y F; Dirksen, R T

    2015-09-01

    ATP is involved in neurosensory processing, including nociceptive transduction. Thus, ATP signaling may participate in dentin hypersensitivity and dental pain. In this study, we investigated whether pannexins, which can form mechanosensitive ATP-permeable channels, are present in human dental pulp. We also assessed the existence and functional activity of ecto-ATPase for extracellular ATP degradation. We further tested if ATP is released from dental pulp upon dentin mechanical or thermal stimulation that induces dentin hypersensitivity and dental pain and if pannexin or pannexin/gap junction channel blockers reduce stimulation-dependent ATP release. Using immunofluorescence staining, we demonstrated immunoreactivity of pannexin 1 and 2 in odontoblasts and their processes extending into the dentin tubules. Using enzymatic histochemistry staining, we also demonstrated functional ecto-ATPase activity within the odontoblast layer, subodontoblast layer, dental pulp nerve bundles, and blood vessels. Using an ATP bioluminescence assay, we found that mechanical or cold stimulation to the exposed dentin induced ATP release in an in vitro human tooth perfusion model. We further demonstrated that blocking pannexin/gap junction channels with probenecid or carbenoxolone significantly reduced external dentin stimulation-induced ATP release. Our results provide evidence for the existence of functional machinery required for ATP release and degradation in human dental pulp and that pannexin channels are involved in external dentin stimulation-induced ATP release. These findings support a plausible role for ATP signaling in dentin hypersensitivity and dental pain.

  11. Space shuttle (ATP configuration) abort staging investigation

    NASA Technical Reports Server (NTRS)

    Rampy, J. M.; Blackwell, K. L.; Allen, E. C., Jr.; Fossler, I.

    1973-01-01

    A wind tunnel test conducted in a 14-inch trisonic wind tunnel to determine the force and moment characteristics of the ATP Orbiter and modified ATP External Tank/SRB combination during abort staging conditions is discussed. Six component aerodynamic force and moment data were recorded for the orbiter and ET/SRB combination. Pitch polars were obtained for an angle of attack range from minus 10 to plus 10 degrees and orbiter incidence angles (orbiter relative to the ET/SRB combination) of 0 and 2 degrees. A limited amount of yaw data were obtained at 0 degree angle of attack and beta range from minus 10 to plus 10 degrees. In addition, orbiter pitch control effectiveness was determined at several grid points. These force and moment data were obtained for Mach numbers of 0.9, 1.2 and 2.0.

  12. Changes in dermal interstitial ATP levels during local heating of human skin

    PubMed Central

    Gifford, Jayson R; Heal, Cory; Bridges, Jarom; Goldthorpe, Scott; Mack, Gary W

    2012-01-01

    Heating skin is believed to activate vanilloid type III and IV transient receptor potential ion channels (TRPV3, TRPV4, respectively), resulting in the release of ATP into the interstitial fluid. We examined the hypothesis that local skin heating would result in an accumulation of ATP in the interstitial fluid that would be related with a rise in skin blood flow (SkBF) and temperature sensation. Two microdialysis probes were inserted into the dermis on the dorsal aspect of the forearm in 15 young, healthy subjects. The probed skin was maintained at 31°C, 35°C, 39°C and 43°C for 8 min periods, during which SkBF was monitored as cutaneous vascular conductance (CVC). Dialysate was collected and analysed for ATP ([ATP]d) using a luciferase-based assay, and ratings of perceived warmth were taken at each temperature. At a skin temperature of 31°C, [ATP]d averaged 18.93 ± 4.06 nm and CVC averaged 12.57 ± 1.59% peak. Heating skin to 35°C resulted in an increase in CVC (17.63 ± 1.27% peak; P < 0.05), but no change in [ATP]d. Heating skin to 39°C and 43°C resulted in a decreased [ATP]d (5.88 ± 1.68 nm and 8.75 ± 3.44 nm, respectively; P < 0.05), which was accompanied by significant elevations in CVC (38.90 ± 1.37% peak and 60.32 ± 1.95% peak, respectively; P < 0.05). Ratings of perceived warmth increased in proportion to the increase in skin temperature (r2 = 0.75, P < 0.05). In conclusion, our data indicate that an accumulation of interstitial ATP does not occur during local heating, and therefore does not have a role in temperature sensation or the dilator response in human skin. Nevertheless, the low threshold of dilatation (35°C) indicates a possible role for the TRPV3, TRPV4 channels or the sensitization of other ion channels in mediating the dilator response. PMID:23045344

  13. Yeast ADP/ATP Carrier Isoform 2

    PubMed Central

    Clémençon, Benjamin; Rey, Martial; Trézéguet, Véronique; Forest, Eric; Pelosi, Ludovic

    2011-01-01

    The mitochondrial ADP/ATP carrier, or Ancp, is a member of the mitochondrial carrier family responsible for exchanging ADP and ATP across the mitochondrial inner membrane. ADP/ATP transport involves Ancp switching between two conformational states. These can be analyzed using specific inhibitors, carboxyatractyloside (CATR) and bongkrekic acid (BA). The high resolution three-dimensional structure of bovine Anc1p (bAnc1p), as a CATR-carrier complex, has been solved. However, because the structure of the BA-carrier complex has not yet been determined, the detailed mechanism of transport remains unknown. Recently, sample processing for hydrogen/deuterium exchange experiments coupled to mass spectrometry was improved, providing novel insights into bAnc1p conformational transitions due to inhibitor binding. In this work we performed both hydrogen/deuterium exchange-mass spectrometry experiments and genetic manipulations. Because these are very difficult to apply with bovine Anc1p, we used Saccharomyces cerevisiae Anc isoform 2 (ScAnc2p). Significant differences in solvent accessibility were observed throughout the amino acid sequence for ScAnc2p complexed to either CATR or BA. Interestingly, in detergent solution, the conformational dynamics of ScAnc2p were dissimilar to those of bAnc1p, in particular for the upper half of the cavity, toward the intermembrane space, and the m2 loop, which is thought to be easily accessible to the solvent from the matrix in bAnc1p. Our study then focused on the methionyl residues of the Ancp signature sequence, RRRMMM. All our results indicate that the methionine cluster is involved in the ADP/ATP transport mechanism and confirm that the Ancp cavity is a highly dynamic structure. PMID:21868387

  14. Uncoupling by (--)-epigallocatechin-3-gallate of ATP-sensitive potassium channels from phosphatidylinositol polyphosphates and ATP.

    PubMed

    Jin, Jun-Yup; Park, Sung-Hee; Bae, Jae-Hoon; Cho, Ho-Chan; Lim, Jeong-Geun; Park, Won Sun; Han, Jin; Lee, Jin Ho; Song, Dae-Kyu

    2007-09-01

    Of green tea catechins, (--)-epigallocatechin-3-gallate (EGCG) and (--)-epicatechin-3-gallate (ECG), but not (--)-epicatechin and (--)-epigallocatechin, inhibit the activity of ATP-sensitive potassium (K(ATP)) channels at tens of micromolar concentrations, ECG being three times more effective than EGCG. Further, we found that by using cloned beta cell-type K(ATP) channels, only EGCG at 1 microM, a readily achievable plasma concentration by oral intake in humans, but not other epicatechins, significantly blocked channel reactivation after ATP wash-out, suggesting that interaction of phosphatidylinositol polyphosphates (PIP) with the channel was impaired by EGCG. In addition, a 10-fold higher concentration of EGCG reduced the channel sensitivity to ATP, but not AMP and ADP. This effect of EGCG was greater in the channel with the sulfonylurea receptor (SUR) than with the inwardly rectifying K(+) channel (Kir6.2) alone. Neomycin, a polycation, profoundly suppressed the effect of EGCG. Expectedly, glucose-stimulated cytosolic Ca(2+) elevation in rat pancreatic beta cells, and insulin secretory responses to high glucose loading in vivo were impaired by EGCG. In rabbit cardiac myocytes, dinitrophenol-induced opening of the channel was delayed by 1 microM EGCG. These results suggest that EGCG may interact with PIP-binding sites on the Kir6.2 subunit. SUR further endows EGCG with an ability to interfere with an interaction of the gamma-phosphate tail of ATP with Kir6.2. The specificity of EGCG possibly implies that 5'-OH of the B-ring on the pyrogallol moiety in the EGCG molecule may be critical for these actions of EGCG on the K(ATP) channel.

  15. H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

    PubMed Central

    Brand, M D; Lehninger, A L

    1977-01-01

    The stoichiometry of H+ ejection by mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) has been reexamined in the light of our recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. We show that earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scaler H+ production and describe a modified method for determination of this ratio which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods. A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and phosphate, and 1 H+ returns through the combined action of the phosphate and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3- and efflux of ATP4-. Thus, up to one-third of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 h+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems. PMID:17116

  16. H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

    PubMed

    Brand, M D; Lehninger, A L

    1977-05-01

    The stoichiometry of H+ ejection by mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) has been reexamined in the light of our recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. We show that earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scaler H+ production and describe a modified method for determination of this ratio which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods. A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and phosphate, and 1 H+ returns through the combined action of the phosphate and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3- and efflux of ATP4-. Thus, up to one-third of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 h+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems.

  17. Regulation of mitochondrial translation of the ATP8/ATP6 mRNA by Smt1p.

    PubMed

    Rak, Malgorzata; Su, Chen Hsien; Xu, Jonathan Tong; Azpiroz, Ricardo; Singh, Angela Mohan; Tzagoloff, Alexander

    2016-03-15

    Expression of the mitochondrially encoded ATP6 and ATP8 genes is translationally regulated by F1 ATPase. We report a translational repressor (Smt1p) of the ATP6/8 mRNA that, when mutated, restores translation of the encoded Atp6p and Atp8p subunits of the ATP synthase. Heterozygous smt1 mutants fail to rescue the translation defect, indicating that the mutations are recessive. Smt1p is an intrinsic inner membrane protein, which, based on its sedimentation, has a native size twice that of the monomer. Affinity purification of tagged Smt1p followed by reverse transcription of the associated RNA and PCR amplification of the resultant cDNA with gene-specific primers demonstrated the presence in mitochondria of Smt1p-ATP8/ATP6 and Smt1p-COB mRNA complexes. These results indicate that Smt1p is likely to be involved in translational regulation of both mRNAs. Applying Occam's principle, we favor a mechanistic model in which translation of the ATP8/ATP6 bicistronic mRNA is coupled to the availability of F1 for subsequent assembly of the Atp6p and Atp8p products into the ATP synthase. The mechanism of this regulatory pathway is proposed to entail a displacement of the repressor from the translationally mute Smt1-ATP8/ATP6 complex by F1, thereby permitting the Atp22p activator to interact with and promote translation of the mRNA.

  18. Suppressors of superoxide production from mitochondrial complex III

    PubMed Central

    Orr, Adam L.; Vargas, Leonardo; Turk, Carolina N.; Baaten, Janine E.; Matzen, Jason T.; Dardov, Victoria J.; Attle, Stephen J.; Li, Jing; Quackenbush, Douglas C.; Goncalves, Renata L. S.; Perevoshchikova, Irina V.; Petrassi, H. Michael; Meeusen, Shelly L.; Ainscow, Edward K.; Brand, Martin D.

    2015-01-01

    Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species (ROS), which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies but its role remains controversial. Using high-throughput screening we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress. PMID:26368590

  19. 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.

  20. Microglial migration mediated by ATP-induced ATP release from lysosomes.

    PubMed

    Dou, Ying; Wu, Hang-jun; Li, Hui-quan; Qin, Song; Wang, Yin-er; Li, Jing; Lou, Hui-fang; Chen, Zhong; Li, Xiao-ming; Luo, Qing-ming; Duan, Shumin

    2012-06-01

    Microglia are highly motile cells that act as the main form of active immune defense in the central nervous system. Attracted by factors released from damaged cells, microglia are recruited towards the damaged or infected site, where they are involved in degenerative and regenerative responses and phagocytotic clearance of cell debris. ATP release from damaged neural tissues has been suggested to mediate the rapid extension of microglial process towards the site of injury. However, the mechanisms of the long-range migration of microglia remain to be clarified. Here, we found that lysosomes in microglia contain abundant ATP and exhibit Ca(2+)-dependent exocytosis in response to various stimuli. By establishing an efficient in vitro chemotaxis assay, we demonstrated that endogenously-released ATP from microglia triggered by local microinjection of ATPγS is critical for the long-range chemotaxis of microglia, a response that was significantly inhibited in microglia treated with an agent inducing lysosome osmodialysis or in cells derived from mice deficient in Rab 27a (ashen mice), a small GTPase required for the trafficking and exocytosis of secretory lysosomes. These results suggest that microglia respond to extracellular ATP by releasing ATP themselves through lysosomal exocytosis, thereby providing a positive feedback mechanism to generate a long-range extracellular signal for attracting distant microglia to migrate towards and accumulate at the site of injury.

  1. 43 CFR 10000.3 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CONSERVATION COMMISSION ORGANIZATION AND FUNCTIONS § 10000.3 Definitions. Act refers to the Central Utah Project Completion Act, Titles II, III, IV, V, and VI of Public Law 102-575, October 30, 1992....

  2. 36 CFR 230.2 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... FORESTRY ASSISTANCE Community Forest and Open Space Conservation Program § 230.2 Definitions. The terms..., (iii) The preservation of open space (including farmland and forest land) where such preservation...

  3. 29 CFR 1983.101 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... tobacco products; (iii) Motor vehicles or motor vehicle equipment (as defined by 49 U.S.C. 30102(a)(6) and.... (o) Any future statutory amendments that affect the definition of a term or terms listed in this...

  4. Real-time luminescence imaging of cellular ATP release.

    PubMed

    Furuya, Kishio; Sokabe, Masahiro; Grygorczyk, Ryszard

    2014-03-15

    Extracellular ATP and other purines are ubiquitous mediators of local intercellular signaling within the body. While the last two decades have witnessed enormous progress in uncovering and characterizing purinergic receptors and extracellular enzymes controlling purinergic signals, our understanding of the initiating step in this cascade, i.e., ATP release, is still obscure. Imaging of extracellular ATP by luciferin-luciferase bioluminescence offers the advantage of studying ATP release and distribution dynamics in real time. However, low-light signal generated by bioluminescence reactions remains the major obstacle to imaging such rapid processes, imposing substantial constraints on its spatial and temporal resolution. We have developed an improved microscopy system for real-time ATP imaging, which detects ATP-dependent luciferin-luciferase luminescence at ∼10 frames/s, sufficient to follow rapid ATP release with sensitivity of ∼10 nM and dynamic range up to 100 μM. In addition, simultaneous differential interference contrast cell images are acquired with infra-red optics. Our imaging method: (1) identifies ATP-releasing cells or sites, (2) determines absolute ATP concentration and its spreading manner at release sites, and (3) permits analysis of ATP release kinetics from single cells. We provide instrumental details of our approach and give several examples of ATP-release imaging at cellular and tissue levels, to illustrate its potential utility.

  5. When Too Much ATP Is Bad for Protein Synthesis.

    PubMed

    Pontes, Mauricio H; Sevostyanova, Anastasia; Groisman, Eduardo A

    2015-08-14

    Adenosine triphosphate (ATP) is the energy currency of living cells. Even though ATP powers virtually all energy-dependent activities, most cellular ATP is utilized in protein synthesis via tRNA aminoacylation and guanosine triphosphate regeneration. Magnesium (Mg(2+)), the most common divalent cation in living cells, plays crucial roles in protein synthesis by maintaining the structure of ribosomes, participating in the biochemistry of translation initiation and functioning as a counterion for ATP. A non-physiological increase in ATP levels hinders growth in cells experiencing Mg(2+) limitation because ATP is the most abundant nucleotide triphosphate in the cell, and Mg(2+) is also required for the stabilization of the cytoplasmic membrane and as a cofactor for essential enzymes. We propose that organisms cope with Mg(2+) limitation by decreasing ATP levels and ribosome production, thereby reallocating Mg(2+) to indispensable cellular processes. Copyright © 2015. Published by Elsevier Ltd.

  6. Changing paradigms from a historical DSM-III and DSM-IV view toward an evidence-based definition of premature ejaculation. Part II--proposals for DSM-V and ICD-11.

    PubMed

    Waldinger, Marcel D; Schweitzer, Dave H

    2006-07-01

    In the Diagnostic and Statistical Manual of Mental Disorders (DSM), a descriptive definition for premature ejaculation (PE) that was based on historical assumptions has been accepted. To formulate a new functional definition of PE in the DSM. A "syndrome" approach instead of a "complaint" approach is applied and evidence-based data from epidemiological and clinical studies are used. A new functional definition of PE should pertain to a cluster of "symptoms" of a distinct "syndrome." A syndrome rather than a descriptive definition should distinguish Lifelong and Acquired PE variants. Evidence-based data also suggest another PE type "Natural Variable PE," which is not a typical syndrome but rather a cluster of inconsistent symptoms of rapid ejaculation. Moreover, in "Natural Variable PE" the occurrence of rapid ejaculation is not based on neurobiological or psychological pathology, but belongs to the normal variability of sexual performance. Its prevalence is probably much higher than that of Lifelong and Acquired PE. We propose three separate operationalized definitions of these three PE types for the pending DSM-V and ICD-11, which include a quantification of the ejaculation time (intravaginal ejaculation latency time), inability of ejaculatory control, and a description of severity of PE in terms of psychological distress. The use of the intravaginal ejaculation latency time into the DSM-V and ICD-11 would mean that statistical evidence becomes accepted as one of the mainstays for establishing an evidence-based definition of the three PE types.

  7. Release of ATP induced by hypertonic solutions in Xenopus oocytes

    PubMed Central

    Aleu, Jordi; Martín-Satué, Mireia; Navarro, Piedad; de Lara, Ivanna Pérez; Bahima, Laia; Marsal, Jordi; Solsona, Carles

    2003-01-01

    ATP mediates intercellular communication. Mechanical stress and changes in cell volume induce ATP release from various cell types, both secretory and non-secretory. In the present study, we stressed Xenopus oocytes with a hypertonic solution enriched in mannitol (300 mm). We measured simultaneously ATP release and ionic currents from a single oocyte. A decrease in cell volume, the activation of an inward current and ATP release were coincident. We found two components of ATP release: the first was associated with granule or vesicle exocytosis, because it was inhibited by tetanus neurotoxin, and the second was related to the inward current. A single exponential described the correlation between ATP release and the hypertonic-activated current. Gadolinium ions, which block mechanically activated ionic channels, inhibited the ATP release and the inward current but did not affect the decrease in volume. Oocytes expressing CFTR (cystic fibrosis transmembrane regulator) released ATP under hypertonic shock, but ATP release was significantly inhibited in the first component: that related to granule exocytosis. Since the ATP measured is the balance between ATP release and ATP degradation by ecto-enzymes, we measured the nucleoside triphosphate diphosphohydrolase (NTPDase) activity of the oocyte surface during osmotic stress, as the calcium-dependent hydrolysis of ATP, which was inhibited by more than 50 % in hypertonic conditions. The best-characterized membrane protein showing NTPDase activity is CD39. Oocytes injected with an antisense oligonucleotide complementary to CD39 mRNA released less ATP and showed a lower amplitude in the inward current than those oocytes injected with water. PMID:12562935

  8. From ATP to PTP and Back: A Dual Function for the Mitochondrial ATP Synthase.

    PubMed

    Bernardi, Paolo; Di Lisa, Fabio; Fogolari, Federico; Lippe, Giovanna

    2015-05-22

    Mitochondria not only play a fundamental role in heart physiology but are also key effectors of dysfunction and death. This dual role assumes a new meaning after recent advances on the nature and regulation of the permeability transition pore, an inner membrane channel whose opening requires matrix Ca(2+) and is modulated by many effectors including reactive oxygen species, matrix cyclophilin D, Pi (inorganic phosphate), and matrix pH. The recent demonstration that the F-ATP synthase can reversibly undergo a Ca(2+)-dependent transition to form a channel that mediates the permeability transition opens new perspectives to the field. These findings demand a reassessment of the modifications of F-ATP synthase that take place in the heart under pathological conditions and of their potential role in determining the transition of F-ATP synthase from and energy-conserving into an energy-dissipating device.

  9. Bringing Definitions into High Definition

    ERIC Educational Resources Information Center

    Mason, John

    2010-01-01

    Why do definitions play such a central role in mathematics? It may seem obvious that precision about the terms one uses is necessary in order to use those terms reasonably (while reasoning). Definitions are chosen so as to be definite about the terms one uses, but also to make both the statement of, and the reasoning to justify, theorems as…

  10. Bringing Definitions into High Definition

    ERIC Educational Resources Information Center

    Mason, John

    2010-01-01

    Why do definitions play such a central role in mathematics? It may seem obvious that precision about the terms one uses is necessary in order to use those terms reasonably (while reasoning). Definitions are chosen so as to be definite about the terms one uses, but also to make both the statement of, and the reasoning to justify, theorems as…

  11. Mitochondrial ATP-Pi exchange complex and the site of uncoupling of oxidative phosphorylation.

    PubMed

    Hatefi, Y; Hanstein, W G; Galante, Y; Stiggall, D L

    1975-07-01

    Five enzyme complexes, which are concerned with electron transport and oxidative phosphorylation, have been isolated from beef heart mitochondria. Enzyme complexes I, II, III and IV are the electron transfer complexes discovered in 1961. Complex V is an energy-conserving complex. It catalyzes ATP-Pi exchange and ATP hydrolysis. The exchange reaction is sensitive to uncouplers, rutamycin, valinomycin plus K-+, dicyclorexylcarboditmide, arsenate, azide, and adenylyl imidodiphosphate. It is also specific for ATP; ITP, GTP and UTP are essentially ineffective. Studies with the photoaffinity labeling uncoupler, 2-azido-4-nitrophenol (NPA), have shown that the mitochondrial uncoupler-binding sites are located exclusively in complex V. Complexes I, III and IV, which carry the three coupling sites of the respiratory chain, had negligible capacity for the binding of NPA, whereas the uncoupler-binding capacity of complex V appeared to be increased two- to threefold as compared to mitochondria. Complexes I, II, III, IV and V are obtained from the same batch of mitochondria by a simple fractionation procedure, which employs cholate, deoxycholate, ammonium acetate and ammonium sulfate. Studies with NPA have shown that mitochondria contain per milligram protein about 0.6 nmole of uniformly reacting uncoupler binding site. All of the uncouplers tested appeared to interact competitively with this site. Photoaffinity labeling with tritiated NPA has shown that a major portion of NPA binds to a polypeptide of molecular weight between 26,000 and 30,000. Other studies on the mechanism of uncoupling have shown that picrate is a membrane-impermeable uncoupler. It cannot uncouple mitochondria. However, it is an effective uncoupler of ATP synthesis and ATP-induced transhydrogenation or reverse electron transfer when used in conjunction with sonicated submitochondrial particles, which have an inside-out orientation of the inner membrane with respect to the medium. In these particles, picrate

  12. Molecular analysis of Wilson patients: direct sequencing and MLPA analysis in the ATP7B gene and Atox1 and COMMD1 gene analysis.

    PubMed

    Bost, Muriel; Piguet-Lacroix, Guénaelle; Parant, François; Wilson, C M R

    2012-06-01

    ATP7B mutations result in Cu storage in the liver and brain in Wilson disease (WD). Atox1 and COMMD1 were found to interact with ATP7B and involved in copper transport in the hepatocyte. To understand the molecular etiology of WD, we analyzed ATP7B, Atox1 and COMMD1 genes. Direct sequencing of (i) ATP7B gene was performed in 112 WD patients to identify the spectrum of disease-causing mutations in the French population, (ii) Atox1 gene was performed to study the known polymorphism 5'UTR-99T>C in 78 WD patients with two ATP7B mutations and (iii) COMMD1 gene was performed to detect the nucleotide change c.492GAT>GAC. MLPA (Multiplex Ligation-dependent Probe Amplification) analysis was performed in WD patients presenting only one ATP7B mutation. Among our 112 WD unrelated patients, 83 different ATP7B gene mutations were identified, 27 of which were novel. Two ATP7B mutations were identified in 98 WD cases, and one mutation was identified in 14 cases. In two of these 14 WD patients, we identified the deletion of exon 4 of the ATP7B gene by MLPA technique. In 78 selected patients of the cohort with two mutations in ATP7B, we have examined genotype-phenotype correlation between the detected changes in Atox1 and COMMD1 genes, and the presentation of the WD patients. Based on the data of this study, no major role can be attributed to Atox1 and COMMD in the pathophysiology or clinical variation of WD.

  13. Prevalence of metabolic syndrome diagnosis in patients with obstructive sleep apnoea syndrome according to adopted definition.

    PubMed

    Kumor, Marta; Bielicki, Piotr; Barnaś, Małgorzata; Przybyłowski, Tadeusz; Zieliński, Jan; Chazan, Ryszarda

    2013-01-01

    Metabolic syndrome (MS), which is connected with enlarged cardiovascular risk, is common in patients with OSAS. The aim of the study was to estimate the prevalence of MS in patients with OSAS according to two definitions of MS (criteria from NCEP-ATP III from 2001 versus criteria from IDF 2005). Materials consisted of 155 males and 18 females with OSAS (mean AHI 44 ± 22 h-1), obesity (BMI 31.8 ± 5.0 kg/m2), aged 53.9 ± 9.3 years (mean ± SD). Serum lipids, glucose, body mass index (BMI), waist circumference (WC) and waist-to-hip ratio (WHR) were measured in all patients. According to first definition (NCEP - ATP III from 2001), MS was diagnosed in 98 patients (56% of the whole group - MS1 group) compared to 120 patients (69% of the whole group - MS2 group) diagnosed according to the second definition (IDF from 2005), p < 0.05. No differences in BMI and WC between the groups were found. Significant differences in WHR were noted (MS1 group: 1.005 ± 0.05 vs. MS2 group: 1.027 ± 0.06, p < 0.05). Patients from the MS2 group had higher cholesterol HDL compared to the MS1 group (52.3 ± 12.1 mg/dl vs. 42.3 ± 12.1 mg/dl, p < 0.05). Serum triglyceride concentrations were significantly higher in the MS1 group than in the MS2 group (228 ± 122 mg/dl vs. 122 ± 49 mg/dl, p < 0.05). There were no differences in OSAS severity between the MS1 and MS2 group. In both groups weak correlations between diagnosis of MS and AHI were found (r = 0.19 for MS1 and r = 0.21 for MS2, p < 0.05) They are, however, clinically insignificant. The IDF definition from 2005 of metabolic syndrome indeed increases the frequency of diagnosis of metabolic syndrome in patients with OSAS. We did not observe essential clinical correlation among the degree of OSAS severity and recognition of metabolic syndrome in the MS1 or in the MS2 group.

  14. Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

    SciTech Connect

    Gruenhagen, Jason Alan

    2003-01-01

    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 activated a G{sub q}-type protein to initiate ATP release from HUVECs. Ca2+ imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca2+ 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

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

    PubMed

    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 (SO4 (2-)), 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 SO4 (2-)-activation and yields activated high-energy compound adenosine-5(')-phosphosulfate that is reduced to sulfide (S(2-)) 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.

  16. Mitochondrial flashes regulate ATP homeostasis in the heart.

    PubMed

    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

    2017-07-10

    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.

  17. 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

  18. A taste for ATP: neurotransmission in taste buds.

    PubMed

    Kinnamon, Sue C; Finger, Thomas E

    2013-12-18

    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.

  19. 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

  20. Release of extracellular ATP by bacteria during growth

    PubMed Central

    2013-01-01

    Background Adenosine triphosphate (ATP) is used as an intracellular energy source by all living organisms. It plays a central role in the respiration and metabolism, and is the most important energy supplier in many enzymatic reactions. Its critical role as the energy storage molecule makes it extremely valuable to all cells. Results We report here the detection of extracellular ATP in the cultures of a variety of bacterial species. The levels of the extracellular ATP in bacterial cultures peaked around the end of the log phase and decreased in the stationary phase of growth. Extracellular ATP levels were dependent on the cellular respiration as bacterial mutants lacking cytochrome bo oxidase displayed lower extracellular ATP levels. We have also shown that Escherichia coli (E. coli) and Salmonella actively depleted extracellular ATP and an ATP supplement in culture media enhanced the stationary survival of E. coli and Salmonella. In addition to E. coli and Salmonella the presence of the extracellular ATP was observed in a variety of bacterial species that contain human pathogens such as Acinetobacter, Pseudomonas, Klebsiella and Staphylococcus. Conclusion Our results indicate that extracellular ATP is produced by many bacterial species during growth and extracellular ATP may serve a role in the bacterial physiology. PMID:24364860

  1. Role of ATP-bound divalent metal ion in the conformation and function of actin. Comparison of Mg-ATP, Ca-ATP, and metal ion-free ATP-actin.

    PubMed

    Valentin-Ranc, C; Carlier, M F

    1991-04-25

    The fluorescence of N-acetyl-N'-(sulfo-1-naphthyl)ethylenediamine (AEDANS) covalently bound to Cys-374 of actin is used as a probe for different conformational states of G-actin according to whether Ca-ATP, Mg-ATP, or unchelated ATP is bound to the nucleotide site. Upon addition of large amounts (greater than 10(2)-fold molar excess) of EDTA to G-actin, metal ion-free ATP-G-actin is obtained with EDTA bound. Metal ion free ATP-G-actin is characterized by a higher AEDANS fluorescence than Mg-ATP-G-actin, which itself has a higher fluorescence than Ca-ATP-G-actin. Evidence for EDTA binding to G-actin is shown using difference spectrophotometry. Upon binding of EDTA, the rate of dissociation of the divalent metal ion from G-actin is increased (2-fold for Ca2+, 10-fold for Mg2+) in a range of pH from 7.0 to 8.0. A model is proposed that quantitatively accounts for the kinetic data. The affinity of ATP is weakened 10(6)-fold upon removal of the metal ion. Metal ion-free ATP-G-actin is in a partially open conformation, as indicated by the greater accessibility of -SH residues, yet it retains functional properties of polymerization and ATP hydrolysis that appear almost identical to those of Ca-ATP-actin, therefore different from those of Mg-ATP-actin. These results are discussed in terms of the role of the ATP-bound metal ion in actin structure and function.

  2. Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP

    PubMed Central

    Blodgett, David M.; De Zutter, Julie K.; Levine, Kara B.; Karim, Pusha; Carruthers, Anthony

    2007-01-01

    Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6–7. PMID:17635959

  3. Thioredoxin-insensitive plastid ATP synthase that performs moonlighting functions

    PubMed Central

    Kohzuma, Kaori; Dal Bosco, Cristina; Kanazawa, Atsuko; Dhingra, Amit; Nitschke, Wolfgang; Meurer, Jörg; Kramer, David M.

    2012-01-01

    The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and acts as a key feedback regulatory component of photosynthesis. Arabidopsis possesses two homologues of the regulatory γ subunit of the ATP synthase, encoded by the ATPC1 and ATPC2 genes. Using a series of mutants, we show that both these subunits can support photosynthetic ATP synthesis in vivo with similar specific activities, but that in wild-type plants, only γ1 is involved in ATP synthesis in photosynthesis. The γ1-containing ATP synthase shows classical light-induced redox regulation, whereas the mutant expressing only γ2-ATP synthase (gamma exchange-revised ATP synthase, gamera) shows equally high ATP synthase activity in the light and dark. In situ redox titrations demonstrate that the regulatory thiol groups on γ2-ATP synthase remain reduced under physiological conditions but can be oxidized by the strong oxidant diamide, implying that the redox potential for the thiol/disulphide transition in γ2 is substantially higher than that for γ1. This regulatory difference may be attributed to alterations in the residues near the redox-active thiols. We propose that γ2-ATP synthase functions to catalyze ATP hydrolysis-driven proton translocation in nonphotosynthetic plastids, maintaining a sufficient transthylakoid proton gradient to drive protein translocation or other processes. Consistent with this interpretation, ATPC2 is predominantly expressed in the root, whereas modifying its expression results in alteration of root hair development. Phylogenetic analysis suggests that γ2 originated from ancient gene duplication, resulting in divergent evolution of functionally distinct ATP synthase complexes in dicots and mosses. PMID:22328157

  4. Definitely Life but not Definitively

    NASA Astrophysics Data System (ADS)

    Oliver, Joan D.; Perry, Randall S.

    2006-12-01

    Although there have been attempts at a definition of life from many disciplines, none is accepted by all as definitive. Some people believe that it is impossible to define ‘life’ adequately at the moment. We agree with this point of view on linguistic grounds, examining the different types of definition, the contexts in which they are used and their relative usefulness as aids to arriving at a scientific definition of life. We look at some of the more recent definitions and analyse them in the light of our criteria for a good definition. We argue that since there are so many linguistic and philosophical difficulties with such a definition of life, what is needed is a series of working descriptions, which are suited to the audience and context in which they are used and useful for the intended purpose. We provide some ideas and examples of the forms these may take.

  5. Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP Binding: Requirement for Establishing Chronic Persistent Infection

    SciTech Connect

    Drumm, J.; Mi, K; Bilder, P; Sun, M; Lim, J; Bielefeldt-Ohmann, H; Basaraba, R; So, M; Zhu, G; et. al.

    2009-01-01

    Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

  6. Low energy costs of F1Fo ATP synthase reversal in colon carcinoma cells deficient in mitochondrial complex IV.

    PubMed

    Zhdanov, Alexander V; Andreev, Dmitry E; Baranov, Pavel V; Papkovsky, Dmitri B

    2017-05-01

    Mitochondrial polarisation is paramount for a variety of cellular functions. Under ischemia, mitochondrial membrane potential (ΔΨm) and proton gradient (ΔpH) are maintained via a reversal of mitochondrial F1Fo ATP synthase (mATPase), which can rapidly deplete ATP and drive cells into energy crisis. We found that under normal conditions in cells with disassembled cytochrome c oxidase complex (COX-deficient HCT116), mATPase maintains ΔΨm at levels only 15-20% lower than in WT cells, and for this utilises relatively little ATP. For a small energy expenditure, mATPase enables mitochondrial ΔpH, protein import, Ca(2+) turnover, and supports free radical detoxication machinery enlarged to protect the cells from oxidative damage. Whereas in COX-deficient cells the main source of ATP is glycolysis, the ΔΨm is still maintained upon inhibition of the adenine nucleotide translocators with bongkrekic acid and carboxyatractyloside, indicating that the role of ANTs is redundant, and matrix substrate level phosphorylation alone or in cooperation with ATP-Mg/Pi carriers can continuously support the mATPase activity. Intriguingly, we found that mitochondrial complex III is active, and it contributes not only to free radical production, but also to ΔΨm maintenance and energy budget of COX-deficient cells. Overall, this study demonstrates that F1Fo ATP synthase can support general mitochondrial and cellular functions, working in extremely efficient 'energy saving' reverse mode and flexibly recruiting free radical detoxication and ATP producing / transporting pathways.

  7. A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    PubMed Central

    Schulz, Sarah; Iglesias-Cans, Marina; Krah, Alexander; Yildiz, Özkan; Leone, Vanessa; Matthies, Doreen; Cook, Gregory M.

    2013-01-01

    The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na+. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1Fo-ATP synthase with a novel Na+ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na+ specificity in physiological settings. Consistently, activity measurements showed Na+ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na+ ionophore monensin. Furthermore, Na+ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na+ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na+ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na+ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen. PMID:23824040

  8. 20 CFR 631.87 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 3 2010-04-01 2010-04-01 false Definitions. 631.87 Section 631.87 Employees' Benefits EMPLOYMENT AND TRAINING ADMINISTRATION, DEPARTMENT OF LABOR PROGRAMS UNDER TITLE III OF THE JOB TRAINING PARTNERSHIP ACT Disaster Relief Employment Assistance § 631.87 Definitions. As used in this...

  9. 40 CFR 350.1 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... INFORMATION: AND TRADE SECRET DISCLOSURES TO HEALTH PROFESSIONALS Trade Secrecy Claims § 350.1 Definitions... secret. Sanitized means a version of a document from which information claimed as trade secret or... support of a claim that chemical identity is a trade secret. Title III means Title III of the...

  10. 40 CFR 350.1 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... INFORMATION: AND TRADE SECRET DISCLOSURES TO HEALTH PROFESSIONALS Trade Secrecy Claims § 350.1 Definitions... secret. Sanitized means a version of a document from which information claimed as trade secret or... support of a claim that chemical identity is a trade secret. Title III means Title III of the...

  11. 40 CFR 350.1 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... INFORMATION: AND TRADE SECRET DISCLOSURES TO HEALTH PROFESSIONALS Trade Secrecy Claims § 350.1 Definitions... secret. Sanitized means a version of a document from which information claimed as trade secret or... support of a claim that chemical identity is a trade secret. Title III means Title III of the...

  12. [Effect of complex therapy including ATP-long on left ventricular diastolic function in patients with ischemic heart disease at rest and under isometric load].

    PubMed

    Amosova, E N; Bereza, N V; Potapkova, I V

    2002-01-01

    The study comprised 34 patients with ischemic heart disease (IHD) stable functional class I-II extertional angina with impaired relaxation type diastolic dysfunction of the left ventricle. Instituted in all patients before and after the combined treatment involving the use of ATP-Long (group I) or ATP solution injectable i.m. (group II) was dopplercardiometry in rest and at the peak of isometric load. The course of ATP treatments administration was ten days in duration. The use in a combined treatment IHD patients of ATP-Long, a new metabolic-action type drug preparation of Ukraine, permits improving parameters of the diastole temporal patterns, as evidenced by results of the studies made.

  13. 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.

  14. Paradox applications integration ATP`s for MAC and mass balance programs

    SciTech Connect

    Russell, V.K.; Mullaney, J.E.

    1994-10-17

    The K Basins Materials Accounting (MAC) and Material Balance (MBA) database system were set up to run under one common applications program. This Acceptance Test Plan (ATP) describes how the code was to be tested to verify its correctness. The scope of the tests is minimal, since both MAC and MBA have already been tested in detail as stand-alone programs.

  15. Aldosterone acts via an ATP autocrine/paracrine system: The Edelman ATP hypothesis revisited

    PubMed Central

    Gorelik, Julia; Zhang, Yanjun; Sánchez, Daniel; Shevchuk, Andrew; Frolenkov, Gregory; Lab, Max; Klenerman, David; Edwards, Christopher; Korchev, Yuri

    2005-01-01

    Aldosterone, the most important sodium-retaining hormone, was first characterized >50 years ago. However, despite numerous studies including the classical work of Isidore S. “Izzy” Edelman showing that aldosterone action depended on ATP production, the mechanism by which it activates sodium reabsorption via the epithelial sodium channel remains unclear. Here, we report experiments that suggest that one of the key steps in aldosterone action is via an autocrine/paracrine system. The hormone stimulates ATP release from the basolateral side of the target kidney cell. Prevention of ATP accumulation or its removal blocks aldosterone action. ATP then acts via a purinergic mechanism to produce contraction of small groups of adjacent epithelial cells. Patch clamping demonstrates that it is these contracted cells that have channel activity. With progressive recruitment of contracting cells, there is then a parallel increase in transepithelial electrical conductance. In common with other stimuli of sodium transport, this pathway involves phosphatidylinositol 3-kinase. Inhibition of phosphatidylinositol 3-kinase blocks both cell contraction and conductance. We put forward the hypothesis that redistribution of the cell volume caused by the lateral contraction results in apical swelling and that this change, in turn, disrupts the epithelial sodium channel interaction with the F-actin cytoskeleton, opening the channel and hence increasing sodium transport. PMID:16230642

  16. ATP alone triggers the outward facing conformation of the maltose ATP-binding cassette transporter.

    PubMed

    Bao, Huan; Duong, Franck

    2013-02-01

    The maltose transporter MalFGK(2) is a study prototype for ABC importers. During catalysis, the MalFG membrane domain alternates between inward and outward facing conformations when the MalK dimer closes and hydrolyzes ATP. Because a rapid ATP hydrolysis depends on MalE and maltose, it has been proposed that closed liganded MalE facilitates the transition to the outward facing conformation. Here we find that, in contrast to the expected, ATP is sufficient for the closure of MalK and for the conversion of MalFG to the outward facing state. The outward facing transporter binds MalE with nanomolar affinity, yet neither MalE nor maltose is necessary or facilitates the transition. Thus, the rapid hydrolysis of ATP observed in the presence of MalE and maltose is not because closed liganded MalE accelerates the formation of the outward facing conformation. These findings have fundamental implications for the description of the transport reaction.

  17. Sequential Action of MalE and Maltose Allows Coupling ATP Hydrolysis to Translocation in the MalFGK2 Transporter.

    PubMed

    Bao, Huan; Dalal, Kush; Cytrynbaum, Eric; Duong, Franck

    2015-10-16

    ATP-binding cassette (ABC) transporters have evolved an ATP-dependent alternating-access mechanism to transport substrates across membranes. Despite important progress, especially in their structural analysis, it is still unknown how the substrate stimulates ATP hydrolysis, the hallmark of ABC transporters. In this study, we measure the ATP turnover cycle of MalFGK2 in steady and pre-steady state conditions. We show that (i) the basal ATPase activity of MalFGK2 is very low because the cleavage of ATP is rate-limiting, (ii) the binding of open-state MalE to the transporter induces ATP cleavage but leaves release of Pi limiting, and (iii) the additional presence of maltose stimulates release of Pi, and therefore increases the overall ATP turnover cycle. We conclude that open-state MalE stabilizes MalFGK2 in the outward-facing conformation until maltose triggers return to the inward-facing state for substrate and Pi release. This concerted action explains why ATPase activity of MalFGK2 depends on maltose, and why MalE is essential for transport.

  18. ATP-sensitive K+ channels in rat pancreatic beta-cells: modulation by ATP and Mg2+ ions.

    PubMed Central

    Ashcroft, F M; Kakei, M

    1989-01-01

    1. The inside-out configuration of the patch-clamp method was used to study the effects of MgATP, free ATP and Mg2+ on single ATP-sensitive K+ channel currents in rat pancreatic beta-cells. 2. Magnesium ions caused a marked reduction of channel activity: 5 mM-free Mg2+ produced a 50% reduction in the activity of inward currents recorded at -60 mV in symmetrical K+ concentrations. 3. Inhibition of channel activity by MgATP does not involve phosphorylation as both free ATP (i.e. ATP in the absence of divalent cations) and non-hydrolysable ATP analogues were effective inhibitors. 4. Magnesium ions produced a striking reduction in the ability of ATP (total) to inhibit channel activity. When channel activity was plotted as a function of the total ATP concentration, the Ki for channel inhibition was 4 microM in Mg2(+)-free solution, compared to a Ki of 26 microM in the presence of 2 mM-Mg2+. The shape of the relationship between channel activity and the total ATP concentration was not changed by Mg2+. When channel activity was plotted as a function of the free ATP concentration, however, Mg2+ had little effect on Ki. This suggests that free ATP is the more potent inhibitor of channel activity and that MgATP has little inhibitory effect. 5. ATP analogues that dissociate only as far as the tribasic form were also able to inhibit channel activity. This suggests that both ATP4- and ATPH3- can block the channel. 6. Like ATP, ADP was more effective at inhibiting channel activity in the absence of Mg2+, that is as the free base. The non-hydrolysable ATP analogues AMP-PNP and AMP-PCP, however, were more effective in the presence of Mg2+. 7. It is suggested that (1) the potency of inhibition is related to the amount of negative charge carried by the ion and (2) the intracellular concentration of free ATP will be an important modulator of channel activity in the intact beta-cell. PMID:2691645

  19. Effects upon metabolic pathways and energy production by Sb(III) and As(III)/Sb(III)-oxidase gene aioA in Agrobacterium tumefaciens GW4

    PubMed Central

    Li, Jingxin; Yang, Birong; Shi, Manman; Yuan, Kai; Guo, Wei; Li, Mingshun

    2017-01-01

    Agrobacterium tumefaciens GW4 is a heterotrophic arsenite [As(III)]/antimonite [Sb(III)]-oxidizing strain. The As(III) oxidase AioAB is responsible for As(III) oxidation in the periplasm and it is also involved in Sb(III) oxidation in Agrobacterium tumefaciens 5A. In addition, Sb(III) oxidase AnoA and cellular H2O2 are also responsible for Sb(III) oxidation in strain GW4. However, the deletion of aioA increased the Sb(III) oxidation efficiency in strain GW4. In the present study, we found that the cell mobility to Sb(III), ATP and NADH contents and heat release were also increased by Sb(III) and more significantly in the aioA mutant. Proteomics and transcriptional analyses showed that proteins/genes involved in Sb(III) oxidation and resistance, stress responses, carbon metabolism, cell mobility, phosphonate and phosphinate metabolism, and amino acid and nucleotide metabolism were induced by Sb(III) and were more significantly induced in the aioA mutant. The results suggested that Sb(III) oxidation may produce energy. In addition, without periplasmic AioAB, more Sb(III) would enter bacterial cells, however, the cytoplasmic AnoA and the oxidative stress response proteins were significantly up-regulated, which may contribute to the increased Sb(III) oxidation efficiency. Moreover, the carbon metabolism was also activated to generate more energy against Sb(III) stress. The generated energy may be used in Sb transportation, DNA repair, amino acid synthesis, and cell mobility, and may be released in the form of heat. PMID:28241045

  20. Effects upon metabolic pathways and energy production by Sb(III) and As(III)/Sb(III)-oxidase gene aioA in Agrobacterium tumefaciens GW4.

    PubMed

    Li, Jingxin; Yang, Birong; Shi, Manman; Yuan, Kai; Guo, Wei; Li, Mingshun; Wang, Gejiao

    2017-01-01

    Agrobacterium tumefaciens GW4 is a heterotrophic arsenite [As(III)]/antimonite [Sb(III)]-oxidizing strain. The As(III) oxidase AioAB is responsible for As(III) oxidation in the periplasm and it is also involved in Sb(III) oxidation in Agrobacterium tumefaciens 5A. In addition, Sb(III) oxidase AnoA and cellular H2O2 are also responsible for Sb(III) oxidation in strain GW4. However, the deletion of aioA increased the Sb(III) oxidation efficiency in strain GW4. In the present study, we found that the cell mobility to Sb(III), ATP and NADH contents and heat release were also increased by Sb(III) and more significantly in the aioA mutant. Proteomics and transcriptional analyses showed that proteins/genes involved in Sb(III) oxidation and resistance, stress responses, carbon metabolism, cell mobility, phosphonate and phosphinate metabolism, and amino acid and nucleotide metabolism were induced by Sb(III) and were more significantly induced in the aioA mutant. The results suggested that Sb(III) oxidation may produce energy. In addition, without periplasmic AioAB, more Sb(III) would enter bacterial cells, however, the cytoplasmic AnoA and the oxidative stress response proteins were significantly up-regulated, which may contribute to the increased Sb(III) oxidation efficiency. Moreover, the carbon metabolism was also activated to generate more energy against Sb(III) stress. The generated energy may be used in Sb transportation, DNA repair, amino acid synthesis, and cell mobility, and may be released in the form of heat.

  1. Single molecule thermodynamics of ATP synthesis by F1-ATPase

    NASA Astrophysics Data System (ADS)

    Toyabe, Shoichi; Muneyuki, Eiro

    2015-01-01

    FoF1-ATP synthase is a factory for synthesizing ATP in virtually all cells. Its core machinery is the subcomplex F1-motor (F1-ATPase) and performs the reversible mechanochemical coupling. The isolated F1-motor hydrolyzes ATP, which is accompanied by unidirectional rotation of its central γ -shaft. When a strong opposing torque is imposed, the γ -shaft rotates in the opposite direction and drives the F1-motor to synthesize ATP. This mechanical-to-chemical free-energy transduction is the final and central step of the multistep cellular ATP-synthetic pathway. Here, we determined the amount of mechanical work exploited by the F1-motor to synthesize an ATP molecule during forced rotations using a methodology combining a nonequilibrium theory and single molecule measurements of responses to external torque. We found that the internal dissipation of the motor is negligible even during rotations far from a quasistatic process.

  2. Snapshots of the maltose transporter during ATP hydrolysis

    SciTech Connect

    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.

  3. ATP6AP1 — EDRN Public Portal

    Cancer.gov

    ATP6AP1 is an integral membrane protein composed of at least 10 subunits. It is responsible for acidifying various eukaryotic intracellular organelles. ATP6AP1, also known as vacuolar ATPase or V-ATPase, has a cytosolic V1 domain and a transmembrane V0 domain. The acidification performed by ATP6AP1 is necessary for intracellular processes such as protein sorting, zymogen activation, and receptor-mediated endocytosis.

  4. ATP7B detoxifies silver in ciliated airway epithelial cells

    SciTech Connect

    Ibricevic, Aida; Brody, Steven L.; Youngs, Wiley J.; Cannon, Carolyn L.

    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 compounds 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.

  5. Intracellular Assessment of ATP Levels in Caenorhabditis elegans

    PubMed Central

    Palikaras, Konstantinos; Tavernarakis, Nektarios

    2017-01-01

    Eukaryotic cells heavily depend on adenosine triphosphate (ATP) generated by oxidative phosphorylation (OXPHOS) within mitochondria. ATP is the major energy currency molecule, which fuels cell to carry out numerous processes, including growth, differentiation, transportation and cell death among others (Khakh and Burnstock, 2009). Therefore, ATP levels can serve as a metabolic gauge for cellular homeostasis and survival (Artal-Sanz and Tavernarakis, 2009; Gomes et al., 2011; Palikaras et al., 2015). In this protocol, we describe a method for the determination of intracellular ATP levels using a bioluminescence approach in the nematode Caenorhabditis elegans. PMID:28194429

  6. Application of luciferase assay for ATP to antimicrobial drug susceptibility

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Vellend, H.; Tuttle, S. A.; Barza, M. J.; Weinstein, L. (Inventor)

    1977-01-01

    The susceptibility of bacteria, particularly those derived from body fluids, to antimicrobial agents is determined in terms of an ATP index measured by culturing a bacterium in a growth medium. The amount of ATP is assayed in a sample of the cultured bacterium by measuring the amount of luminescent light emitted when the bacterial ATP is reacted with a luciferase-luciferin mixture. The sample of the cultured bacterium is subjected to an antibiotic agent. The amount of bacterial adenosine triphosphate is assayed after treatment with the antibiotic by measuring the luminescent light resulting from the reaction. The ATP index is determined from the values obtained from the assay procedures.

  7. A hierarchy of ATP-consuming processes in mammalian cells.

    PubMed Central

    Buttgereit, F; Brand, M D

    1995-01-01

    The rates of different ATP-consuming reactions were measured in concanavalin A-stimulated thymocytes, a model system in which more than 80% of the ATP consumption can be accounted for. There was a clear hierarchy of the responses of different energy-consuming reactions to changes in energy supply: pathways of macromolecule biosynthesis (protein synthesis and RNA/DNA synthesis) were most sensitive to energy supply, followed by sodium cycling and then calcium cycling across the plasma membrane. Mitochondrial proton leak was the least sensitive to energy supply. Control analysis was used to quantify the relative control over ATP production exerted by the individual groups of ATP-consuming reactions. Control was widely shared; no block of reactions had more than one-third of the control. A fuller control analysis showed that there appeared to be a hierarchy of control over the flux through ATP: protein synthesis > RNA/DNA synthesis and substrate oxidation > Na+ cycling and Ca2+ cycling > other ATP consumers and mitochondrial proton leak. Control analysis also indicated that there was significant control over the rates of individual ATP consumers by energy supply. Each ATP consumer had strong control over its own rate but very little control over the rates of the other ATP consumers. Images Figure 3 PMID:7492307

  8. Rows of ATP Synthase Dimers in Native Mitochondrial Inner Membranes

    PubMed Central

    Buzhynskyy, Nikolay; Sens, Pierre; Prima, Valerie; Sturgis, James N.; Scheuring, Simon

    2007-01-01

    The ATP synthase is a nanometric rotary machine that uses a transmembrane electrochemical gradient to form ATP. The structures of most components of the ATP synthase are known, and their organization has been elucidated. However, the supramolecular assembly of ATP synthases in biological membranes remains unknown. Here we show with submolecular resolution the organization of ATP synthases in the yeast mitochondrial inner membranes. The atomic force microscopy images we have obtained show how these molecules form dimers with characteristic 15 nm distance between the axes of their rotors through stereospecific interactions of the membrane embedded portions of their stators. A different interaction surface is responsible for the formation of rows of dimers. Such an organization elucidates the role of the ATP synthase in mitochondrial morphology. Some dimers have a different morphology with 10 nm stalk-to-stalk distance, in line with ATP synthases that are accessible to IF1 inhibition. Rotation torque compensation within ATP synthase dimers stabilizes the ATP synthase structure, in particular the stator-rotor interaction. PMID:17557793

  9. Binding of ATP by pertussis toxin and isolated toxin subunits

    SciTech Connect

    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; 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.

  10. Understanding structure, function, and mutations in the mitochondrial ATP synthase

    PubMed Central

    Xu, Ting; Pagadala, Vijayakanth; Mueller, David M.

    2015-01-01

    The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs) in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs. PMID:25938092

  11. Imaging changes in the cytosolic ATP-to-ADP ratio

    PubMed Central

    Tantama, Mathew; Yellen, Gary

    2015-01-01

    Adenosine triphosphate (ATP) is a central metabolite that plays fundamental roles as an energy transfer molecule, a phosphate donor, and a signaling molecule inside cells. The phosphoryl group transfer potential of ATP provides a thermodynamic driving force for many metabolic reactions, and phosphorylation of both small metabolites and large proteins can serve as a regulatory modification. In the process of phosphoryl transfer from ATP, the diphosphate ADP is produced, and as a result, the ATP-to-ADP ratio is an important physiological control parameter. The ATP-to-ADP ratio is directly proportional to cellular energy charge and phosphorylation potential. Furthermore, several ATP-dependent enzymes and signaling proteins are regulated by ADP, and their activation profiles are a function of the ATP-to-ADP ratio. Finally, regeneration of ATP from ADP can serve as an important readout of energy metabolism and mitochondrial function. We therefore developed a genetically-encoded fluorescent biosensor tuned to sense ATP-to-ADP ratios in the physiological range of healthy mammalian cells. Here we present a protocol for using this biosensor to visualize energy status using live-cell fluorescence microscopy. PMID:25416365

  12. Diversity and regulation of ATP sulfurylase in photosynthetic organisms

    PubMed Central

    Prioretti, Laura; Gontero, Brigitte; Hell, Ruediger; Giordano, Mario

    2014-01-01

    ATP sulfurylase (ATPS) catalyzes the first committed step in the sulfate assimilation pathway, the activation of sulfate prior to its reduction. ATPS has been studied in only a few model organisms and even in these cases to a much smaller extent than the sulfate reduction and cysteine synthesis enzymes. This is possibly because the latter were considered of greater regulatory importance for sulfate assimilation. Recent evidences (reported in this paper) challenge this view and suggest that ATPS may have a crucial regulatory role in sulfate assimilation, at least in algae. In the ensuing text, we summarize the current knowledge on ATPS, with special attention to the processes that control its activity and gene(s) expression in algae. Special attention is given to algae ATPS proteins. The focus on algae is the consequence of the fact that a comprehensive investigation of ATPS revealed that the algal enzymes, especially those that are most likely involved in the pathway of sulfate reduction to cysteine, possess features that are not present in other organisms. Remarkably, algal ATPS proteins show a great diversity of isoforms and a high content of cysteine residues, whose positions are often conserved. According to the occurrence of cysteine residues, the ATPS of eukaryotic algae is closer to that of marine cyanobacteria of the genera Synechococcus and Prochlorococcus and is more distant from that of freshwater cyanobacteria. These characteristics might have evolved in parallel with the radiation of algae in the oceans and the increase of sulfate concentration in seawater. PMID:25414712

  13. ATP25, a New Nuclear Gene of Saccharomyces cerevisiae Required for Expression and Assembly of the Atp9p Subunit of Mitochondrial ATPase

    PubMed Central

    Zeng, Xiaomei; Barros, Mario H.; Shulman, Theodore

    2008-01-01

    We report a new nuclear gene, designated ATP25 (reading frame YMR098C on chromosome XIII), required for expression of Atp9p (subunit 9) of the Saccharomyces cerevisiae mitochondrial proton translocating ATPase. Mutations in ATP25 elicit a deficit of ATP9 mRNA and of its translation product, thereby preventing assembly of functional F0. Unlike Atp9p, the other mitochondrial gene products, including ATPase subunits Atp6p and Atp8p, are synthesized normally in atp25 mutants. Northern analysis of mitochondrial RNAs in an atp25 temperature-sensitive mutant confirmed that Atp25p is required for stability of the ATP9 mRNA. Atp25p is a mitochondrial inner membrane protein with a predicted mass of 70 kDa. The primary translation product of ATP25 is cleaved in vivo after residue 292 to yield a 35-kDa C-terminal polypeptide. The C-terminal half of Atp25p is sufficient to stabilize the ATP9 mRNA and restore synthesis of Atp9p. Growth on respiratory substrates, however, depends on both halves of Atp25p, indicating that the N-terminal half has another function, which we propose to be oligomerization of Atp9p into a proper size ring structure. PMID:18216280

  14. Research conference summary from the 2014 International Task Force on ATP1A3-Related Disorders

    PubMed Central

    Rosewich, Hendrik; DeBrosse, Suzanne; Ess, Kevin; Ozelius, Laurie; Andermann, Eva; Andermann, Frederick; Andrasco, Gene; Belgrade, Alice; Brashear, Allison; Ciccodicola, Sharon; Egan, Lynn; George, Alfred L.; Lewelt, Aga; Magelby, Joshua; Merida, Mario; Newcomb, Tara; Platt, Vicky; Poncelin, Dominic; Reyna, Sandra; Sasaki, Masayuki; Sotero de Menezes, Marcio; Sweadner, Kathleen; Viollet, Louis; Zupanc, Mary; Silver, Kenneth; Swoboda, Kathryn

    2017-01-01

    Objective: ATP1A3-related neurologic disorders encompass a broad range of phenotypes that extend well beyond initial phenotypic criteria associated with alternating hemiplegia of childhood (AHC) and rapid-onset dystonia parkinsonism. Methods: In 2014, the Alternating Hemiplegia of Childhood Foundation hosted a multidisciplinary workshop intended to address fundamental challenges surrounding the diagnosis and management of individuals with ATP1A3-related disorders. Results: Workshop attendees were charged with the following: (1) to achieve consensus on expanded diagnostic criteria to facilitate the identification of additional patients, intended to supplement existing syndrome-specific diagnostic paradigms; (2) to standardize definitions for the broad range of paroxysmal manifestations associated with AHC to disseminate to families; (3) to create clinical recommendations for common recurrent issues facing families and medical care providers; (4) to review data related to the death of individuals in the Alternating Hemiplegia of Childhood Foundation database to guide future efforts in identifying at-risk subjects and potential preventative measures; and (5) to identify critical gaps where we most need to focus national and international research efforts. Conclusions: This report summarizes recommendations of the workshop committee, highlighting the key phenotypic features to facilitate the diagnosis of possible ATP1A3 mutations, providing recommendations for genetic testing, and outlining initial acute management for common recurrent clinical conditions, including epilepsy. PMID:28293679

  15. ATP5H/KCTD2 locus is associated with Alzheimer's disease risk

    PubMed Central

    Boada, M; Antúnez, C; Ramírez-Lorca, R; DeStefano, A L; González-Pérez, A; Gayán, J; López-Arrieta, J; Ikram, M A; Hernández, I; Marín, J; Galán, J J; Bis, J C; Mauleón, A; Rosende-Roca, M; Moreno-Rey, C; Gudnasson, V; Morón, F J; Velasco, J; Carrasco, J M; Alegret, M; Espinosa, A; Vinyes, G; Lafuente, A; Vargas, L; Fitzpatrick, A L; Launer, L J; Sáez, M E; Vázquez, E; Becker, J T; López, O L; Serrano-Ríos, M; Tárraga, L; van Duijn, C M; Real, L M; Seshadri, S; Ruiz, A

    2014-01-01

    To identify loci associated with Alzheimer disease, we conducted a three-stage analysis using existing genome-wide association studies (GWAS) and genotyping in a new sample. In Stage I, all suggestive single-nucleotide polymorphisms (at P<0.001) in a previously reported GWAS of seven independent studies (8082 Alzheimer's disease (AD) cases; 12 040 controls) were selected, and in Stage II these were examined in an in silico analysis within the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium GWAS (1367 cases and 12904 controls). Six novel signals reaching P<5 × 10−6 were genotyped in an independent Stage III sample (the Fundació ACE data set) of 2200 sporadic AD patients and 2301 controls. We identified a novel association with AD in the adenosine triphosphate (ATP) synthase, H+ transporting, mitochondrial F0 (ATP5H)/Potassium channel tetramerization domain-containing protein 2 (KCTD2) locus, which reached genome-wide significance in the combined discovery and genotyping sample (rs11870474, odds ratio (OR)=1.58, P=2.6 × 10−7 in discovery and OR=1.43, P=0.004 in Fundació ACE data set; combined OR=1.53, P=4.7 × 10−9). This ATP5H/KCTD2 locus has an important function in mitochondrial energy production and neuronal hyperpolarization during cellular stress conditions, such as hypoxia or glucose deprivation. PMID:23857120

  16. 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.

  17. 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.

  18. Genetic diversity of ATP8 and ATP6 genes is associated with high-altitude adaptation in yak.

    PubMed

    Wang, Jie; Shi, Yu; Elzo, Mauricio A; Dang, Shuzhang; Jia, Xianbo; Lai, Songjia

    2017-03-17

    ATP synthase 8 (ATP8) and ATPase synthase 6 (ATP6) play an important role in mitochondrial ATPase assembly. Mutations in either of these units could affect the ATP processing and the respiration chain in mitochondria. To find out if there were differences in gene diversity between Tibetan yaks and domestic cattle, we sequenced the ATP8 and ATP6 genes in 66 Tibetan yaks and 81 domestic cattle. We identified 20 SNPs in the ATP8 gene and 60 SNPs in the ATP6 gene. Ten SNPs detected in ATP8 were probably positively associated with high-altitude adaptation, of which SNPs m.8164 G > A, m.8210 G > A, m.8231 C > T and m. 8249 C > T resulted in amino acid changes. Similarly, SNPs m.8308A > G, m.8370A > C, m.8514G > A of ATP6 also appeared to be associated with high-altitude adaptability. Specifically, m.8308 A > G, located in the overlap region, might bring in a conserved region found in cytochrome b561 which play an important role in iron regulation, thus it might help the Tibetan yaks with this mutation to utilize rare oxygen efficiently. Considering all mutations, three of eight haplotypes identified in gene ATP8 were present only in Tibetan yaks, and six (H3 to H8) out of 21 haplotypes (H1 to H21) in gene ATP6 were restricted to Tibetan yaks. Haplotypes present only in Tibetan yaks could be positively associated with high-altitude adaptation.

  19. Boron-containing aptamers to ATP

    PubMed Central

    Lato, Susan M.; Ozerova, Nicole D. S.; He, Kaizhang; Sergueeva, Zinaida; Shaw, Barbara Ramsay; Burke, Donald H.

    2002-01-01

    Boron neutron capture therapy (BNCT), an experimental treatment for certain cancers, destroys only cells near the boron; however, there is a need to develop highly specific delivery agents. As nucleic acid aptamers recognize specific molecular targets, we investigated the influence of boronated nucleotide analogs on RNA function and on the systematic evolution of ligands by exponential enrichment (SELEX) process. Substitution of guanosine 5′-(α-P-borano) triphosphate (bG) for GTP or uridine 5′-(α-P-borano) triphosphate (bU) for UTP in several known aptamers diminished or eliminated target recognition by those RNAs. Specifically, ATP-binding aptamers containing the ζ-fold, which appears in several selections for adenosine aptamers, became inactive upon bG substitution but were only moderately affected by bU substitution. Selections were carried out using the bG or bU analogs with C8-linked ATP agarose as the binding target. The selections with bU and normal NTP yielded some ζ-fold aptamers, while the bG selection yielded none of this type. Non-ζ aptamers from bU and bG populations tolerated the borano substitution and many required it. The borano nucleotide requirement is specific; bU could not be used in bG-dependent aptamers nor vice versa. The borano group plays an essential role, as yet undefined, in target recognition or RNA structure. We conclude that the bG and bU nucleotides are fully compatible with SELEX, and that these analogs could be used to make boronated aptamers as therapeutics for BNCT. PMID:11884639

  20. Substantia nigra osmoregulation: taurine and ATP involvement.

    PubMed

    Morales, Ingrid; Dopico, Jose G; Sabate, Magdalena; Gonzalez-Hernandez, Tomas; Rodriguez, Manuel

    2007-05-01

    An extracellular nonsynaptic taurine pool of glial origin was recently reported in the substantia nigra (SN). There is previous evidence showing taurine as an inhibitory neurotransmitter in the SN, but the physiological role of this nonsynaptic pool of taurine has not been explored. By using microdialysis methods, we studied the action of local osmolarity on the nonsynaptic taurine pool in the SN of the rat. Hypoosmolar pulses (285-80 mosM) administered in the SN by the microdialysis probe increased extrasynaptic taurine in a dose-dependent way, a response that was counteracted by compensating osmolarity with choline. The opposite effect (taurine decrease) was observed when osmolarity was increased. Under basal conditions, the blockade of either the AMPA-kainate glutamate receptors with 6-cyano-7-nitroquinoxaline-2,3-dionine disodium or the purinergic receptors with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid modified the taurine concentration, suggesting that both receptors modulate the extrasynaptic pool of taurine. In addition, these drugs decreased the taurine response to hypoosmolar pulses, suggesting roles for glutamatergic and purinergic receptors in the taurine response to osmolarity. The participation of purinergic receptors was also supported by the fact that ATP (which, under basal conditions, increased the extrasynaptic taurine in a dose-dependent way) administered in doses saturating purinergic receptors also decreased the taurine response to hypoosmolarity. Taken together, present data suggest osmoregulation as a role of the nonsynaptic taurine pool of the SN, a function that also involves glutamate and ATP and that could influence the nigral cell vulnerability in Parkinson's disease.

  1. Functional Analysis of the Streptomyces coelicolor NrdR ATP-Cone Domain: Role in Nucleotide Binding, Oligomerization, and DNA Interactions▿ †

    PubMed Central

    Grinberg, Inna; Shteinberg, Tatyana; Hassan, A. Quamrul; Aharonowitz, Yair; Borovok, Ilya; Cohen, Gerald

    2009-01-01

    Ribonucleotide reductases (RNRs) are essential enzymes in all living cells, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides (dNTPs), the immediate precursors of DNA synthesis and repair. RNRs catalyze the controlled reduction of all four ribonucleotides to maintain a balanced pool of dNTPs during the cell cycle. Streptomyces species contain genes, nrdAB and nrdJ, coding for oxygen-dependent class I and oxygen-independent class II RNRs, either of which is sufficient for vegetative growth. Both sets of genes are transcriptionally repressed by NrdR. NrdR contains a zinc ribbon DNA-binding domain and an ATP-cone domain similar to that present in the allosteric activity site of many class I and class III RNRs. Purified NrdR contains up to 1 mol of tightly bound ATP or dATP per mol of protein and binds to tandem 16-bp sequences, termed NrdR-boxes, present in the upstream regulatory regions of bacterial RNR operons. Previously, we showed that the ATP-cone domain alone determines nucleotide binding and that an NrdR mutant defective in nucleotide binding was unable to bind to DNA probes containing NrdR-boxes. These observations led us to propose that when NrdR binds ATP/dATP it undergoes a conformational change that affects DNA binding and hence RNR gene expression. In this study, we analyzed a collection of ATP-cone mutant proteins containing changes in residues inferred to be implicated in nucleotide binding and show that they result in pleiotrophic effects on ATP/dATP binding, on protein oligomerization, and on DNA binding. A model is proposed to integrate these observations. PMID:19047342

  2. 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.

  3. 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.

  4. ATP7A trafficking and mechanisms underlying the distal motor neuropathy induced by mutations in ATP7A

    PubMed Central

    Yi, Ling; Kaler, Stephen

    2014-01-01

    Diverse mutations in the gene encoding the copper transporter ATP7A lead to X-linked recessive Menkes disease or occipital horn syndrome. Recently, two unique ATP7A mutations, T994I and P1386S, were shown to cause isolated adult-onset distal motor neuropathy. These mutations induce subtle defects in ATP7A intracellular trafficking resulting in preferential accumulation at the plasma membrane compared to wild-type ATP7A. Immunoprecipitation assays revealed abnormal interaction between ATP7AT994I and p97/VCP, a protein mutated in two autosomal dominant forms of motor neuron disease. Small-interfering RNA knockdown of p97/VCP corrected ATP7AT994I mislocalization. For ATP7AP1386S, flow cytometry documented that non-permeabilized fibroblasts bound a C-terminal ATP7A antibody, suggesting unstable insertion of the 8th transmembrane segment due to a helix-breaker effect of the amino acid substitution. This could sabotage interaction of ATP7AP1386S with adaptor protein complexes. These molecular events appear to selectively disturb normal motor neuron function and lead to neurologic illness that takes years and sometimes decades to develop. PMID:24754450

  5. ATP dependent charge movement in ATP7B Cu+-ATPase is demonstrated by pre-steady state electrical measurements.

    PubMed

    Tadini-Buoninsegni, Francesco; Bartolommei, Gianluca; Moncelli, Maria Rosa; Pilankatta, Rajendra; Lewis, David; Inesi, Giuseppe

    2010-11-19

    ATP7B is a copper dependent P-type ATPase, required for copper homeostasis. Taking advantage of high yield heterologous expression of recombinant protein, we investigated charge transfer in ATP7B. We detected charge displacement within a single catalytic cycle upon ATP addition and formation of phosphoenzyme intermediate. We attribute this charge displacement to movement of bound copper within ATP7B. Based on specific mutations, we demonstrate that enzyme activation by copper requires occupancy of a site in the N-terminus extension which is not present in other transport ATPases, as well as of a transmembrane site corresponding to the cation binding site of other ATPases.

  6. 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.

  7. 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.

  8. Bcl-2 delays cell cycle through mitochondrial ATP and ROS.

    PubMed

    Du, Xing; Fu, Xufeng; Yao, Kun; Lan, Zhenwei; Xu, Hui; Cui, Qinghua; Yang, Elizabeth

    2017-02-22

    Bcl-2 inhibits cell proliferation by delaying G0/G1 to S phase entry. We tested the hypothesis that Bcl-2 regulates S phase entry through mitochondrial pathways. Existing evidence indicates mitochondrial adenosine tri-phosphate (ATP) and reactive oxygen species (ROS) are important signals in cell survival and cell death, however, the molecular details of how these 2 processes are linked remain unknown. In this study, 2 cell lines stably expressing Bcl-2, 3T3Bcl-2 and C3HBcl-2, and vector-alone PB controls were arrested in G0/G1 phase by serum starvation and contact inhibition, and ATP and ROS were measured during re-stimulation of cell cycle entry. Both ATP and ROS levels were decreased in G0/G1 arrested cells compared with normal growing cells. In addition, ROS levels were significant lower in synchronized Bcl-2 cells than those in PB controls. After re-stimulation, ATP levels increased with time, reaching peak value 1-3 hours ahead of S phase entry for both Bcl-2 cells and PB controls. Consistent with 2 hours of S phase delay, Bcl-2 cells reached ATP peaks 2 hours later than PB control, which suggests a rise in ATP levels is required for S phase entry. To examine the role of ATP and ROS in cell cycle regulation, ATP and ROS level were changed. We observed that elevation of ATP accelerated cell cycle progression in both PB and Bcl-2 cells, and decrease of ATP and ROS to the level equivalent to Bcl-2 cells delayed S phase entry in PB cells. Our results support the hypothesis that Bcl-2 protein regulates mitochondrial metabolism to produce less ATP and ROS, which contributes to S phase entry delay in Bcl-2 cells. These findings reveal a novel mechanistic basis for understanding the link between mitochondrial metabolism and tumor-suppressive function of Bcl-2.

  9. ATP storage and uptake by isolated pancreatic zymogen granules.

    PubMed

    Haanes, Kristian A; Novak, Ivana

    2010-07-15

    ATP is released from pancreatic acini in response to cholinergic and hormonal stimulation. The same stimuli cause exocytosis of ZG (zymogen granules) and release of digestive enzymes. The aim of the present study was to determine whether ZG stored ATP and to characterize the uptake mechanism for ATP transport into the ZG. ZG were isolated and the ATP content was measured using luciferin/luciferase assays and was related to protein in the sample. The estimate of ATP concentration in freshly isolated granules was 40-120 microM. The ATP uptake had an apparent Km value of 4.9+/-2.1 mM when granules were incubated without Mg2+ and a Km value of 0.47+/-0.05 mM in the presence of Mg2+, both in pH 6.0 buffers. The uptake of ATP was significantly higher at pH 7.2 compared with pH 6.0 solutions. The anion transport blockers DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate) and Evans Blue inhibited ATP transport. Western blot analysis on the ZG showed the presence of VNUT (vesicular nucleotide transporter). Together, these findings indicate that VNUT may be responsible for the ATP uptake into ZG. Furthermore, the present study shows the presence of ATP together with digestive enzymes in ZG. This indicates that co-released ATP would regulate P2 receptors in pancreatic ducts and, thus, ductal secretion, and this would aid delivery of enzymes to the duodenum.

  10. Mechanically driven ATP synthesis by F1-ATPase

    NASA Astrophysics Data System (ADS)

    Itoh, Hiroyasu; Takahashi, Akira; Adachi, Kengo; Noji, Hiroyuki; Yasuda, Ryohei; Yoshida, Masasuke; Kinosita, Kazuhiko

    2004-01-01

    ATP, the main biological energy currency, is synthesized from ADP and inorganic phosphate by ATP synthase in an energy-requiring reaction. The F1 portion of ATP synthase, also known as F1-ATPase, functions as a rotary molecular motor: in vitro its γ-subunit rotates against the surrounding α3β3 subunits, hydrolysing ATP in three separate catalytic sites on the β-subunits. It is widely believed that reverse rotation of the γ-subunit, driven by proton flow through the associated Fo portion of ATP synthase, leads to ATP synthesis in biological systems. Here we present direct evidence for the chemical synthesis of ATP driven by mechanical energy. We attached a magnetic bead to the γ-subunit of isolated F1 on a glass surface, and rotated the bead using electrical magnets. Rotation in the appropriate direction resulted in the appearance of ATP in the medium as detected by the luciferase-luciferin reaction. This shows that a vectorial force (torque) working at one particular point on a protein machine can influence a chemical reaction occurring in physically remote catalytic sites, driving the reaction far from equilibrium.

  11. Effect of chemical sanitizing agents on ATP bioluminescence measurements.

    PubMed

    Green, T A; Russell, S M; Fletcher, D L

    1998-08-01

    Experiments were conducted to determine the effects of sodium hypochlorite (SH), quaternary ammonium (QA), trisodium phosphate (TSP), lactic acid (LA), hydrogen peroxide (HP), and trichlosan (TR) on ATP bioluminescence measurements. Each sanitizer was tested at three different levels and compared to a control group containing no sanitizer. ATP from three sources was analyzed, Escherichia coli, chicken blood, and a pure ATP standard. The effect of each sanitizer was reported as a percent decrease in log10 relative light units (LRLU) for the treatment groups when compared to LRLU from the control groups. SH concentrations of 30, 50, and 70 ppm and quaternary ammonium at concentrations of 10, 20, and 30 ppm had no effect on LRLU measurements, regardless of the ATP source. LA concentrations of 0.5% and higher reduced LRLU by 75%. LRLU measurements were significantly (P < or = 0.05) reduced by approximately 60% when levels of TSP exceeded 1%. HP had no effect on LRLU measurements from any of the ATP sources at 0.1%; however, at 1% HP significantly (P < or = 0.05) decreased LRLU measurements by approximately 60% for all ATP sources. TR at 0.25% had no significant effect on LRLU measurements from any of the ATP sources. TR at 0.5 and 1% reduced LRLU measurements by 30 and 50%, respectively. These results indicate that commercial sanitizers containing LA, TSP, HP, or TR may negatively affect LRLU measurements if the sanitizer is allowed to come into direct contact with the ATP bioluminescence reagents.

  12. The relationship of granulocyte ATP to chemotactic response during storage.

    PubMed

    McCullough, J J; Weiblen, B J

    1979-01-01

    During the storage of granulocytes, bactericidal activity declines more slowly than does chemotactic response (CTR). Bacterial killing involves increased activity of the hexose monophosphate shunt, oxygen utilization and the generation of toxic products of oxygen. Chemotaxis is probably a contractile process involving myosin and actin filaments and possibly ATP as a source of energy. Thus, maintainance of ATP may be important in granulocyte preservation. During storage at 1 to 6 C of granulocytes collected by continuous and intermittent flow centrifuge leukapheresis, both CTR and ATP decreased approximately 33 percent. Decreases in CTR and ATP were 12 and 10 percent respectively when cells were stored at 20 to 24 C. Further decreases in CTR and ATP occurred between 24 and 48 hours of storage, although levels of both were higher in cells stored at 20 to 24 C compared with those stored at 1 to 6 C. When the results from all storage conditions were combined, the overall coefficient of correlation between CTR and ATP was 0.71 (p less than .05). Although ATP is probably not the only important variable in granulocyte preservation, granulocytes may resemble red blood cells in that a minimal level of ATP may be necessary for adequate function.

  13. The dark side of extracellular ATP in kidney diseases.

    PubMed

    Solini, Anna; Usuelli, Vera; Fiorina, Paolo

    2015-05-01

    Intracellular ATP is the most vital source of cellular energy for biologic systems, whereas extracellular ATP is a multifaceted mediator of several cell functions via its interaction, in an autocrine or paracrine manner, with P2 purinergic receptors expressed on the cell surface. These ionotropic and metabotropic P2 purinergic receptors modulate a variety of physiologic events upon the maintenance of a highly sensitive "set point," the derangement of which may lead to the development of key pathogenic mechanisms during acute and chronic diseases. Growing evidence suggests that extracellular ATP signaling via P2 purinergic receptors may be involved in different renal pathologic conditions. For these reasons, investigators and pharmaceutical companies are actively exploring novel strategies to antagonize or block these receptors with the goal of reducing extracellular ATP production or accelerating extracellular ATP clearance. Targeting extracellular ATP signaling, particularly through the P2X7 receptor, has considerable translational potential, given that novel P2X7-receptor inhibitors are already available for clinical use (e.g., CE224,535, AZD9056, and GSK1482160). This review summarizes the current evidence regarding the involvement of extracellular ATP and its P2 purinergic receptor-mediated signaling in physiologic and pathologic processes in the kidney; potential therapeutic options targeting extracellular ATP purinergic receptors are analyzed as well.

  14. Investigation of the association between ATP2B4 and ATP5B genes with colorectal cancer.

    PubMed

    Geyik, Esra; Igci, Yusuf Ziya; Pala, Elif; Suner, Ali; Borazan, Ersin; Bozgeyik, Ibrahim; Bayraktar, Emine; Bayraktar, Recep; Ergun, Sercan; Cakmak, Ecir Ali; Gokalp, Avni; Arslan, Ahmet

    2014-05-01

    Colorectal cancer (CRC) develops as a multi-step process which results from gradual accumulation of mutations in proto-oncogenes, tumor suppressor, and DNA repair genes. Mortality rate of CRC is very high. Therefore, development of alternative diagnostic methods which can be used in the early diagnosis is crucial. ATP2B4 gene encodes one of the four isoforms of p-type ATPase PMCA enzyme and bears critical importance in maintaining the balance of intracellular calcium homeostasis by providing the export of calcium ions out of the cell. ATP5B encodes a subunit of the mitochondrial ATP synthase which is an f-type ATPase. In this study, the relationship between ATP2B4 and ATP5B genes and CRC regarding gene expression was investigated. Study groups were constructed from a number of 50 patients (25 males, 25 females) with the mean age of 55.68 ± 9.4 and the gene expression levels in the healthy and cancerous tissues of the patients were compared by using semi-quantitative PCR and Real-Time PCR methods. As a result, in patients with rectum tumors, there was a significant relationship between ATP2B4 gene expression and the tumor location and in patients younger than 45 years, ATP5B gene expressions were detected significantly higher in tumor tissues by using RT-PCR. However, no significant relationship was detected in terms of expression differences of ATP2B4 and ATP5B genes between cancerous and healthy tissues of the CRC patients. ATP2B4 and ATP5B genes might have indirect associations in CRC pathogenesis and the investigation of their interactions with DNA repair and other related genes may help in understanding of CRC formation. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Radioprotective effects of ATP in human blood ex vivo

    SciTech Connect

    Swennen, Els L.R. Dagnelie, Pieter C.; Van den Beucken, Twan; Bast, Aalt

    2008-03-07

    Damage to healthy tissue is a major limitation of radiotherapy treatment of cancer patients, leading to several side effects and complications. Radiation-induced release of pro-inflammatory cytokines is thought to be partially responsible for the radiation-associated complications. The aim of the present study was to investigate the protective effects of extracellular ATP on markers of oxidative stress, radiation-induced inflammation and DNA damage in irradiated blood ex vivo. ATP inhibited radiation-induced TNF-{alpha} release and increased IL-10 release. The inhibitory effect of ATP on TNF- {alpha} release was completely reversed by adenosine 5'-O-thiomonophosphate, indicating a P2Y{sub 11} mediated effect. Furthermore, ATP attenuated radiation-induced DNA damage immediate, 3 and 6 h after irradiation. Our study indicates that ATP administration alleviates radiation-toxicity to blood cells, mainly by inhibiting radiation-induced inflammation and DNA damage.

  16. Intracellular ATP can regulate afferent arteriolar tone via ATP-sensitive K+ channels in the rabbit.

    PubMed Central

    Lorenz, J N; Schnermann, J; Brosius, F C; Briggs, J P; Furspan, P B

    1992-01-01

    Studies were performed to assess whether ATP-sensitive K+ (KATP) channels on rabbit preglomerular vessels can influence afferent arteriolar (AA) tone. K+ channels with a slope conductance of 258 +/- 13 (n = 7) pS and pronounced voltage dependence were demonstrated in excised patches from vascular smooth muscle cells of microdissected preglomerular segments. Channel activity was markedly reduced by 1 mM ATP and in a dose-dependent fashion by glibenclamide (10(-9) M to 10(-6) M), a specific antagonist of KATP channels. 10(-5) M diazoxide, a K+ channel opener, activated these channels in the presence of ATP, and this effect was also blocked by glibenclamide. To determine the role of these KATP channels in the control of vascular tone, diazoxide was tested on isolated perfused AA. After preconstriction from a control diameter of 13.1 +/- 1.1 to 3.5 +/- 2.1 microns with phenylephrine (PE), addition of 10(-5) M diazoxide dilated vessels to 11.2 +/- 0.7 microns, which was not different from control. Further addition of 10(-5) M glibenclamide reconstricted the vessels to 5.8 +/- 1.5 microns (n = 5; P less than 0.03). In support of its specificity for KATP channels, glibenclamide did not reverse verapamil induced dilation in a separate series of experiments. To determine whether intracellular ATP levels can effect AA tone, studies were conducted to test the effect of the glycolytic inhibitor 2-deoxy-D-glucose. After preconstriction from 13.4 +/- 3.2 to 7.7 +/- 1.3 microns with PE, bath glucose was replaced with 6 mM 2-deoxy-D-glucose. Within 10 min, the arteriole dilated to a mean value of 11.8 +/- 1.4 microns (n = 6; NS compared to control). Subsequent addition of 10(-5) M glibenclamide significantly reconstricted the vessels to a diameter of 8.6 +/- 0.5 micron (P less than 0.04). These data demonstrate that KATP channels are present on the preglomerular vasculature and that changes in intracellular ATP can directly influence afferent arteriolar tone via these channels

  17. Neuroretinitis -- definition

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/007624.htm Neuroretinitis - definition To use the sharing features on this page, ... this important distinction for online health information and services. Learn more about A.D.A.M.'s editorial ...

  18. Electrophysiological effects of extracellular ATP on Necturus gallbladder epithelium

    PubMed Central

    1991-01-01

    The effects of addition of ATP to the mucosal bathing solution on transepithelial, apical, and basolateral membrane voltages and resistances in Necturus gallbladder epithelium were determined. Mucosal ATP (100 microM) caused a rapid hyperpolarization of both apical (Vmc) and basolateral (Vcs) cell membrane voltages (delta Vm = 18 +/- 1 mV), a fall in transepithelial resistance (Rt) from 142 +/- 8 to 122 +/- 7 omega.cm2, and a decrease in fractional apical membrane resistance (fRa) from 0.93 +/- 0.02 to 0.83 +/- 0.03. The rapid initial hyperpolarization of Vmc and Vcs was followed by a slower depolarization of cell membrane voltages and a lumen-negative change in transepithelial voltage (Vms). This phase also included an additional decrease in fRa. Removal of the ATP caused a further depolarization of membrane voltages followed by a hyperpolarization and then a return to control values. fRa fell to a minimum after removal of ATP and then returned to control values as the cell membrane voltages repolarized. Similar responses could be elicited by ADP but not by adenosine. The results of two-point cable experiments revealed that ATP induced an initial increase in cell membrane conductance followed by a decrease. Transient elevations of mucosal solution [K+] induced a larger depolarization of Vmc and Vcs during exposure to ATP than under control conditions. Reduction of mucosal solution [Cl-] induced a slow hyperpolarization of Vmc and Vcs before exposure to ATP and a rapid depolarization during exposure to ATP. We conclude that ATP4- is the active agent and that it causes a concentration-dependent increase in apical and basolateral membrane K+ permeability. In addition, an apical membrane electrodiffusive Cl- permeability is activated by ATP4-. PMID:1713948

  19. 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

  20. Nanomolar ambient ATP decelerates P2X3 receptor kinetics.

    PubMed

    Grote, Alexander; Hans, Michael; Boldogkoi, Zsolt; Zimmer, Andreas; Steinhäuser, Christian; Jabs, Ronald

    2008-12-01

    Homomeric P2X receptors differ in their electrophysiological and pharmacological profiles. The rapidly activating and desensitizing P2X3 receptors are known for their involvement in pain signalling pathways. Modulatory effects on P2X3 receptors have been reported for low concentrations of ATP ([ATP]). This includes both, enhancement and reduction of receptor currents. The first has been reported to be mediated by activation of ectoprotein kinases and high affinity desensitization (HAD), respectively. Both processes influence receptor current amplitudes. Here we describe a new phenomenon, the modulatory influence of ambient low [ATP] on P2X3 receptor kinetics. First, we studied in HEK cells whether persistent ATP affects current decay. To this end, P2X3 receptor mediated currents, elicited by pressure application of saturating [ATP], were analyzed after pre-application of low [ATP]. Second, UV-flash photolysis of ATP was employed to investigate whether submicromolar [ATP] affects receptor activation. Finally we confirmed the action of nanomolar [ATP] on native P2X3 receptors of neurons freshly isolated from rat dorsal root ganglia. We found that persistent low [ATP] caused pronounced deceleration of receptor current activation and decay. This priming effect indicates a mechanism different from HAD. It could be explained by a pre-opening receptor isomerization, induced by the occupation of a high affinity binding site already at the resting state. The observed modulation of the receptor kinetics could be considered as a physiological fine tuning mechanism of the nociceptive system, driven by the actual ambient agonist concentration.

  1. Regulation of ClC-2 gating by intracellular ATP.

    PubMed

    Stölting, Gabriel; Teodorescu, Georgeta; Begemann, Birgit; Schubert, Julian; Nabbout, Rima; Toliat, Mohammad Reza; Sander, Thomas; Nürnberg, Peter; Lerche, Holger; Fahlke, Christoph

    2013-10-01

    ClC-2 is a voltage-dependent chloride channel that activates slowly at voltages negative to the chloride reversal potential. Adenosine triphosphate (ATP) and other nucleotides have been shown to bind to carboxy-terminal cystathionine-ß-synthase (CBS) domains of ClC-2, but the functional consequences of binding are not sufficiently understood. We here studied the effect of nucleotides on channel gating using single-channel and whole-cell patch clamp recordings on transfected mammalian cells. ATP slowed down macroscopic activation and deactivation time courses in a dose-dependent manner. Removal of the complete carboxy-terminus abolishes the effect of ATP, suggesting that CBS domains are necessary for ATP regulation of ClC-2 gating. Single-channel recordings identified long-lasting closed states of ATP-bound channels as basis of this gating deceleration. ClC-2 channel dimers exhibit two largely independent protopores that are opened and closed individually as well as by a common gating process. A seven-state model of common gating with altered voltage dependencies of opening and closing transitions for ATP-bound states correctly describes the effects of ATP on macroscopic and microscopic ClC-2 currents. To test for a potential pathophysiological impact of ClC-2 regulation by ATP, we studied ClC-2 channels carrying naturally occurring sequence variants found in patients with idiopathic generalized epilepsy, G715E, R577Q, and R653T. All naturally occurring sequence variants accelerate common gating in the presence but not in the absence of ATP. We propose that ClC-2 uses ATP as a co-factor to slow down common gating for sufficient electrical stability of neurons under physiological conditions.

  2. Evolution of killer cell Ig-like receptor (KIR) genes: definition of an orangutan KIR haplotype reveals expansion of lineage III KIR associated with the emergence of MHC-C.

    PubMed

    Guethlein, Lisbeth A; Older Aguilar, Anastazia M; Abi-Rached, Laurent; Parham, Peter

    2007-07-01

    Orangutan (Pongo pygmaeus) MHC-C appears less evolved than human HLA-C: Popy-C is not fixed and its alleles encode only one (C1) of the two motifs for killer cell Ig-like receptor (KIR) ligands. To assess the structure and complexity of the orangutan KIR locus, the complete nucleotide sequence of an orangutan KIR haplotype was determined. The PopyKIR locus is flanked by LILR and FCAR and consists of seven genes and pseudogenes, two novel and five corresponding to known cDNA. Distinguishing all KIRs in this rapidly evolving KIR locus from the KIR3DX1 gene is an LTR33A/MLT1D element in intron 3. These two forms of KIR represent lineages that originated by duplication of a common ancestor. The conserved, framework regions of primate KIR loci comprise the 5' part of a lineage V KIR, the 3' part of a pseudogene, the complete 2DL4 gene, and the 3' part of a lineage II KIR. Although previously defined PopyKIR2DL4 alleles contain premature termination codons, the sequenced haplotype's PopyKIR2DL4 allele encodes a full-length protein. A model for KIR evolution is proposed. Distinguishing the orangutan KIR haplotype from the proposed common ancestor of primate KIR haplotypes is an increased number to give three lineage III KIR genes in the centromeric part of the locus, the site for most human lineage III genes encoding HLA-C specific KIR. Thus, expansion of lineage III KIR is associated with emergence of MHC-C.

  3. Possible Involvement of F1F0-ATP synthase and Intracellular ATP in Keratinocyte Differentiation in normal skin and skin lesions

    PubMed Central

    Xiaoyun, Xie; Chaofei, Han; Weiqi, Zeng; Chen, Chen; Lixia, Lu; Queping, Liu; Cong, Peng; Shuang, Zhao; Juan, Su; Xiang, Chen

    2017-01-01

    The F1F0-ATP synthase, an enzyme complex, is mainly located on the mitochondrial inner membrane or sometimes cytomembrane to generate or hydrolyze ATP, play a role in cell proliferation. This study focused on the role of F1F0-ATP synthase in keratinocyte differentiation, and its relationship with intracellular and extracellular ATP (InATP and ExATP). The F1F0-ATP synthase β subunit (ATP5B) expression in various skin tissues and confluence-dependent HaCaT differentiation models was detected. ATP5B expression increased with keratinocyte and HaCaT cell differentiation in normal skin, some epidermis hyper-proliferative diseases, squamous cell carcinoma, and the HaCaT cell differentiation model. The impact of InATP and ExATP content on HaCaT differentiation was reflected by the expression of the differentiation marker involucrin. Inhibition of F1F0-ATP synthase blocked HaCaT cell differentiation, which was associated with a decrease of InATP content, but not with changes of ExATP. Our results revealed that F1F0-ATP synthase expression is associated with the process of keratinocyte differentiation which may possibly be related to InATP synthesis. PMID:28209970

  4. ATP7A Gene Addition to the Choroid Plexus Results in Long-term Rescue of the Lethal Copper Transport Defect in a Menkes Disease Mouse Model

    PubMed Central

    Donsante, Anthony; Yi, Ling; Zerfas, Patricia M; Brinster, Lauren R; Sullivan, Patricia; Goldstein, David S; Prohaska, Joseph; Centeno, Jose A; Rushing, Elisabeth; Kaler, Stephen G

    2011-01-01

    Menkes disease is a lethal infantile neurodegenerative disorder of copper metabolism caused by mutations in a P-type ATPase, ATP7A. Currently available treatment (daily subcutaneous copper injections) is not entirely effective in the majority of affected individuals. The mottled-brindled (mo-br) mouse recapitulates the Menkes phenotype, including abnormal copper transport to the brain owing to mutation in the murine homolog, Atp7a, and dies by 14 days of age. We documented that mo-br mice on C57BL/6 background were not rescued by peripheral copper administration, and used this model to evaluate brain-directed therapies. Neonatal mo-br mice received lateral ventricle injections of either adeno-associated virus serotype 5 (AAV5) harboring a reduced-size human ATP7A (rsATP7A) complementary DNA (cDNA), copper chloride, or both. AAV5-rsATP7A showed selective transduction of choroid plexus epithelia and AAV5-rsATP7A plus copper combination treatment rescued mo-br mice; 86% survived to weaning (21 days), median survival increased to 43 days, 37% lived beyond 100 days, and 22% survived to the study end point (300 days). This synergistic treatment effect correlated with increased brain copper levels, enhanced activity of dopamine-β-hydroxylase, a copper-dependent enzyme, and correction of brain pathology. Our findings provide the first definitive evidence that gene therapy may have clinical utility in the treatment of Menkes disease. PMID:21878905

  5. Distance of myofilament sliding per ATP molecule in skeletal muscle fibers studied using laser flash photolysis of caged ATP.

    PubMed

    Yamada, T; Abe, O; Kobayashi, T; Sugi, H

    1993-01-01

    We studied the distance of myofilament sliding per hydrolysis of one ATP molecule by recording shortening of single glycerinated muscle fibers induced by laser flash photolysis of caged ATP, diffusion of photochemically released ATP out of the fiber being prevented by surrounding the fiber with silicone oil. With 75 microM ATP released (one half of the total myosin head concentration within the fiber), the fiber showed the minimum shortening (10 +/- 2 nm/half sarcomere, n = 10) taking place uniformly in each sarcomere in the fiber. Comparison of the initial flash-induced shortening velocity with the force-velocity relation of maximally Ca(2+)-activated fibers indicated that the above minimum fiber shortening took place under an internal load nearly equal to Po. These results may be taken to indicate that, under a nearly isometric condition, the distance of myofilament sliding per hydrolysis of one ATP molecule is of the order of 10 nm.

  6. NIF Title III engineering plan

    SciTech Connect

    Deis, G

    1998-06-01

    The purpose of this document is to define the work that must be accomplished by the NIF Project during Title III Engineering. This definition is intended to be sufficiently detailed to provide a framework for yearly planning, to clearly identify the specific deliverables so that the Project teams can focus on them, and to provide a common set of objectives and processes across the Project. This plan has been preceded by similar documents for Title I and Title II design and complements the Site Management Plan, the Project Control Manual, the Quality Assurance Program Plan, the RM Parsons NIF Title III Configuration Control Plan, the Integrated Project Schedule, the Preliminary Safety Analysis Report, the Configuration Management Plan, and the Transition Plan.

  7. A1Ao-ATP Synthase of Methanobrevibacter ruminantium Couples Sodium Ions for ATP Synthesis under Physiological Conditions*

    PubMed Central

    McMillan, Duncan G. G.; Ferguson, Scott A.; Dey, Debjit; Schröder, Katja; Aung, Htin Lin; Carbone, Vincenzo; Attwood, Graeme T.; Ronimus, Ron S.; Meier, Thomas; Janssen, Peter H.; Cook, Gregory M.

    2011-01-01

    An unresolved question in the bioenergetics of methanogenic archaea is how the generation of proton-motive and sodium-motive forces during methane production is used to synthesize ATP by the membrane-bound A1Ao-ATP synthase, with both proton- and sodium-coupled enzymes being reported in methanogens. To address this question, we investigated the biochemical characteristics of the A1Ao-ATP synthase (MbbrA1Ao) of Methanobrevibacter ruminantium M1, a predominant methanogen in the rumen. Growth of M. ruminantium M1 was inhibited by protonophores and sodium ionophores, demonstrating that both ion gradients were essential for growth. To study the role of these ions in ATP synthesis, the ahaHIKECFABD operon encoding the MbbrA1Ao was expressed in Escherichia coli strain DK8 (Δatp) and purified yielding a 9-subunit protein with an SDS-stable c oligomer. Analysis of the c subunit amino acid sequence revealed that it consisted of four transmembrane helices, and each hairpin displayed a complete Na+-binding signature made up of identical amino acid residues. The purified MbbrA1Ao was stimulated by sodium ions, and Na+ provided pH-dependent protection against inhibition by dicyclohexylcarbodiimide but not tributyltin chloride. ATP synthesis in inverted membrane vesicles lacking sodium ions was driven by a membrane potential that was sensitive to cyanide m-chlorophenylhydrazone but not to monensin. ATP synthesis could not be driven by a chemical gradient of sodium ions unless a membrane potential was imposed. ATP synthesis under these conditions was sensitive to monensin but not cyanide m-chlorophenylhydrazone. These data suggest that the M. ruminantium M1 A1Ao-ATP synthase exhibits all the properties of a sodium-coupled enzyme, but it is also able to use protons to drive ATP synthesis under conditions that favor proton coupling, such as low pH and low levels of sodium ions. PMID:21953465

  8. Coupling DNA-binding and ATP hydrolysis in Escherichia coli RecQ: role of a highly conserved aromatic-rich sequence.

    PubMed

    Zittel, Morgan C; Keck, James L

    2005-01-01

    RecQ enzymes are broadly conserved Superfamily-2 (SF-2) DNA helicases that play critical roles in DNA metabolism. RecQ proteins use the energy of ATP hydrolysis to drive DNA unwinding; however, the mechanisms by which RecQ links ATPase activity to DNA-binding/unwinding are unknown. In many Superfamily-1 (SF-1) DNA helicases, helicase sequence motif III links these activities by binding both single-stranded (ss) DNA and ATP. However, the ssDNA-binding aromatic-rich element in motif III present in these enzymes is missing from SF-2 helicases, raising the question of how these enzymes link ATP hydrolysis to DNA-binding/unwinding. We show that Escherichia coli RecQ contains a conserved aromatic-rich loop in its helicase domain between motifs II and III. Although placement of the RecQ aromatic-rich loop is topologically distinct relative to the SF-1 enzymes, both loops map to similar tertiary structural positions. We examined the functions of the E.coli RecQ aromatic-rich loop using RecQ variants with single amino acid substitutions within the segment. Our results indicate that the aromatic-rich loop in RecQ is critical for coupling ATPase and DNA-binding/unwinding activities. Our studies also suggest that RecQ's aromatic-rich loop might couple ATP hydrolysis to DNA-binding in a mechanistically distinct manner from SF-1 helicases.

  9. Variations in carbachol- and ATP-induced contractions of the rat detrusor: effects of gender, mucosa and contractile direction.

    PubMed

    Liang, Willmann; Leung, Ping Chung

    2012-12-01

    Contractile characteristics of the bladder may depend on variables such as gender, mucosa (MU) and direction of the contractions. However, definitive information is not yet available despite earlier studies on the effects of one variable or another. Here, we explored the differences in the rat detrusor attributable to gender, mucosa and contractile direction. K+, carbachol (CCh) and ATP were used as contractile stimuli on rat detrusor strips with and without MU. Contractility was monitored using a myograph system. Both tonic and phasic contractile activities were analyzed. MU-independent contractions induced by CCh were more potent in females, an effect specific to the longitudinal direction only. The maximal CCh response was larger also in females when MU was removed, suggesting a stronger MU-independent component in the contraction. The larger area under curves of the females under ATP stimulation showed dependence on MU and contractile direction as well. ATP-induced contractions in the males were affected more by MU in the transverse direction than in the females. Direction- and MU-dependent variability of ATP responses was also observed in the males but not in females. Findings here added new information to the understanding of bladder contractile physiology, providing insights into the quest for better drugs in managing bladder disorders.

  10. Intrarenal localization of the plasma membrane ATP channel pannexin1.

    PubMed

    Hanner, Fiona; Lam, Lisa; Nguyen, Mien T X; Yu, Alan; Peti-Peterdi, János

    2012-11-15

    In the renal tubules, ATP released from epithelial cells stimulates purinergic receptors, regulating salt and water reabsorption. However, the mechanisms by which ATP is released into the tubular lumen are multifaceted. Pannexin1 (Panx1) is a newly identified. ubiquitously expressed protein that forms connexin-like channels in the plasma membrane, which have been demonstrated to function as a mechanosensitive ATP conduit. Here, we report on the localization of Panx1 in the mouse kidney. Using immunofluorescence, strong Panx1 expression was observed in renal tubules, including proximal tubules, thin descending limbs, and collecting ducts, along their apical cell membranes. In the renal vasculature, Panx1 expression was localized to vascular smooth muscle cells in renal arteries, including the afferent and efferent arterioles. Additionally, we tested whether Panx1 channels expressed in renal epithelial cells facilitate luminal ATP release by measuring the ATP content of urine samples freshly collected from wild-type and Panx1(-/-) mice. Urinary ATP levels were reduced by 30% in Panx1(-/-) compared with wild-type mice. These results suggest that Panx1 channels in the kidney may regulate ATP release and via purinergic signaling may participate in the control of renal epithelial fluid and electrolyte transport and vascular functions.

  11. Intrarenal localization of the plasma membrane ATP channel pannexin1

    PubMed Central

    Hanner, Fiona; Lam, Lisa; Nguyen, Mien T. X.; Yu, Alan

    2012-01-01

    In the renal tubules, ATP released from epithelial cells stimulates purinergic receptors, regulating salt and water reabsorption. However, the mechanisms by which ATP is released into the tubular lumen are multifaceted. Pannexin1 (Panx1) is a newly identified. ubiquitously expressed protein that forms connexin-like channels in the plasma membrane, which have been demonstrated to function as a mechanosensitive ATP conduit. Here, we report on the localization of Panx1 in the mouse kidney. Using immunofluorescence, strong Panx1 expression was observed in renal tubules, including proximal tubules, thin descending limbs, and collecting ducts, along their apical cell membranes. In the renal vasculature, Panx1 expression was localized to vascular smooth muscle cells in renal arteries, including the afferent and efferent arterioles. Additionally, we tested whether Panx1 channels expressed in renal epithelial cells facilitate luminal ATP release by measuring the ATP content of urine samples freshly collected from wild-type and Panx1−/− mice. Urinary ATP levels were reduced by 30% in Panx1−/− compared with wild-type mice. These results suggest that Panx1 channels in the kidney may regulate ATP release and via purinergic signaling may participate in the control of renal epithelial fluid and electrolyte transport and vascular functions. PMID:22952282

  12. Authentic role of ATP signaling in micturition reflex

    PubMed Central

    Takezawa, Kentaro; Kondo, Makoto; Kiuchi, Hiroshi; Ueda, Norichika; Soda, Tetsuji; Fukuhara, Shinichiro; Takao, Tetsuya; Miyagawa, Yasushi; Tsujimura, Akira; Matsumoto-Miyai, Kazumasa; Ishida, Yusuke; Negoro, Hiromitsu; Ogawa, Osamu; Nonomura, Norio; Shimada, Shoichi

    2016-01-01

    Adenosine triphosphate (ATP) is a signaling molecule that regulates cellular processes. Based on previous studies of bladder function over the past decade, bladder ATP signaling was thought to have an essential role in the normal micturition reflex. In this study, we performed detailed analyses of bladder function in purinergic receptor-deficient mice using the automated voided stain on paper method and video-urodynamics. Unexpectedly, a lack of P2X2 or P2X3 receptors did not affect bladder function under normal physiological conditions, indicating that bladder ATP signaling is not essential for normal micturition reflex. In contrast, we found that lipopolysaccharide (LPS) induced markedly high levels of ATP release from the urothelium. In addition, LPS-induced rapid bladder hyperactivity was attenuated in P2X2−/− and P2X3−/− mice. Contrary to the previous interpretation, our present findings indicate that bladder ATP signaling has a fundamental role in the micturition reflex, especially in bladder dysfunction, under pathological conditions. Therefore, the bladder ATP signaling pathway might be a highly promising therapeutic target for functional bladder disorders. This study newly defines an authentic role for bladder ATP signaling in the micturition reflex. PMID:26795755

  13. Authentic role of ATP signaling in micturition reflex.

    PubMed

    Takezawa, Kentaro; Kondo, Makoto; Kiuchi, Hiroshi; Ueda, Norichika; Soda, Tetsuji; Fukuhara, Shinichiro; Takao, Tetsuya; Miyagawa, Yasushi; Tsujimura, Akira; Matsumoto-Miyai, Kazumasa; Ishida, Yusuke; Negoro, Hiromitsu; Ogawa, Osamu; Nonomura, Norio; Shimada, Shoichi

    2016-01-22

    Adenosine triphosphate (ATP) is a signaling molecule that regulates cellular processes. Based on previous studies of bladder function over the past decade, bladder ATP signaling was thought to have an essential role in the normal micturition reflex. In this study, we performed detailed analyses of bladder function in purinergic receptor-deficient mice using the automated voided stain on paper method and video-urodynamics. Unexpectedly, a lack of P2X2 or P2X3 receptors did not affect bladder function under normal physiological conditions, indicating that bladder ATP signaling is not essential for normal micturition reflex. In contrast, we found that lipopolysaccharide (LPS) induced markedly high levels of ATP release from the urothelium. In addition, LPS-induced rapid bladder hyperactivity was attenuated in P2X2(-/-) and P2X3(-/-) mice. Contrary to the previous interpretation, our present findings indicate that bladder ATP signaling has a fundamental role in the micturition reflex, especially in bladder dysfunction, under pathological conditions. Therefore, the bladder ATP signaling pathway might be a highly promising therapeutic target for functional bladder disorders. This study newly defines an authentic role for bladder ATP signaling in the micturition reflex.

  14. ATP and potassium ions: a deadly combination for astrocytes

    NASA Astrophysics Data System (ADS)

    Jackson, David G.; Wang, Junjie; Keane, Robert W.; Scemes, Eliana; Dahl, Gerhard

    2014-04-01

    The ATP release channel Pannexin1 (Panx1) is self-regulated, i.e. the permeant ATP inhibits the channel from the extracellular space. The affinity of the ATP binding site is lower than that of the purinergic P2X7 receptor allowing a transient activation of Panx1 by ATP through P2X7R. Here we show that the inhibition of Panx1 by ATP is abrogated by increased extracellular potassium ion concentration ([K+]o) in a dose-dependent manner. Since increased [K+]o is also a stimulus for Panx1 channels, it can be expected that a combination of ATP and increased [K+]o would be deadly for cells. Indeed, astrocytes did not survive exposure to these combined stimuli. The death mechanism, although involving P2X7R, does not appear to strictly follow a pyroptotic pathway. Instead, caspase-3 was activated, a process inhibited by Panx1 inhibitors. These data suggest that Panx1 plays an early role in the cell death signaling pathway involving ATP and K+ ions. Additionally, Panx1 may play a second role once cells are committed to apoptosis, since Panx1 is also a substrate of caspase-3.

  15. Unidirectional Transport Mechanism in an ATP Dependent Exporter

    PubMed Central

    2017-01-01

    ATP-binding cassette (ABC) transporters use the energy of ATP binding and hydrolysis to move a large variety of compounds across biological membranes. P-glycoprotein, involved in multidrug resistance, is the most investigated eukaryotic family member. Although a large number of biochemical and structural approaches have provided important information, the conformational dynamics underlying the coupling between ATP binding/hydrolysis and allocrite transport remains elusive. To tackle this issue, we performed molecular dynamic simulations for different nucleotide occupancy states of Sav1866, a prokaryotic P-glycoprotein homologue. The simulations reveal an outward-closed conformation of the transmembrane domain that is stabilized by the binding of two ATP molecules. The hydrolysis of a single ATP leads the X-loop, a key motif of the ATP binding cassette, to interfere with the transmembrane domain and favor its outward-open conformation. Our findings provide a structural basis for the unidirectionality of transport in ABC exporters and suggest a ratio of one ATP hydrolyzed per transport cycle. PMID:28386603

  16. 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.

  17. Modeling K,ATP-Dependent Excitability in Pancreatic Islets

    PubMed Central

    Silva, Jonathan R.; Cooper, Paige; Nichols, Colin G.

    2014-01-01

    In pancreatic β-cells, K,ATP channels respond to changes in glucose to regulate cell excitability and insulin release. Confirming a high sensitivity of electrical activity to K,ATP activity, mutations that cause gain of K,ATP function cause neonatal diabetes. Our aim was to quantitatively assess the contribution of K,ATP current to the regulation of glucose-dependent bursting by reproducing experimentally observed changes in excitability when K,ATP conductance is altered by genetic manipulation. A recent detailed computational model of single cell pancreatic β-cell excitability reproduces the β-cell response to varying glucose concentrations. However, initial simulations showed that the model underrepresents the significance of K,ATP activity and was unable to reproduce K,ATP conductance-dependent changes in excitability. By altering the ATP and glucose dependence of the L-type Ca2+ channel and the Na-K ATPase to better fit experiment, appropriate dependence of excitability on K,ATP conductance was reproduced. Because experiments were conducted in islets, which contain cell-to-cell variability, we extended the model from a single cell to a three-dimensional model (10×10×10 cell) islet with 1000 cells. For each cell, the conductance of the major currents was allowed to vary as was the gap junction conductance between cells. This showed that single cell glucose-dependent behavior was then highly variable, but was uniform in coupled islets. The study highlights the importance of parameterization of detailed models of β-cell excitability and suggests future experiments that will lead to improved characterization of β-cell excitability and the control of insulin secretion. PMID:25418087

  18. Performance and Specificity of the Covalently Linked Immunomagnetic Separation-ATP Method for Rapid Detection and Enumeration of Enterococci in Coastal Environments

    PubMed Central

    Zimmer-Faust, Amity G.; Thulsiraj, Vanessa; Ferguson, Donna

    2014-01-01

    The performance and specificity of the covalently linked immunomagnetic separation-ATP (Cov-IMS/ATP) method for the detection and enumeration of enterococci was evaluated in recreational waters. Cov-IMS/ATP performance was compared with standard methods: defined substrate technology (Enterolert; IDEXX Laboratories), membrane filtration (EPA Method 1600), and an Enterococcus-specific quantitative PCR (qPCR) assay (EPA Method A). We extend previous studies by (i) analyzing the stability of the relationship between the Cov-IMS/ATP method and culture-based methods at different field sites, (ii) evaluating specificity of the assay for seven ATCC Enterococcus species, (iii) identifying cross-reacting organisms binding the antibody-bead complexes with 16S rRNA gene sequencing and evaluating specificity of the assay to five nonenterococcus species, and (iv) conducting preliminary tests of preabsorption as a means of improving the assay. Cov-IMS/ATP was found to perform consistently and with strong agreement rates (based on exceedance/compliance with regulatory limits) of between 83% and 100% compared to the culture-based Enterolert method at a variety of sites with complex inputs. The Cov-IMS/ATP method is specific to five of seven different Enterococcus spp. tested. However, there is potential for nontarget bacteria to bind the antibody, which may be reduced by purification of the IgG serum with preabsorption at problematic sites. The findings of this study help to validate the Cov-IMS/ATP method, suggesting a predictable relationship between the Cov-IMS/ATP method and traditional culture-based methods, which will allow for more widespread application of this rapid and field-portable method for coastal water quality assessment. PMID:24561583

  19. Twisting and subunit rotation in single FOF1-ATP synthase

    PubMed Central

    Sielaff, Hendrik; Börsch, Michael

    2013-01-01

    FOF1-ATP synthases are ubiquitous proton- or ion-powered membrane enzymes providing ATP for all kinds of cellular processes. The mechanochemistry of catalysis is driven by two rotary nanomotors coupled within the enzyme. Their different step sizes have been observed by single-molecule microscopy including videomicroscopy of fluctuating nanobeads attached to single enzymes and single-molecule Förster resonance energy transfer. Here we review recent developments of approaches to monitor the step size of subunit rotation and the transient elastic energy storage mechanism in single FOF1-ATP synthases. PMID:23267178

  20. Synthesis and in vitro microbial evaluation of La(III), Ce(III), Sm(III) and Y(III) metal complexes of vitamin B6 drug

    NASA Astrophysics Data System (ADS)

    Refat, Moamen S.; Al-Azab, Fathi M.; Al-Maydama, Hussein M. A.; Amin, Ragab R.; Jamil, Yasmin M. S.

    2014-06-01

    Metal complexes of pyridoxine mono hydrochloride (vitamin B6) are prepared using La(III), Ce(III), Sm(III) and Y(III). The resulting complexes are investigated. Some physical properties, conductivity, analytical data and the composition of the four pyridoxine complexes are discussed. The elemental analysis shows that the formed complexes of La(III), Ce(III), Sm(III) and Y(III) with pyridoxine are of 1:2 (metal:PN) molar ratio. All the synthesized complexes are brown in color and possess high melting points. These complexes are partially soluble in hot methanol, dimethylsulfoxide and dimethylformamide and insoluble in water and some other organic solvents. Elemental analysis data, spectroscopic (IR, UV-vis. and florescence), effective magnetic moment in Bohr magnetons and the proton NMR suggest the structures. However, definite particle size is determined by invoking the X-ray powder diffraction and scanning electron microscopy data. The results obtained suggested that pyridoxine reacted with metal ions as a bidentate ligand through its phenolate oxygen and the oxygen of the adjacent group at the 4‧-position. The molar conductance measurements proved that the pyridoxine complexes are electrolytic in nature. The kinetic and thermodynamic parameters such as: Ea, ΔH*, ΔS* and ΔG* were estimated from the DTG curves. The antibacterial evaluation of the pyridoxine and their complexes were also performed against some gram positive, negative bacteria as well as fungi.

  1. 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.

  2. Differences in G-actin containing bound ATP or ADP: the Mg2+-induced conformational change requires ATP.

    PubMed

    Frieden, C; Patane, K

    1985-07-16

    The role of adenosine 5'-triphosphate (ATP) in the Mg2+-induced conformational change of rabbit skeletal muscle G-actin has been investigated by comparing actin containing bound ADP with actin containing bound ATP. As previously described [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886], N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled G-actin containing ATP undergoes a time-dependent Mg2+-induced fluorescence change that reflects a conformational change in the actin. Addition of Mg2+ to labeled G-actin containing ADP gives no fluorescence change, suggesting that the conformational change does not occur. The fluorescence change can be restored on the addition of ATP. Examination of the time courses of these experiments suggests that ATP must replace ADP prior to the Mg2+-induced change. The Mg2+-induced polymerization of actin containing ADP is extraordinarily slow compared to that of actin containing ATP. The lack of the Mg2+-induced conformational change, which is an essential step in the Mg2+-induced polymerization, is probably the cause for the very slow polymerization of actin containing ADP. On the other hand, at 20 degrees C, at pH 8, and in 2 mM Mg2+, the elongation rate from the slow growing end of an actin filament, measured by using the protein brevin to block growth at the fast growing end, is only 4 times slower for actin containing ADP than for actin containing ATP.

  3. Extracellular ATP a New Player in Cancer Metabolism: NSCLC Cells Internalize ATP In Vitro and In Vivo Using Multiple Endocytic Mechanisms.

    PubMed

    Qian, Yanrong; Wang, Xuan; Li, Yunsheng; Cao, Yanyang; Chen, Xiaozhuo

    2016-11-01

    Intratumoral extracellular ATP concentrations are 1000 times higher than those in normal tissues of the same cell origin. However, whether or not cancer cells use the abundant extracellular ATP was unknown until we recently reported that cancer cells internalize ATP. The internalized ATP was found to substantially increase intracellular ATP concentration and promote cell proliferation and drug resistance in cancer cells. Here, using a nonhydrolyzable fluorescent ATP (NHF-ATP), radioactive and regular ATP, coupled with high and low molecular weight dextrans as endocytosis tracers and fluorescence microscopy and ATP assays, cultured human NSCLC A549 and H1299 cells as well as A549 tumor xenografts were found to internalize extracellular ATP at concentrations within the reported intratumoral extracellular ATP concentration range. In addition to macropinocytosis, both clathrin- and caveolae-mediated endocytosis significantly contribute to the ATP internalization, which led to an approximately 30% (within 45 minutes) or more than 50% (within 4 hours) increase in intracellular ATP levels after ATP incubation. This increase could not be accounted for by either purinergic receptor signaling or increased intracellular ATP synthesis rates in the ATP-treated cancer cells. These new findings significantly deepen our understanding of the Warburg effect by shedding light on how cancer cells in tumors, which are heterogeneous for oxygen and nutrition supplies, take up extracellular ATP and use the internalized ATP to perform multiple previously unrecognized functions of biological importance. They strongly suggest the existence of ATP sharing among cancer and stromal cells in tumors and simultaneously identify multiple new anticancer targets.

  4. Distinct neurological disorders with ATP1A3 mutations

    PubMed Central

    Heinzen, Erin L.; Arzimanoglou, Alexis; Brashear, Allison; Clapcote, Steven J.; Gurrieri, Fiorella; Goldstein, David B.; Jóhannesson, Sigurður H.; Mikati, Mohamad A.; Neville, Brian; Nicole, Sophie; Ozelius, Laurie J.; Poulsen, Hanne; Schyns, Tsveta; Sweadner, Kathleen J.; van den Maagdenberg, Arn; Vilsen, Bente

    2014-01-01

    Genetic research has shown that mutations that modify the protein-coding sequence of ATP1A3, the gene encoding the α3 subunit of Na+/K+-ATPase, cause both rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood. These discoveries link two clinically distinct neurological diseases to the same gene, however, ATP1A3 mutations are, with one exception, disease-specific. Although the exact mechanism of how these mutations lead to disease is still unknown, much knowledge has been gained about functional consequences of ATP1A3 mutations using a range of in vitro and animal model systems, and the role of Na+/K+-ATPases in the brain. Researchers and clinicians are attempting to further characterise neurological manifestations associated with mutations in ATP1A3, and to build on the existing molecular knowledge to understand how specific mutations can lead to different diseases. PMID:24739246

  5. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth.

    PubMed

    Ahmad, Zulfiqar; Laughlin, Thomas F; Kady, Ismail O

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.

  6. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

    PubMed Central

    Ahmad, Zulfiqar; Laughlin, Thomas F.; Kady, Ismail O.

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase. PMID:25996607

  7. ATP economy of force maintenance in human tibialis anterior muscle.

    PubMed

    Nakagawa, Yoshinao; Ratkevicius, Aivaras; Mizuno, Masao; Quistorff, Bjorn

    2005-06-01

    The aim of this study was investigate ATP economy of force maintenance in the human tibialis anterior muscle during 60 s of anaerobic voluntary contraction at 50% of maximum voluntary contraction (MVC). ATP turnover rate was evaluated using P magnetic resonance spectroscopy (P-MRS). The total volume of ankle dorsiflexor muscles was assessed by H magnetic resonance imaging (MRI) (H-MRI), and the fiber type composition of the tibialis anterior muscle was evaluated using histochemical analysis of muscle biopsies. The tibialis anterior muscle occupied 59.7 +/- 0.6% (mean +/- SEM) of the total ankle dorsiflexor muscle volume, which was 267 +/- 10 cm. Relative cross-sectional areas occupied by Type I, IIA, and IIB fibers in the tibialis anterior were 69.3 +/- 2.2, 27.4 +/- 2.76, and 3.2 +/- 1.0%, respectively. ATP economy of force maintenance did not change significantly during the 60-s contraction. It averaged at 4.81 +/- 0.42 N.s.micromol-1, and correlated with the relative cross-sectional area of the muscle occupied by Type I fiber (r = 0.73, P < 0.01). For the second half of the contraction, subjects dropping in force showed lower ATP economy compared with those maintaining the force (3.7 +/- 0.6 vs 5.3 +/- 0.6 N.s.micromol-1; P < 0.05). It is argued that the unchanged ATP economy of force maintenance during the voluntary contraction could be due to an increase in the ATP economy of contracting muscle fibers offsetting the effects of increased temperature and low ATP economy of Type II fibers. Mechanical interaction between motor units could also act to improve ATP economy of force maintenance.

  8. Sleep and brain energy levels: ATP changes during sleep.

    PubMed

    Dworak, Markus; McCarley, Robert W; Kim, Tae; Kalinchuk, Anna V; Basheer, Radhika

    2010-06-30

    Sleep is one of the most pervasive biological phenomena, but one whose function remains elusive. Although many theories of function, indirect evidence, and even common sense suggest sleep is needed for an increase in brain energy, brain energy levels have not been directly measured with modern technology. We here report that ATP levels, the energy currency of brain cells, show a surge in the initial hours of spontaneous sleep in wake-active but not in sleep-active brain regions of rat. The surge is dependent on sleep but not time of day, since preventing sleep by gentle handling of rats for 3 or 6 h also prevents the surge in ATP. A significant positive correlation was observed between the surge in ATP and EEG non-rapid eye movement delta activity (0.5-4.5 Hz) during spontaneous sleep. Inducing sleep and delta activity by adenosine infusion into basal forebrain during the normally active dark period also increases ATP. Together, these observations suggest that the surge in ATP occurs when the neuronal activity is reduced, as occurs during sleep. The levels of phosphorylated AMP-activated protein kinase (P-AMPK), well known for its role in cellular energy sensing and regulation, and ATP show reciprocal changes. P-AMPK levels are lower during the sleep-induced ATP surge than during wake or sleep deprivation. Together, these results suggest that sleep-induced surge in ATP and the decrease in P-AMPK levels set the stage for increased anabolic processes during sleep and provide insight into the molecular events leading to the restorative biosynthetic processes occurring during sleep.

  9. Mechanism of actin polymerization in cellular ATP depletion.

    PubMed

    Atkinson, Simon J; Hosford, Melanie A; Molitoris, Bruce A

    2004-02-13

    Cellular ATP depletion in diverse cell types results in the net conversion of monomeric G-actin to polymeric F-actin and is an important aspect of cellular injury in tissue ischemia. We propose that this conversion results from altering the ratio of ATP-G-actin and ADP-G-actin, causing a net decrease in the concentration of thymosinactin complexes as a consequence of the differential affinity of thymosin beta4 for ATP- and ADP-G-actin. To test this hypothesis we examined the effect of ATP depletion induced by antimycin A and substrate depletion on actin polymerization, the nucleotide state of the monomer pool, and the association of actin monomers with thymosin and profilin in the kidney epithelial cell line LLC-PK1. ATP depletion for 30 min increased F-actin content to 145% of the levels under physiological conditions, accompanied by a corresponding decrease in G-actin content. Cytochalasin D treatment did not reduce F-actin formation during ATP depletion, indicating that it was predominantly not because of barbed end monomer addition. ATP-G-actin levels decreased rapidly during depletion, but there was no change in the concentration of ADP-G-actin monomers. The decrease in ATP-G-actin levels could be accounted for by dissociation of the thymosin-G-actin binary complex, resulting in a rise in the concentration of free thymosin beta4 from 4 to 11 microm. Increased detection of profilin-actin complexes during depletion indicated that profilin may participate in catalyzing nucleotide exchange during depletion. This mechanism provides a biochemical basis for the accumulation of F-actin aggregates in ischemic cells.

  10. Control and monitoring the effectiveness of different biocides with the use of free ATP

    SciTech Connect

    Chalut, J.; Small, G.; Payton, J.

    1996-12-01

    The Adenosine Triphosphate (ATP) technology can be used as a measurement of the total living biomass. However, care must be exercised in its application and its interpretation. The use of this technique on samples from cooling water systems clearly indicate that there are three distinct pools of ATP, classified as bacterial ATP, free ATP and total ATP, the latter being the sum of the free ATP and bacterial ATP. The mode of action of certain biocidal agents is by disruption of cell membranes, a process which does not decrease the pool of total ATP, but does move ATP from the bacterial ATP pool to the free ATP pool. As a consequence, it is important that for a realistic interpretation to be made, it is necessary to know which biocidal agents are being used and to fully understand their mode of action.

  11. Follow the ATP: tumor energy production: a perspective.

    PubMed

    Oronsky, Bryan T; Oronsky, Neil; Fanger, Gary R; Parker, Christopher W; Caroen, Scott Z; Lybeck, Michelle; Scicinski, Jan J

    2014-01-01

    As early as the 1920s, the eminent physician and chemist, Otto Warburg, nominated for a second Nobel Prize for his work on fermentation, observed that the core metabolic signature of cancer cells is a high glycolytic flux. Warburg averred that the prime mover of cancer is defective mitochondrial respiration, which drives a switch to an alternative energy source, aerobic glycolysis in lieu of Oxidative Phosphorylation (OXPHOS), in an attempt to maintain cellular viability and support critical macromolecular needs. The cell, deprived of mitochondrial ATP production, must reprogram its metabolism as a secondary survival mechanism to maintain sufficient ATP and NADH levels for macromolecule production, membrane integrity and DNA synthesis as well as maintenance of membrane ionic gradients. A time-tested method to identify and disrupt criminal activity is to "follow the money" since the illicit proceeds from crime are required to underwrite it. By analogy, strategies to target cancer involve following and disrupting the flow of ATP and NADH, the energetic and redox "currencies" of the cell, respectively, since the tumor requires high levels of ATP and NADH, not only for metastasis and proliferation, but also, on a more basic level, for survival. Accordingly, four broad ATP reduction strategies to impact and potentially derail cancer energy production are highlighted herein: 1) small molecule energy-restriction mimetic agents (ERMAs) that target various aspects of energy metabolism, 2) reduction of energy 'subsidization' with autophagy inhibitors, 3) acceleration of ATP turnover to increase energy inefficiency, and 4) dietary energy restriction to limit the energy supply.

  12. ATP-dependent degradation of ubiquitin-protein conjugates.

    PubMed Central

    Hershko, A; Leshinsky, E; Ganoth, D; Heller, H

    1984-01-01

    Previous studies have indicated that the ATP-requiring conjugation of ubiquitin with proteins plays a role in the energy-dependent degradation of intracellular proteins. To examine whether such conjugates are indeed intermediates in protein breakdown, conjugates of 125I-labeled lysozyme with ubiquitin were isolated and incubated with a fraction of reticulocyte extract that lacks the enzymes that carry out ubiquitin-protein conjugation. ATP markedly stimulated degradation of the lysozyme moiety of ubiquitin conjugates to products soluble in trichloroacetic acid. By contrast, free 125I-labeled lysozyme was not degraded under these conditions, unless ubiquitin and the three enzymes required for ubiquitin conjugation were supplemented. Mg2+ was absolutely required for conjugate breakdown. Of various nucleotides, only CTP replaced ATP. Nonhydrolyzable analogs of ATP were not effective. In the absence of ATP, free lysozyme is released from ubiquitin-lysozyme conjugates by isopeptidases present in the extract. Thus, ATP is involved in both the formation and the breakdown of ubiquitin-protein conjugates. Images PMID:6324208

  13. Phospho-oligosaccharide dependent phosphorylation of ATP citrate lyase.

    PubMed

    Puerta, J; Mato, J M; Alemany, S

    1990-01-01

    The effect of insulin on ATP citrate lyase phosphorylation has been shown to be mimicked by a phospho-oligosaccharide in intact adipocytes. We demonstrate that the addition of phospho-oligosaccharide to intact adipocytes enhances the phosphorylation of ATP citrate lyase in the same tryptic peptide as insulin does. The addition of phospho-oligosaccharide to an adipocyte extract also results in an increase in ATP citrate lyase phosphorylation but in a different site than that observed in intact cells. The phospho-oligosaccharide-dependent incorporation of phosphate into ATP citrate lyase in intact cells is resistant to isopropanol and acetic acid, but the phosphoenzyme phosphorylated in cell extracts is acid labile. In cell extracts, the addition of phospho-oligosaccharide markedly inhibits ATP hydrolysis, which may explain the effect of this molecule on ATP citrate lyase phosphorylation in broken cells. These results support the hypothesis that this phospho-oligosaccharide mediates some of the effects of insulin on protein phosphorylation. They also indicate that caution should be exercised in interpreting the results obtained by adding phospho-oligosaccharide to broken cell preparations.

  14. A genetically encoded fluorescent reporter of ATP/ADP ratio

    PubMed Central

    Berg, Jim; Hung, Yin Pun; Yellen, Gary

    2008-01-01

    A fluorescent sensor of adenylate nucleotides was constructed by combining a circularly permuted variant of green fluorescent protein with a bacterial regulatory protein, GlnK1, from Methanococcus jannaschii. The affinity for Mg-ATP is below 100 nM, as seen for the other members of the bacterial PII regulator family – a surprisingly high affinity given normal intracellular [ATP] in the millimolar range. ADP binds to the same site, competing with Mg-ATP but producing a smaller change in fluorescence. With normal physiological concentrations of ATP and ADP, the binding site is saturated, but competition between the two substrates causes the sensor to behave as a nearly ideal reporter of the ATP/ADP concentration ratio. This principle for sensing the ratio of two analytes by competition at a high affinity site probably underlies the normal functioning of PII regulatory proteins. The engineered sensor, Perceval, can be used to monitor the ATP/ADP ratio during live cell imaging. PMID:19122669

  15. 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.

  16. Piezo1 regulates mechanotransductive release of ATP from human RBCs

    PubMed Central

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

    2015-01-01

    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 (Ca2+) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca2+ influx decrease significantly. Remarkably, a critical extracellular Ca2+ 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. PMID:26351678

  17. Catalytic Mechanism of the Maltose Transporter Hydrolyzing ATP.

    PubMed

    Huang, Wenting; Liao, Jie-Lou

    2016-01-12

    We use quantum mechanical and molecular mechanical (QM/MM) simulations to study ATP hydrolysis catalyzed by the maltose transporter. This protein is a prototypical member of a large family that consists of ATP-binding cassette (ABC) transporters. The ABC proteins catalyze ATP hydrolysis to perform a variety of biological functions. Despite extensive research efforts, the precise molecular mechanism of ATP hydrolysis catalyzed by the ABC enzymes remains elusive. In this work, the reaction pathway for ATP hydrolysis in the maltose transporter is evaluated using a QM/MM implementation of the nudged elastic band method without presuming reaction coordinates. The potential of mean force along the reaction pathway is obtained with an activation free energy of 19.2 kcal/mol in agreement with experiments. The results demonstrate that the reaction proceeds via a dissociative-like pathway with a trigonal bipyramidal transition state in which the cleavage of the γ-phosphate P-O bond occurs and the O-H bond of the lytic water molecule is not yet broken. Our calculations clearly show that the Walker B glutamate as well as the switch histidine stabilizes the transition state via electrostatic interactions rather than serving as a catalytic base. The results are consistent with biochemical and structural experiments, providing novel insight into the molecular mechanism of ATP hydrolysis in the ABC proteins.

  18. [ATP pool and bioluminescence in psychrophilic bacteria Photobacterium phosphoreum].

    PubMed

    Alekserova, L É; Alenina, K A; Efremenko, E N; Mazhul', M M; Piskunova, N F; Ismailov, A D

    2014-01-01

    Bioluminescence activity and ATP pool were investigated in the culture of psychrophilic bacteria Photobacterium phosphoreum collected-from the exponential and stationary growth phases, as well as immobilized in polyvinyl alcohol (PVA) cryogel. In liquid culture, ATP pool remained at an almost a constant level throughout the luminescence cycle (over 100 h). The ATP pool in the stationary-phase and PVA-immobilizedl cells remained constant throughout their incubation in the medium (over 200 h) and in 3% NaCl solution (over 100 h): Quantitative assessment of integral photon yield and ATP pool indicated that bioluminescence decay in growing or stationary cells was not caused by limitation by the energy substrates of the luciferase reaction. Kinetic and quantitative parameters of emission activity and ATP pool excluded the possibility of formation of the aldehyde substrate for luciferase via reduction of the relevant fatty acids in NADPH and ATP-dependent reductase reaction and its oxidation in the monooxygenase reaction. Our results indicate that the aliphatic aldehyde is not utilized in the process of light emission.

  19. A new type of Na(+)-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif.

    PubMed

    Schulz, Sarah; Iglesias-Cans, Marina; Krah, Alexander; Yildiz, Ozkan; Leone, Vanessa; Matthies, Doreen; Cook, Gregory M; Faraldo-Gómez, José D; Meier, Thomas

    2013-01-01

    The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na⁺. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F₁F₀-ATP synthase with a novel Na⁺ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na⁺ specificity in physiological settings. Consistently, activity measurements showed Na⁺ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na⁺ ionophore monensin. Furthermore, Na⁺ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na⁺ coupling is provided by two identical crystal structures of the c₁₁ ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na⁺ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na⁺ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.

  20. High affinity ATP/ADP analogues as new tools for studying CFTR gating

    PubMed Central

    Zhou, Zhen; Wang, Xiaohui; Li, Min; Sohma, Yoshiro; Zou, Xiaoqin; Hwang, Tzyh-Chang

    2005-01-01

    Previous studies using non-hydrolysable ATP analogues and hydrolysis-deficient cystic fibrosis transmembrane conductance regulator (CFTR) mutants have indicated that ATP hydrolysis precedes channel closing. Our recent data suggest that ATP binding is also important in modulating the closing rate. This latter hypothesis predicts that ATP analogues with higher binding affinities should stabilize the open state more than ATP. Here we explore the possibility of using N6-modified ATP/ADP analogues as high-affinity ligands for CFTR gating, since these analogues have been shown to be more potent than native ATP/ADP in other ATP-binding proteins. Among the three N6-modified ATP analogues tested, N6-(2-phenylethyl)-ATP (P-ATP) was the most potent, with a K½ of 1.6 ± 0.4 μm (>50-fold more potent than ATP). The maximal open probability (Po) in the presence of P-ATP was ∼30% higher than that of ATP, indicating that P-ATP also has a higher efficacy than ATP. Single-channel kinetic analysis showed that as [P-ATP] was increased, the opening rate increased, whereas the closing rate decreased. The fact that these two kinetic parameters have different sensitivities to changes of [P-ATP] suggests an involvement of two different ATP-binding sites, a high-affinity site modulating channel closing and a low affinity site controlling channel opening. The effect of P-ATP on the stability of open states was more evident when ATP hydrolysis was abolished, either by mutating the nucleotide-binding domain 2 (NBD2) Walker B glutamate (i.e. E1371) or by using the non-hydrolysable ATP analogue AMP-PNP. Similar strategies to develop nucleotide analogues with a modified adenine ring could be valuable for future studies of CFTR gating. PMID:16223764

  1. Global Positioning System III (GPS III)

    DTIC Science & Technology

    2015-12-01

    modernization of the constellation . GPS III complies with 10 United States Code (USC) § 2281, ensuring the continued sustainment and operation of GPS for... constellations , further increasing the accuracy and availability of user PNT solutions. GPS III December 2015 SAR March 23, 2016 16:15:29 UNCLASSIFIED

  2. ATP and heat production in human skeletal muscle during dynamic exercise: higher efficiency of anaerobic than aerobic ATP resynthesis

    PubMed Central

    Krustrup, Peter; Ferguson, Richard A; Kjær, Michael; Bangsbo, Jens

    2003-01-01

    The aim of the present study was to simultaneously examine skeletal muscle heat production and ATP turnover in humans during dynamic exercise with marked differences in aerobic metabolism. This was done to test the hypothesis that efficiency is higher in anaerobic than aerobic ATP resynthesis. Six healthy male subjects performed 90 s of low intensity knee-extensor exercise with (OCC) and without thigh occlusion (CON-LI) as well as 90 s of high intensity exercise (CON-HI) that continued from the CON-LI bout. Muscle heat production was determined by continuous measurements of muscle heat accumulation and heat release to the blood. Muscle ATP production was quantified by repeated measurements of thigh oxygen uptake as well as blood and muscle metabolite changes. All temperatures of the thigh were equalized to ≈37 °C prior to exercise by a water-perfused heating cuff. Oxygen uptake accounted for 80 ± 2 and 59 ± 4 %, respectively, of the total ATP resynthesis in CON-LI and CON-HI, whereas it was negligible in OCC. The rise in muscle temperature was lower (P < 0.05) in OCC than CON-LI (0.32 ± 0.04 vs. 0.37 ± 0.03 °C). The mean rate of heat production was also lower (P < 0.05) in OCC than CON-LI (36 ± 4 vs. 57 ± 4 J s−1). Mechanical efficiency was 52 ± 4 % after 15 s of OCC and remained constant, whereas it decreased (P < 0.05) from 56 ± 5 to 32 ± 3 % during CON-LI. During CON-HI, mechanical efficiency transiently increased (P < 0.05) to 47 ± 4 %, after which it decreased (P < 0.05) to 36 ± 3 % at the end of CON-HI. Assuming a fully coupled mitochondrial respiration, the ATP turnover per unit of work was calculated to be unaltered during OCC (≈20 mmol ATP kJ−1), whereas it increased (P < 0.05) from 21 ± 4 to 29 ± 3 mmol ATP kJ−1 during CON-LI and further (P < 0.05) to 37 ± 3 mmol ATP kJ−1 during CON-HI. The present data confirm the hypothesis that heat loss is lower in anaerobic ATP resynthesis than in oxidative phosphorylation and can in part

  3. cDNA, genomic sequence cloning and overexpression of giant panda (Ailuropoda melanoleuca) mitochondrial ATP synthase ATP5G1.

    PubMed

    Hou, W-R; Hou, Y-L; Ding, X; Wang, T

    2012-09-03

    The ATP5G1 gene is one of the three genes that encode mitochondrial ATP synthase subunit c of the proton channel. We cloned the cDNA and determined the genomic sequence of the ATP5G1 gene from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology and touchdown-PCR, respectively. The cloned cDNA fragment contains an open reading frame of 411 bp encoding 136 amino acids; the length of the genomic sequence is of 1838 bp, containing three exons and two introns. Alignment analysis revealed that the nucleotide sequence and the deduced protein sequence are highly conserved compared to Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus, and Sus scrofa. The homologies for nucleotide sequences of the giant panda ATP5G1 to those of these species are 93.92, 92.21, 92.46, 93.67, and 92.46%, respectively, and the homologies for amino acid sequences are 90.44, 95.59, 93.38, 94.12, and 91.91%, respectively. Topology prediction showed that there is one protein kinase C phosphorylation site, one casein kinase II phosphorylation site, five N-myristoylation sites, and one ATP synthase c subunit signature in the ATP5G1 protein of the giant panda. The cDNA of ATP5G1 was transfected into Escherichia coli, and the ATP5G1 fused with the N-terminally GST-tagged protein gave rise to accumulation of an expected 40-kDa polypeptide, which had the characteristics of the predicted protein.

  4. Conformational change induced by ATP binding in the multidrug ATP-binding cassette transporter BmrA.

    PubMed

    Orelle, Cédric; Gubellini, Francesca; Durand, Anne; Marco, Sergio; Lévy, Daniel; Gros, Philippe; Di Pietro, Attilio; Jault, Jean-Michel

    2008-02-26

    ATP-binding cassette (ABC) transporters are involved in the transport of a wide variety of substrates, and ATP-driven dimerization of their nucleotide binding domains (NBDs) has been suggested to be one of the most energetic steps of their catalytic cycle. Taking advantage of the propensity of BmrA, a bacterial multidrug resistance ABC transporter, to form stable, highly ordered ring-shaped structures [Chami et al. (2002) J. Mol. Biol. 315, 1075-1085], we show here that addition of ATP in the presence of Mg2+ prevented ring formation or destroyed the previously formed rings. To pinpoint the catalytic step responsible for such an effect, two classes of hydrolysis-deficient mutants were further studied. In contrast to hydrolytically inactive glutamate mutants that behaved essentially as the wild-type, lysine Walker A mutants formed ring-shaped structures even in the presence of ATP-Mg. Although the latter mutants still bound ATP-Mg, and even slowly hydrolyzed it for the K380R mutant, they were most likely unable to undergo a proper NBD dimerization upon ATP-Mg addition. The ATP-driven dimerization step, which was still permitted in glutamate mutants and led to a stable conformation suitable to monitor the growth of 2D crystals, appeared therefore responsible for destabilization of the BmrA ring structures. Our results provide direct visual evidence that the ATP-induced NBD dimerization triggers a conformational change large enough in BmrA to destabilize the rings, which is consistent with the assumption that this step might constitute the "power stroke" for ABC transporters.

  5. Dexamethasone Enhances ATP-Induced Inflammatory Responses in Endothelial Cells

    PubMed Central

    Ding, Yi; Gao, Zhan-Guo; Jacobson, Kenneth A.

    2010-01-01

    The purinergic nucleotide ATP is released from stressed cells and is implicated in vascular inflammation. Glucocorticoids are essential to stress responses and are used therapeutically, yet little information is available that describes the effects of glucocorticoids on ATP-induced inflammation. In a human microvascular endothelial cell line, extracellular ATP-induced interleukin (IL)-6 secretion in a dose- and time-dependent manner. When cells were pretreated with dexamethasone, a prototypic glucocorticoid, ATP-induced IL-6 production was enhanced in a time- and dose-dependent manner. Mifepristone, a glucocorticoid receptor antagonist, blocked these effects. ATP-induced IL-6 release was significantly inhibited by a phospholipase C inhibitor [1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] (63.2 ± 3%, p < 0.001) and abolished by a p38 mitogen-activated protein kinase inhibitor [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580)] (88 ± 1%, p < 0.001). Cells treated with dexamethasone induced mRNA expression of the purinergic P2Y2 receptor (P2Y2R) 1.8- ± 0.1-fold and, when stimulated with ATP, enhanced Ca2+ release and augmented IL-6 mRNA expression. Silencing of the P2Y2R by its small interfering RNA decreased ATP-induced IL-6 production by 81 ± 1% (p < 0.001). Dexamethasone enhanced the transcription rate of P2Y2R mRNA and induced a dose-related increase in the activity of the P2Y2R promoter. Furthermore, dexamethasone-enhanced ATP induction of adhesion molecule transcription and augmented the release of IL-8. Dexamethasone leads to an unanticipated enhancement of endothelial inflammatory mediator production by extracellular ATP via a P2Y2R-dependent mechanism. These data define a novel positive feedback loop of glucocorticoids and ATP-induced endothelial inflammation. PMID:20826566

  6. Revisiting the thermodynamic theory of optimal ATP stoichiometries by analysis of various ATP-producing metabolic pathways.

    PubMed

    Werner, Sarah; Diekert, Gabriele; Schuster, Stefan

    2010-12-01

    The stoichiometry of ATP-producing metabolic pathways had been analysed theoretically by several authors by using evolutionary arguments and optimality principles. Waddell et al. (Biochem Educ 27:12-13, 1999) analysed (lactate-producing) glycolysis and used linear irreversible thermodynamics. The result was that half of the free-energy difference should be converted into free-energy of ATP and the remaining half should be used to drive the pathway. The calculated stoichiometry is in agreement with the observed yield of two moles of ATP per mole of glucose. Using the same approach, we here analyse eight other metabolic pathways. Although the deviation is not very large, the calculated values do not fit as nicely as for glycolysis as leading to lactate. For example, for O₂ respiration, the theoretical ATP yield equals 27.9. The real value varies among organisms between 26 and 38. For mixed-acid fermentation in Escherichia coli, the theoretical and experimental values are 2.24 and 2, respectively. For arginine degradation in M. pneumoniae, the calculated value is 2.43 mol of ATP, while in vivo only one mole is produced. During evolution, some pathways may not have reached their optimal ATP net production because energy yield is not their only function. Moreover, it should be acknowledged that the approach by linear irreversible thermodynamics is a rough approximation.

  7. 40 CFR Appendix III to Part 1042 - Not-to-Exceed Zones

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ..., App. III Appendix III to Part 1042—Not-to-Exceed Zones (a) The following definitions apply for this Appendix III: (1) Percent power means the percentage of the maximum power achieved at Maximum Test Speed (or at Maximum Test Torque for constant-speed engines). (2) Percent speed means the percentage of...

  8. 40 CFR Appendix III to Part 1042 - Not-to-Exceed Zones

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ..., App. III Appendix III to Part 1042—Not-to-Exceed Zones (a) The following definitions apply for this Appendix III: (1) Percent power means the percentage of the maximum power achieved at Maximum Test Speed (or at Maximum Test Torque for constant-speed engines). (2) Percent speed means the percentage of...

  9. 40 CFR Appendix III to Part 1042 - Not-to-Exceed Zones

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ..., App. III Appendix III to Part 1042—Not-to-Exceed Zones (a) The following definitions apply for this Appendix III: (1) Percent power means the percentage of the maximum power achieved at Maximum Test Speed (or at Maximum Test Torque for constant-speed engines). (2) Percent speed means the percentage of...

  10. 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...

  11. ATP drives lamina propria T(H)17 cell differentiation.

    PubMed

    Atarashi, Koji; Nishimura, Junichi; Shima, Tatsuichiro; Umesaki, Yoshinori; Yamamoto, Masahiro; Onoue, Masaharu; Yagita, Hideo; Ishii, Naoto; Evans, Richard; Honda, Kenya; Takeda, Kiyoshi

    2008-10-09

    Interleukin (IL)-17-producing CD4(+) T lymphocytes (T(H)17 cells) constitute a subset of T-helper cells involved in host defence and several immune disorders. An intriguing feature of T(H)17 cells is their selective and constitutive presence in the intestinal lamina propria. Here we show that adenosine 5'-triphosphate (ATP) that can be derived from commensal bacteria activates a unique subset of lamina propria cells, CD70(high)CD11c(low) cells, leading to the differentiation of T(H)17 cells. Germ-free mice exhibit much lower concentrations of luminal ATP, accompanied by fewer lamina propria T(H)17 cells, compared to specific-pathogen-free mice. Systemic or rectal administration of ATP into these germ-free mice results in a marked increase in the number of lamina propria T(H)17 cells. A CD70(high)CD11c(low) subset of the lamina propria cells expresses T(H)17-prone molecules, such as IL-6, IL-23p19 and transforming-growth-factor-beta-activating integrin-alphaV and -beta8, in response to ATP stimulation, and preferentially induces T(H)17 differentiation of co-cultured naive CD4(+) T cells. The critical role of ATP is further underscored by the observation that administration of ATP exacerbates a T-cell-mediated colitis model with enhanced T(H)17 differentiation. These observations highlight the importance of commensal bacteria and ATP for T(H)17 differentiation in health and disease, and offer an explanation of why T(H)17 cells specifically present in the intestinal lamina propria.

  12. Impaired ATP Kinetics in Failing in vivo Mouse Hearts

    PubMed Central

    Gupta, Ashish; Chacko, Vadappuram P.; Schär, Michael; Akki, Ashwin; Weiss, Robert G.

    2011-01-01

    Background The hypothesis that the failing heart may be energy starved is supported, in part, by observations of reduced rates of ATP synthesis through the creatine kinase (CK) reaction, the primary myocardial energy reservoir, in heart failure (HF) patients. Although murine models have been used to probe HF pathophysiology, it has not been possible to non-invasively measure the rate of ATP synthesis through CK in the in vivo mouse heart. The purpose of this work was to exploit non-invasive spatially-localized magnetic resonance spectroscopy (MRS) techniques to measure ATP flux through CK in in vivo mouse hearts and determine the extent of any reductions in murine HF. Methods and Results The Triple Repetition Time Saturation Transfer (TRiST) MRS method of measuring ATP kinetics was first validated in skeletal muscle, rendering similar results to conventional saturation transfer MRS. In normal mouse hearts the in vivo CK pseudo-first-order-rate constant, kf, was 0.32±0.03 s−1 (mean±SD) and the rate of ATP synthesis through CK was 3.16±0.47 µmol/g/s. Thoracic aortic constriction (TAC) reduced kf by 31% (0.23±0.03 s−1, p<0.0001) and ATP synthesis through CK by 51% (1.54±0.25 µmol/g/s, p<0.0001), analogous values to those in failing human hearts. Conclusions Despite the small size and high murine heart rate, the ATP synthesis rate through CK is similar in vivo in murine and human hearts and comparably reduced in HF. Because murine TAC shares fundamental energetic similarities with human HF, this model and new MRS approach promise a powerful means to non-invasively probe altered energetics in HF. PMID:20926788

  13. Analysis of aberrant pre-messenger RNA splicing resulting from mutations in ATP8B1 and efficient in vitro rescue by adapted U1 small nuclear RNA.

    PubMed

    van der Woerd, Wendy L; Mulder, Johanna; Pagani, Franco; Beuers, Ulrich; Houwen, Roderick H J; van de Graaf, Stan F J

    2015-04-01

    ATP8B1 deficiency is a severe autosomal recessive liver disease resulting from mutations in the ATP8B1 gene characterized by a continuous phenotypical spectrum from intermittent (benign recurrent intrahepatic cholestasis; BRIC) to progressive familial intrahepatic cholestasis (PFIC). Current therapeutic options are insufficient, and elucidating the molecular consequences of mutations could lead to personalized mutation-specific therapies. We investigated the effect on pre-messenger RNA splicing of 14 ATP8B1 mutations at exon-intron boundaries using an in vitro minigene system. Eleven mutations, mostly associated with a PFIC phenotype, resulted in aberrant splicing and a complete absence of correctly spliced product. In contrast, three mutations led to partially correct splicing and were associated with a BRIC phenotype. These findings indicate an inverse correlation between the level of correctly spliced product and disease severity. Expression of modified U1 small nuclear RNAs (snRNA) complementary to the splice donor sites strongly improved or completely rescued splicing for several ATP8B1 mutations located at donor, as well as acceptor, splice sites. In one case, we also evaluated exon-specific U1 snRNAs that, by targeting nonconserved intronic sequences, might reduce possible off-target events. Although very effective in correcting exon skipping, they also induced retention of the short downstream intron. We systematically characterized the molecular consequences of 14 ATP8B1 mutations at exon-intron boundaries associated with ATP8B1 deficiency and found that the majority resulted in total exon skipping. The amount of correctly spliced product inversely correlated with disease severity. Compensatory modified U1 snRNAs, complementary to mutated donor splice sites, were able to improve exon definition very efficiently and could be a novel therapeutic strategy in ATP8B1 deficiency as well as other genetic diseases. © 2014 by the American Association for the Study

  14. A1Ao-ATP synthase of Methanobrevibacter ruminantium couples sodium ions for ATP synthesis under physiological conditions.

    PubMed

    McMillan, Duncan G G; Ferguson, Scott A; Dey, Debjit; Schröder, Katja; Aung, Htin Lin; Carbone, Vincenzo; Attwood, Graeme T; Ronimus, Ron S; Meier, Thomas; Janssen, Peter H; Cook, Gregory M

    2011-11-18

    An unresolved question in the bioenergetics of methanogenic archaea is how the generation of proton-motive and sodium-motive forces during methane production is used to synthesize ATP by the membrane-bound A(1)A(o)-ATP synthase, with both proton- and sodium-coupled enzymes being reported in methanogens. To address this question, we investigated the biochemical characteristics of the A(1)A(o)-ATP synthase (MbbrA(1)A(o)) of Methanobrevibacter ruminantium M1, a predominant methanogen in the rumen. Growth of M. ruminantium M1 was inhibited by protonophores and sodium ionophores, demonstrating that both ion gradients were essential for growth. To study the role of these ions in ATP synthesis, the ahaHIKECFABD operon encoding the MbbrA(1)A(o) was expressed in Escherichia coli strain DK8 (Δatp) and purified yielding a 9-subunit protein with an SDS-stable c oligomer. Analysis of the c subunit amino acid sequence revealed that it consisted of four transmembrane helices, and each hairpin displayed a complete Na(+)-binding signature made up of identical amino acid residues. The purified MbbrA(1)A(o) was stimulated by sodium ions, and Na(+) provided pH-dependent protection against inhibition by dicyclohexylcarbodiimide but not tributyltin chloride. ATP synthesis in inverted membrane vesicles lacking sodium ions was driven by a membrane potential that was sensitive to cyanide m-chlorophenylhydrazone but not to monensin. ATP synthesis could not be driven by a chemical gradient of sodium ions unless a membrane potential was imposed. ATP synthesis under these conditions was sensitive to monensin but not cyanide m-chlorophenylhydrazone. These data suggest that the M. ruminantium M1 A(1)A(o)-ATP synthase exhibits all the properties of a sodium-coupled enzyme, but it is also able to use protons to drive ATP synthesis under conditions that favor proton coupling, such as low pH and low levels of sodium ions.

  15. An enhanced molecular dynamics study of HPPK-ATP conformation space exploration and ATP binding to HPPK.

    PubMed

    Su, Li; Cukier, Robert I

    2009-03-12

    HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) catalyzes the transfer of pyrophosphate from ATP to HP (6-hydroxymethyl-7,8-dihydropterin). This first reaction in the folate biosynthetic pathway is an important target for potential antimicrobial agents. In this work, the mechanism by which HPPK traps and binds ATP is studied by molecular dynamics (MD)-based methods. Based on the ternary crystal structure of HPPK with an ATP mimic and HP, a complex of ATPMg(2) and HPPK is simulated and found to undergo small conformational changes with conventional MD, as does also conventional MD when started from the apo crystal structure. The introduction of restraints in the MD that serve to move HPPK-ATP from its ternary complex (closed) to apo-like (open) forms shows that throughout the restraint path ATP remains bound to HPPK. That ATP remains bound suggests that there is an ensemble of conformations with ATP bound to HPPK that span the apo to more ligand-bound-like conformations, consistent with the pre-existing equilibrium hypothesis of ligand binding, whereby a ligand can select from and bind to a broad range of protein conformations. In the apo-like conformations, ATPMg(2) remains bound to HPPK through a number of mainly salt-bridge-like interactions between several negatively charged residues and the two magnesium cations. The introduction of a reweight method that enhances the sampling of MD by targeting explicit terms in the force field helps define the interactions that bind ATP to HPPK. Using the reweight method, conformational and center of mass motions of ATP, driven by the breaking and making of hydrogen bonds and salt bridges, are identified that lead to ATP separating from HPPK. An elastic normal mode (ENM) approach to opening the ternary complex and closing the apo crystal structures was carried out. The ENM analysis of the apo structure analysis shows one mode that does have a closing motion of HPPK loops, but the direction does not correlate

  16. A Shigella effector dampens inflammation by regulating epithelial release of danger signal ATP through production of the lipid mediator PtdIns5P.

    PubMed

    Puhar, Andrea; Tronchère, Hélène; Payrastre, Bernard; Nhieu, Guy Tran Van; Sansonetti, Philippe J

    2013-12-12

    Upon infection with Shigella flexneri, epithelial cells release ATP through connexin hemichannels. However, the pathophysiological consequence and the regulation of this process are unclear. Here we showed that in intestinal epithelial cell ATP release was an early alert response to infection with enteric pathogens that eventually promoted inflammation of the gut. Shigella evolved to escape this inflammatory reaction by its type III secretion effector IpgD, which blocked hemichannels via the production of the lipid PtdIns5P. Infection with an ipgD mutant resulted in rapid hemichannel-dependent accumulation of extracellular ATP in vitro and in vivo, which preceded the onset of inflammation. At later stages of infection, ipgD-deficient Shigella caused strong intestinal inflammation owing to extracellular ATP. We therefore describe a new paradigm of host-pathogen interaction based on endogenous danger signaling and identify extracellular ATP as key regulator of mucosal inflammation during infection. Our data provide new angles of attack for the development of anti-inflammatory molecules.

  17. Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones.

    PubMed

    Johansson, Renzo; Jonna, Venkateswara Rao; Kumar, Rohit; Nayeri, Niloofar; Lundin, Daniel; Sjöberg, Britt-Marie; Hofer, Anders; Logan, Derek T

    2016-06-07

    Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone.

  18. Dorsal horn neurons release extracellular ATP in a VNUT-dependent manner that underlies neuropathic pain.

    PubMed

    Masuda, Takahiro; Ozono, Yui; Mikuriya, Satsuki; Kohro, Yuta; Tozaki-Saitoh, Hidetoshi; Iwatsuki, Ken; Uneyama, Hisayuki; Ichikawa, Reiko; Salter, Michael W; Tsuda, Makoto; Inoue, Kazuhide

    2016-08-12

    Activation of purinergic receptors in the spinal cord by extracellular ATP is essential for neuropathic hypersensitivity after peripheral nerve injury (PNI). However, the cell type responsible for releasing ATP within the spinal cord after PNI is unknown. Here we show that PNI increases expression of vesicular nucleotide transporter (VNUT) in the spinal cord. Extracellular ATP content ([ATP]e) within the spinal cord was increased after PNI, and this increase was suppressed by exocytotic inhibitors. Mice lacking VNUT did not show PNI-induced increase in [ATP]e and had attenuated hypersensitivity. These phenotypes were recapitulated in mice with specific deletion of VNUT in spinal dorsal horn (SDH) neurons, but not in mice lacking VNUT in primary sensory neurons, microglia or astrocytes. Conversely, ectopic VNUT expression in SDH neurons of VNUT-deficient mice restored PNI-induced increase in [ATP]e and pain. Thus, VNUT is necessary for exocytotic ATP release from SDH neurons which contributes to neuropathic pain.

  19. A terbium(III)-organic framework for highly selective sensing of cytidine triphosphate.

    PubMed

    Zhao, Xi Juan; He, Rong Xing; Li, Yuan Fang

    2012-11-21

    Highly selective sensing of cytidine triphosphate (CTP) against other triphosphate nucleosides including ATP, GTP and UTP is successfully achieved with a luminescent terbium(III)-organic framework (TbOF) of [Tb(2)(2,3-pzdc)(2)(ox)(H(2)O)(2)](n) (2,3-pzdc(2-) = 2,3-pyrazinedicarboxylate, ox(2-) = oxalate).

  20. ATP synthases: cellular nanomotors characterized by LILBID mass spectrometry

    PubMed Central

    Hoffmann, Jan; Sokolova, Lucie; Preiss, Laura; Hicks, David B.; Krulwich, Terry A.; Morgner, Nina; Wittig, Ilka; Schägger, Hermann; Meier, Thomas; Brutschy, Bernd

    2010-01-01

    Mass spectrometry of membrane protein complexes is still a methodological challenge due to hydrophobic and hydrophilic parts of the species and the fact that all subunits are bound non-covalently together. The present study with the novel laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) reports on the determination of the subunit composition of the F1Fo-ATP synthase from Bacillus pseudofirmus OF4, that of both bovine heart and, for the first time, of human heart mitochondrial F1Fo-ATP synthases. Under selected buffer conditions the mass of the intact F1Fo-ATP synthase of B. pseudofirmus OF4 could be measured, allowing the analysis of complex subunit stoichiometry. The agreement with theoretical masses derived from sequence databases is very good. A comparison of the ATP synthase subunit composition of 5 different ATPases reveals differences in the complexity of eukaryotic and bacterial ATP synthases. However, whereas the overall construction of eukaryotic enzymes is more complex than the bacterial ones, functionally important subunits are conserved among all ATPases. PMID:20820587

  1. Cardiac Metabolism in Heart Failure - Implications beyond ATP production

    PubMed Central

    Doenst, Torsten; Nguyen, T. Dung; Abel, E. Dale

    2013-01-01

    The heart has a high rate of ATP production and turnover which is required to maintain its continuous mechanical work. Perturbations in ATP generating processes may therefore affect contractile function directly. Characterizing cardiac metabolism in heart failure revealed several metabolic alterations termed metabolic remodeling, ranging from changes in substrate utilization to mitochondrial dysfunction, ultimately resulting in ATP deficiency and impaired contractility. However, ATP depletion is not the only relevant consequence of metabolic remodeling during heart failure. By providing cellular building blocks and signaling molecules, metabolic pathways control essential processes such as cell growth and regeneration. Thus, alterations in cardiac metabolism may also affect the progression to heart failure by mechanisms beyond ATP supply. Our aim is therefore to highlight that metabolic remodeling in heart failure not only results in impaired cardiac energetics, but also induces other processes implicated in the development of heart failure such as structural remodeling and oxidative stress. Accordingly, modulating cardiac metabolism in heart failure may have significant therapeutic relevance that goes beyond the energetic aspect. PMID:23989714

  2. Differentiating connexin hemichannels and pannexin channels in cellular ATP release

    PubMed Central

    Lohman, Alexander W; Isakson, Brant E

    2014-01-01

    Adenosine triphosphate (ATP) plays a fundamental role in cellular communication, with its extracellular accumulation triggering purinergic signaling cascades in a diversity of cell types. While the roles for purinergic signaling in health and disease have been well established, identification and differentiation of the specific mechanisms controlling cellular ATP release is less well understood. Multiple mechanisms have been proposed to regulate ATP release with connexin (Cx) hemichannels and pannexin (Panx) channels receiving major focus. However, segregating the specific roles of Panxs and Cxs in ATP release in a plethora of physiological and pathological contexts has remained enigmatic. This multifaceted problem has arisen from the selectivity of pharmacological inhibitors for Panxs and Cxs, methodological differences in assessing Panx and Cx function and the potential compensation by other isoforms in gene silencing and genetic knockout models. Consequently, there remains a void in the current understanding of specific contributions of Panxs and Cxs in releasing ATP during homeostasis and disease. Differentiating the distinct signaling pathways that regulate these two channels will advance our current knowledge of cellular communication and aid in the development of novel rationally-designed drugs for modulation of Panx and Cx activity, respectively. PMID:24548565

  3. Differential modulation by extracellular ATP of carotid chemosensory responses.

    PubMed

    Spergel, D; Lahiri, S

    1993-06-01

    The possibility that the carotid body has ATP surface receptors that mediate O2 chemoreception was tested. To distinguish between the event(s) initiating chemoreception and those at the neurotransmitter level, we also tested the chemosensory response to nicotine before and after ATP administration. Carotid bodies from cats anesthetized with pentobarbital sodium were perfused and superfused in vitro with modified Tyrode solution (PCO2 < 1 Torr, pH 7.4, 36 degrees C) equilibrated at PO2 > 400 or approximately 150 Torr while chemosensory discharge was recorded extracellularly. ATP and adenosine 5'-[gamma-thio]triphosphate stimulated discharge with similar dose dependence, whereas adenosine had little effect. ATP infusion for > or = 2 min evoked an initial stimulation of discharge followed by a decline to baseline (desensitization). Desensitization did not affect the response to hypoxia (perfusate flow interruption) but inhibited the response to nicotine (4-nmol pulse). Therefore, 1) the carotid body has surface ATP receptors that may mediate the chemosensory response to nicotine but not to hypoxia and 2) nicotinic receptors are not required for carotid body O2 chemoreception.

  4. Rotation and structure of FoF1-ATP synthase.

    PubMed

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2011-06-01

    F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, F(o)F(1)-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of F(o)F(1)-ATP synthase as well as a brief historical background.

  5. Flagellar rotation in the archaeon Halobacterium salinarum depends on ATP.

    PubMed

    Streif, Stefan; Staudinger, Wilfried Franz; Marwan, Wolfgang; Oesterhelt, Dieter

    2008-12-05

    Halobacterium salinarum swims with the help of a polarly inserted flagellar bundle. In energized cells, the flagellar motors rotate continuously, occasionally switching the rotational sense. Starving cells become immotile as the energy level drops. Presumably, there is a threshold of energy required for flagellar rotation. When starved, immotile cells are energized by exposure to light, the speed of flagellar rotation increases gradually to its steady state over several minutes. Since the light-driven proton pump bacteriorhodopsin energizes the cell membrane to the maximal level within a fraction of a second, the delay in reaching the maximal swimming speed suggests that the halobacterial flagellar motor may not be driven directly by proton motive force. Swimming cells, which obtain their energy exclusively through light-driven proton pumping, become immotile within 20 min when treated with N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the proton translocating ATP synthase. However, flagellar motility in DCCD-treated cells can be restored by the addition of L-arginine, which serves as a fermentative energy source and restores the cytoplasmic ATP level in the presence of DCCD. This suggests that flagellar motor rotation depends on ATP, and this is confirmed by the observation that motility is increased strongly by L-arginine at zero proton motive force levels. The flagellar motor may be driven either by ATP directly or by an ATP-generated ion gradient that is not coupled directly to the proton gradient or the proton motive force of the cell.

  6. ATP-Dependent Persister Formation in Escherichia coli

    PubMed Central

    Shan, Yue; Brown Gandt, Autumn; Rowe, Sarah E.; Deisinger, Julia P.; Conlon, Brian P.

    2017-01-01

    ABSTRACT Persisters are dormant variants that form a subpopulation of cells tolerant to antibiotics. Persisters are largely responsible for the recalcitrance of chronic infections to therapy. In Escherichia coli, one widely accepted model of persister formation holds that stochastic accumulation of ppGpp causes activation of the Lon protease that degrades antitoxins; active toxins then inhibit translation, resulting in dormant, drug-tolerant persisters. We found that various stresses induce toxin-antitoxin (TA) expression but that induction of TAs does not necessarily increase persisters. The 16S rRNA promoter rrnB P1 was proposed to be a persister reporter and an indicator of toxin activation regulated by ppGpp. Using fluorescence-activated cell sorting (FACS), we confirmed the enrichment for persisters in the fraction of rrnB P1-gfp dim cells; however, this is independent of toxin-antitoxins. rrnB P1 is coregulated by ppGpp and ATP. We show that rrnB P1 can report persisters in a relA/spoT deletion background, suggesting that rrnB P1 is a persister marker responding to ATP. Consistent with this finding, decreasing the level of ATP by arsenate treatment causes drug tolerance. Lowering ATP slows translation and prevents the formation of DNA double-strand breaks upon fluoroquinolone treatment. We conclude that variation in ATP levels leads to persister formation by decreasing the activity of antibiotic targets. PMID:28174313

  7. On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

    PubMed Central

    Birchmeier, W; Singer, SJ

    1977-01-01

    In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell. PMID:194904

  8. 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

  9. Regulation of an ATP-conductive large-conductance anion channel and swelling-induced ATP release by arachidonic acid

    PubMed Central

    Dutta, Amal K; Okada, Yasunobu; Sabirov, Ravshan Z

    2002-01-01

    Mouse mammary C127 cells responded to hypotonic stimulation with activation of the volume-dependent ATP-conductive large conductance (VDACL) anion channel and massive release of ATP. Arachidonic acid downregulated both VDACL currents and swelling-induced ATP release in the physiological concentration range with Kd of 4– 6 μm. The former effect observed in the whole-cell or excised patch mode was more prominent than the latter effect observed in intact cells. The arachidonate effects were direct and not mediated by downstream metabolic products, as evidenced by their insensitivity to inhibitors of arachidonate-metabolizing oxygenases, and by the observation that they were mimicked by cis-unsaturated fatty acids, which are not substrates for oxygenases. A membrane-impermeable analogue, arachidonyl coenzyme A was effective only from the cytosolic side of membrane patches suggesting that the binding site is localized intracellularly. Non-charged arachidonate analogues as well as trans-unsaturated and saturated fatty acids had no effect on VDACL currents and ATP release, indicating the importance of arachidonate's negative charge and specific hydrocarbon chain conformation in the inhibitory effect. VDACL anion channels were inhibited by arachidonic acid in two different ways: channel shutdown (Kd of 4– 5 μm) and reduced unitary conductance (Kd of 13–14 μm) without affecting voltage dependence of open probability. ATP4--conducting inward currents measured in the presence of 100 mm ATP in the bath were reversibly inhibited by arachidonic acid. Thus, we conclude that swelling-induced ATP release and its putative pathway, the VDACL anion channel, are under a negative control by intracellular arachidonic acid signalling in mammary C127 cells. PMID:12154180

  10. From ATP to AZD6140: the discovery of an orally active reversible P2Y12 receptor antagonist for the prevention of thrombosis.

    PubMed

    Springthorpe, Brian; Bailey, Andrew; Barton, Patrick; Birkinshaw, Timothy N; Bonnert, Roger V; Brown, Roger C; Chapman, David; Dixon, John; Guile, Simon D; Humphries, Robert G; Hunt, Simon F; Ince, Francis; Ingall, Anthony H; Kirk, Ian P; Leeson, Paul D; Leff, Paul; Lewis, Richard J; Martin, Barrie P; McGinnity, Dermot F; Mortimore, Michael P; Paine, Stuart W; Pairaudeau, Garry; Patel, Anil; Rigby, Aaron J; Riley, Robert J; Teobald, Barry J; Tomlinson, Wendy; Webborn, Peter J H; Willis, Paul A

    2007-11-01

    Starting from adenosine triphosphate (ATP), the identification of a novel series of P2Y(12) receptor antagonists and exploitation of their SAR is described. Modifications of the acidic side chain and the purine core and investigation of hydrophobic substituents led to a series of neutral molecules. The leading compound, 17 (AZD6140), is currently in a large phase III clinical trial for the treatment of acute coronary syndromes and prevention of thromboembolic clinical sequelae.

  11. Structural Basis for Substrate Binding and the Catalytic Mechanism of Type III Pantothenate Kinase

    SciTech Connect

    Yang, Kun; Strauss, Erick; Huerta, Carlos; Zhang, Hong

    2008-07-15

    Pantothenate kinase (PanK) catalyzes the first step of the universal five-step coenzyme A (CoA) biosynthetic pathway. The recently characterized type III PanK (PanK-III, encoded by the coaX gene) is distinct in sequence, structure and enzymatic properties from both the long-known bacterial type I PanK (PanK-I, exemplified by the Escherichia coli CoaA protein) and the predominantly eukaryotic type II PanK (PanK-II). PanK-III enzymes have an unusually high K{sub m} for ATP, are resistant to feedback inhibition by CoA, and are unable to utilize the N-alkylpantothenamide family of pantothenate analogues as alternative substrates, thus making type III PanK ineffective in generating CoA analogues as antimetabolites in vivo. Previously, we reported the crystal structure of the PanK-III from Thermotoga maritima and identified it as a member of the 'acetate and sugar kinase/heat shock protein 70/actin' (ASKHA) superfamily. Here we report the crystal structures of the same PanK-III in complex with one of its substrates (pantothenate), its product (phosphopantothenate) as well as a ternary complex structure of PanK-III with pantothenate and ADP. These results are combined with isothermal titration calorimetry experiments to present a detailed structural and thermodynamic characterization of the interactions between PanK-III and its substrates ATP and pantothenate. Comparison of substrate binding and catalytic sites of PanK-III with that of eukaryotic PanK-II revealed drastic differences in the binding modes for both ATP and pantothenate substrates, and suggests that these differences may be exploited in the development of new inhibitors specifically targeting PanK-III.

  12. 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

  13. ATP level and caffeine efficiency on cytokinesis inhibition in plants.

    PubMed

    López-Sáez, J F; Mingo, R; González-Fernández, A

    1982-06-01

    Plant cytokinesis appears to be a topographically organized process of exocytosis. Golgi vesicles which contain cell wall precursors are translocated during telophase, by interzonal microtubules, to the equatorial region of the mitotic apparatus where they fuse with each other giving rise to the new cell wall. Caffeine inhibits cytokinesis by hindering Golgi vesicle coalescence. The present results demonstrate that treatments which increase the cellular ATP level (adenosine, cycloheximide and anisomycin) counteract caffein-induced cytokinesis inhibition in meristem cells of onion root tips (Allium cepa L.), while treatments which decrease ATP level potentiate this caffeine effect (dinitrophenol, fluoroacetate, low oxygen tensions, etc.). We postulate that caffeine, in competition with the cellular ATP level, blocks cell plate formation by inhibiting a certain ATPase activity required for membrane fusion of Golgi vesicles.

  14. Release of Adenosine and ATP During Ischemia and Epilepsy

    PubMed Central

    Dale, Nicholas; Frenguelli, Bruno G

    2009-01-01

    Eighty years ago Drury & Szent-Györgyi described the actions of adenosine, AMP (adenylic acid) and ATP (pyrophosphoric or diphosphoric ester of adenylic acid) on the mammalian cardiovascular system, skeletal muscle, intestinal and urinary systems. Since then considerable insight has been gleaned on the means by which these compounds act, not least of which in the distinction between the two broad classes of their respective receptors, with their many subtypes, and the ensuing diversity in cellular consequences their activation invokes. These myriad actions are of course predicated on the release of the purines into the extracellular milieu, but, surprisingly, there is still considerable ambiguity as to how this occurs in various physiological and pathophysiological conditions. In this review we summarise the release of ATP and adenosine during seizures and cerebral ischemia and discuss mechanisms by which the purines adenosine and ATP may be released from cells in the CNS under these conditions. PMID:20190959

  15. Preservative efficacy screening of pharmaceutical formulations using ATP bioluminescence.

    PubMed

    Kramer, Mateja; Suklje-Debeljak, Helena; Kmetec, Vojko

    2008-05-01

    The preservative challenge test is a method used to determine the efficacy of a preservation system in a pharmaceutical or cosmetic formulation. However, such testing is a labor-intensive, repetitive task often requiring days before results can be generated. Several alternatives to traditional colony-count techniques have been developed. A study using pure suspensions of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Candida albicans, and Aspergillus niger showed that the accuracy, repeatability, and linearity of the Pallchek luminometer ATP bioluminescence (ATP-B) system was equivalent to the traditional colony-count method. In any case, the method proved sensitive enough to follow the effect of preservatives on a number of test microorganisms, indicating the applicability of the ATP-B method for preservative screening studies in various pharmaceutical formulations.

  16. Comparing the catalytic strategy of ATP hydrolysis in biomolecular motors.

    PubMed

    Kiani, Farooq Ahmad; Fischer, Stefan

    2016-07-27

    ATP-driven biomolecular motors utilize the chemical energy obtained from the ATP hydrolysis to perform vital tasks in living cells. Understanding the mechanism of enzyme-catalyzed ATP hydrolysis reaction has substantially progressed lately thanks to combined quantum/classical molecular mechanics (QM/MM) simulations. Here, we present a comparative summary of the most recent QM/MM results for myosin, kinesin and F1-ATPase motors. These completely different motors achieve the acceleration of ATP hydrolysis through a very similar catalytic mechanism. ATP hydrolysis has high activation energy because it involves the breaking of two strong bonds, namely the Pγ-Oβγ bond of ATP and the H-O bond of lytic water. The key to the four-fold decrease in the activation barrier by the three enzymes is that the breaking of the Pγ-Oβγ bond precedes the deprotonation of the lytic water molecule, generating a metaphosphate hydrate complex. The resulting singly charged trigonal planar PγO3(-) metaphosphate is a better electrophilic target for attack by an OaH(-) hydroxyl group. The formation of this OaH(-) is promoted by a strong polarization of the lytic water: in all three proteins, this water is forming a hydrogen-bond with a backbone carbonyl group and interacts with the carboxylate group of glutamate (either directly or via an intercalated water molecule). This favors the shedding of one proton by the attacking water. The abstracted proton is transferred to the γ-phosphate via various proton wires, resulting in a H2PγO4(-)/ADP(3-) product state. This catalytic strategy is so effective that most other nucleotide hydrolyzing enzymes adopt a similar approach, as suggested by their very similar triphosphate binding sites.

  17. 5 CFR 831.603 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Definitions. As used in this subpart— CSRS means subchapter III of chapter 83 of title 5, United States Code... credit deposit or redeposit under sections 8334(c) or (d) of title 5, United States Code. First regular... jurisdiction is contrary to the public policy of the United States. If a jurisdiction would recognize more...

  18. 43 CFR 10005.2 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CONSERVATION COMMISSION POLICIES AND PROCEDURES FOR DEVELOPING AND IMPLEMENTING THE COMMISSION'S MITIGATION AND CONSERVATION PLAN § 10005.2 Definitions. The Act refers to the Central Utah Project Completion Act, Titles II, III, IV, V, and VI of Public Law 102-575, October 30, 1992. Applicant refers to an...

  19. 47 CFR 1.1621 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Mass Media Services General Procedures § 1.1621 Definitions. (a) Medium of mass communications means..., (iii) An FM radio station, (iv) A direct broadcast satellite transponder under the editorial control of... preference to such entities only the other ownership interests of those with a 1% or more beneficial interest...

  20. 47 CFR 1.1803 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ..., organic brain syndrome, emotional or mental illness, and specific learning disabilities; (iii) Diseases... means any individual who has a physical or mental impairment that substantially limits one or more major... used in this definition, the phrase: (1) Physical or mental impairment includes, but is not limited to...

  1. 7 CFR 786.102 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS DAIRY DISASTER ASSISTANCE PAYMENT PROGRAM (DDAP-III) § 786.102 Definitions. The... dairy herd during each applicable disaster year. County committee means the FSA county committee....

  2. 7 CFR 786.102 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS DAIRY DISASTER ASSISTANCE PAYMENT PROGRAM (DDAP-III) § 786.102 Definitions. The... dairy herd during each applicable disaster year. County committee means the FSA county committee....

  3. 7 CFR 786.102 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS DAIRY DISASTER ASSISTANCE PAYMENT PROGRAM (DDAP-III) § 786.102 Definitions. The... dairy herd during each applicable disaster year. County committee means the FSA county committee....

  4. 7 CFR 786.102 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS DAIRY DISASTER ASSISTANCE PAYMENT PROGRAM (DDAP-III) § 786.102 Definitions. The... dairy herd during each applicable disaster year. County committee means the FSA county committee....

  5. 7 CFR 786.102 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS DAIRY DISASTER ASSISTANCE PAYMENT PROGRAM (DDAP-III) § 786.102 Definitions. The... dairy herd during each applicable disaster year. County committee means the FSA county committee....

  6. 15 CFR 2301.2 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., mobile equipment, satellite communications equipment, instructional television fixed service equipment..., optical fiber communications equipment, and other means of transmitting, emitting, storing, and receiving... PROGRAM General § 2301.2 Definitions. Act means Part IV of Title III of the Communications Act of 1934, 47...

  7. 15 CFR 2301.2 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ..., mobile equipment, satellite communications equipment, instructional television fixed service equipment..., optical fiber communications equipment, and other means of transmitting, emitting, storing, and receiving... PROGRAM General § 2301.2 Definitions. Act means Part IV of Title III of the Communications Act of 1934, 47...

  8. 15 CFR 2301.2 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ..., mobile equipment, satellite communications equipment, instructional television fixed service equipment..., optical fiber communications equipment, and other means of transmitting, emitting, storing, and receiving... PROGRAM General § 2301.2 Definitions. Act means Part IV of Title III of the Communications Act of 1934, 47...

  9. 15 CFR 2301.2 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ..., mobile equipment, satellite communications equipment, instructional television fixed service equipment..., optical fiber communications equipment, and other means of transmitting, emitting, storing, and receiving... PROGRAM General § 2301.2 Definitions. Act means Part IV of Title III of the Communications Act of 1934, 47...

  10. 15 CFR 2301.2 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., mobile equipment, satellite communications equipment, instructional television fixed service equipment..., optical fiber communications equipment, and other means of transmitting, emitting, storing, and receiving... PROGRAM General § 2301.2 Definitions. Act means Part IV of Title III of the Communications Act of 1934, 47...

  11. 21 CFR 814.3 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES PREMARKET APPROVAL OF MEDICAL DEVICES General § 814.3 Definitions. Link to an amendment published at 79 FR... a medical device. (e) PMA means any premarket approval application for a class III medical...

  12. 31 CFR 1010.605 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... foreign bank. (2) For purposes of this definition: (i) Members of the same family shall be considered to be one person. (ii) The term same family means parents, spouses, children, siblings, uncles, aunts... the foregoing. (iii) Each member of the same family who has an ownership interest in a foreign...

  13. 16 CFR 435.1 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Definitions. For purposes of this part: (a) Mail or telephone order sales shall mean sales in which the buyer... pursuant to paragraph (d)(1) or (2)(iii) of this section, a refund sent to the buyer by first class mail within seven (7) working days of the date on which the buyer's right to refund vests under the...

  14. 16 CFR 435.1 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Definitions. For purposes of this part: (a) Mail or telephone order sales shall mean sales in which the buyer... pursuant to paragraph (d)(1) or (2)(iii) of this section, a refund sent to the buyer by first class mail within seven (7) working days of the date on which the buyer's right to refund vests under the...

  15. 16 CFR 435.1 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Definitions. For purposes of this part: (a) Mail or telephone order sales shall mean sales in which the buyer... pursuant to paragraph (d)(1) or (2)(iii) of this section, a refund sent to the buyer by first class mail within seven (7) working days of the date on which the buyer's right to refund vests under the...

  16. 15 CFR 325.2 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... TRADE ADMINISTRATION, DEPARTMENT OF COMMERCE MISCELLANEOUS REGULATIONS EXPORT TRADE CERTIFICATES OF REVIEW § 325.2 Definitions. As used in this part: (a) Act means title III of Pub. L. 97-290, Export Trade...) Export conduct means specified export trade activities and methods of operation carried out in...

  17. 47 CFR 301.2 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...-ANALOG CONVERTER BOX COUPON PROGRAM § 301.2 Definitions. Act means Title III of the Deficit Reduction Act... means a seller of Coupon-Eligible Converter Boxes directly to consumers that has met the requirements... Agency to Eligible Households which only may be used to purchase a Coupon-Eligible Converter Box from...

  18. 7 CFR 1781.4 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 12 2014-01-01 2013-01-01 true Definitions. 1781.4 Section 1781.4 Agriculture... Watershed Protection and Flood Prevention Act, August 4, 1954, Public Law 83-566 as amended. (b) Resource... III of the Bankhead-Jones Farm Tenant Act (7 U.S.C. 1011). (c) Watershed plan. A plan agreed upon by...

  19. 7 CFR 1781.4 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 12 2010-01-01 2010-01-01 false Definitions. 1781.4 Section 1781.4 Agriculture... Watershed Protection and Flood Prevention Act, August 4, 1954, Public Law 83-566 as amended. (b) Resource... III of the Bankhead-Jones Farm Tenant Act (7 U.S.C. 1011). (c) Watershed plan. A plan agreed upon by...

  20. 7 CFR 1781.4 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 12 2012-01-01 2012-01-01 false Definitions. 1781.4 Section 1781.4 Agriculture... Watershed Protection and Flood Prevention Act, August 4, 1954, Public Law 83-566 as amended. (b) Resource... III of the Bankhead-Jones Farm Tenant Act (7 U.S.C. 1011). (c) Watershed plan. A plan agreed upon by...

  1. 7 CFR 1781.4 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 12 2013-01-01 2013-01-01 false Definitions. 1781.4 Section 1781.4 Agriculture... Watershed Protection and Flood Prevention Act, August 4, 1954, Public Law 83-566 as amended. (b) Resource... III of the Bankhead-Jones Farm Tenant Act (7 U.S.C. 1011). (c) Watershed plan. A plan agreed upon by...

  2. 7 CFR 1781.4 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 12 2011-01-01 2011-01-01 false Definitions. 1781.4 Section 1781.4 Agriculture... Watershed Protection and Flood Prevention Act, August 4, 1954, Public Law 83-566 as amended. (b) Resource... III of the Bankhead-Jones Farm Tenant Act (7 U.S.C. 1011). (c) Watershed plan. A plan agreed upon by...

  3. 5 CFR 892.101 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... eligible family members as follows: (i) Marriage, divorce, annulment, legal separation; (ii) Birth, adoption, acquiring a foster child that meets the definition in § 890.101(a) or a stepchild, issuance of a court order requiring an employee to provide coverage for a child; (iii) Last dependent child...

  4. 26 CFR 521.104 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 19 2014-04-01 2010-04-01 true Definitions. 521.104 Section 521.104 Internal... within the meaning of the convention and his liability to United States tax is not affected by Article... are not governed by the provisions of Article III of the convention. ...

  5. 48 CFR 225.871-2 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 3 2010-10-01 2010-10-01 false Definitions. 225.871-2... support) of certain defense articles; (ii) Concurrent production in the United States and in another member country of a defense article jointly developed; or (iii) Acquisition by the United States of a...

  6. Functional studies of ATP sulfurylase from Penicillium chrysogenum

    SciTech Connect

    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. 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.

  7. Proteomic analysis of extracellular ATP-regulated proteins identifies ATP synthase beta-subunit as a novel plant cell death regulator.

    PubMed

    Chivasa, Stephen; Tomé, Daniel F A; Hamilton, John M; Slabas, Antoni R

    2011-03-01

    Extracellular ATP is an important signal molecule required to cue plant growth and developmental programs, interactions with other organisms, and responses to environmental stimuli. The molecular targets mediating the physiological effects of extracellular ATP in plants have not yet been identified. We developed a well characterized experimental system that depletes Arabidopsis cell suspension culture extracellular ATP via treatment with the cell death-inducing mycotoxin fumonisin B1. This provided a platform for protein profile comparison between extracellular ATP-depleted cells and fumonisin B1-treated cells replenished with exogenous ATP, thus enabling the identification of proteins regulated by extracellular ATP signaling. Using two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption-time of flight MS analysis of microsomal membrane and total soluble protein fractions, we identified 26 distinct proteins whose gene expression is controlled by the level of extracellular ATP. An additional 48 proteins that responded to fumonisin B1 were unaffected by extracellular ATP levels, confirming that this mycotoxin has physiological effects on Arabidopsis that are independent of its ability to trigger extracellular ATP depletion. Molecular chaperones, cellular redox control enzymes, glycolytic enzymes, and components of the cellular protein degradation machinery were among the extracellular ATP-responsive proteins. A major category of proteins highly regulated by extracellular ATP were components of ATP metabolism enzymes. We selected one of these, the mitochondrial ATP synthase β-subunit, for further analysis using reverse genetics. Plants in which the gene for this protein was knocked out by insertion of a transfer-DNA sequence became resistant to fumonisin B1-induced cell death. Therefore, in addition to its function in mitochondrial oxidative phosphorylation, our study defines a new role for ATP synthase β-subunit as a pro-cell death

  8. Application of the Principle of Linked Functions to ATP-Driven Ion Pumps: Kinetics of Activation by ATP

    NASA Astrophysics Data System (ADS)

    Reynolds, Jacqueline A.; Johnson, Edward A.; Tanford, Charles

    1985-06-01

    If a ligand binds with unequal affinity to two distinct states of a protein, then the equilibrium between the two states becomes a function of the concentration of the ligand. A necessary consequence is that the ligand must also affect the forward and/or reverse rate constants for transition between the two states. For an enzyme or transport protein with such a transition as a slow step in the catalytic cycle, the overall rate also becomes a function of ligand concentration. These conclusions are independent of whether or not the ligand is a direct participant in the reaction. If it is a direct partitipant, then the kinetic effect arising from the principle of linked functions is distinct from the direct catalytic effect. These principles suffice to account for the biphasic response of the hydrolytic activity of ATP-driven ion pumps to the concentration of ATP, without the need to invoke more than one ATP binding site per catalytic center.

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

    PubMed Central

    James, Kimberly L.; Ríos-Hernández, Luis A.; Wofford, Neil Q.; Mouttaki, Housna; Sieber, Jessica R.; Sheik, Cody S.; Nguyen, Hong H.; Yang, Yanan; Xie, Yongming; Erde, Jonathan; Rohlin, Lars; Karr, Elizabeth A.; Loo, Joseph A.; Ogorzalek Loo, Rachel R.; Hurst, Gregory B.; Gunsalus, Robert P.; Szweda, Luke I.

    2016-01-01

    ABSTRACT Syntrophus aciditrophicus is 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 by S. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves 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 that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and 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) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. PMID:27531911

  10. [Cloning and expression of atp6 and atp9 genes from ramie (Boehmeria nivea (L.) Gaud.) and their relationship with cytoplasmic male sterility].

    PubMed

    Duan, Ji-Qiang; DU, Guang-Hui; Li, Jian-Yong; Liang, Xue-Ni; Liu, Fei-Hu

    2008-11-01

    The atp6 and apt9 gene fragments associated with cytoplasmic male sterility (CMS) were cloned from the mitochondrial DNA of a ramie (Boehmeria nivea (L.) Gaud.) cytoplasmic male sterile line and its maintainer and restorer lines using PCR and degenerated primer strategy. The primers were designed according to the reserved sequences in the encoding region of mitochondrial genes atp6 and atp9 of some dicotyledons from GenBank. These fragments did not have complete encoding region but showed the homology of 94% and 85% with atp6 and atp9 genes from the referred dicotyledons in GenBank. The complete atp6 and atp9 genes including the complete open reading frames were cloned by means of amplifying the 3' and 5'end unknown sequences of these gene fragments using DNA Walking method. The atp6 gene showed no difference among ramie male sterile line, maintainer and restorer lines at mtDNA sequence, transcription and translation control and protein level. However, compared to the maintainer and restorer lines, the atp9 gene of the male sterile line was different and deletion in several bases at the 3' end of the encoding region. An abnormally high expression of atp9 gene in the male sterile line at the budding stage and full-bloom stage was analyzed by RT-PCR analysis. These results indicated that the variation in DNA sequence and/or abnormality in expression of atp9 gene in the male sterile line maybe closely related to ramie CMS.

  11. Extracellular ATP signaling via P2X(4) receptor and cAMP/PKA signaling mediate ATP oscillations essential for prechondrogenic condensation.

    PubMed

    Kwon, Hyuck Joon

    2012-09-01

    Prechondrogenic condensation is the most critical process in skeletal patterning. A previous study demonstrated that ATP oscillations driven by Ca(2+) oscillations play a critical role in prechondrogenic condensation by inducing oscillatory secretion. However, it remains unknown what mechanisms initiate the Ca(2+)-driven ATP oscillations, mediate the link between Ca(2+) and ATP oscillations, and then result in oscillatory secretion in chondrogenesis. This study has shown that extracellular ATP signaling was required for both ATP oscillations and prechondrogenic condensation. Among P2 receptors, the P2X(4) receptor revealed the strongest expression level and mediated ATP oscillations in chondrogenesis. Moreover, blockage of P2X(4) activity abrogated not only chondrogenic differentiation but also prechondrogenic condensation. In addition, both ATP oscillations and secretion activity depended on cAMP/PKA signaling but not on K(ATP) channel activity and PKC or PKG signaling. This study proposes that Ca(2+)-driven ATP oscillations essential for prechondrogenic condensation is initiated by extracellular ATP signaling via P2X(4) receptor and is mediated by cAMP/PKA signaling and that cAMP/PKA signaling induces oscillatory secretion to underlie prechondrogenic condensation, in cooperation with Ca(2+) and ATP oscillations.

  12. Structural changes during ATP hydrolysis activity of the ATP synthase from Escherichia coli as revealed by fluorescent probes.

    PubMed

    Turina, P

    2000-08-01

    F1F0-ATPase complexes undergo several changes in their tertiary and quaternary structure during their functioning. As a possible way to detect some of these different conformations during their activity, an environment-sensitive fluorescence probe was bound to cysteine residues, introduced by site-directed mutagenesis, in the gamma subunit of the Escherichia coli enzyme. Fluorescence changes and ATP hydrolysis rates were compared under various conditions in F1 and in reconstituted F1F0. The results are discussed in terms of possible modes of operation of the ATP synthases.

  13. Design and development of robust ATP subsystem for the Altair UAV-to-ground lasercomm 2.5-Gbps demonstration

    NASA Astrophysics Data System (ADS)

    Ortiz, Gerardo G.; Lee, Shinhak; Monacos, Steve P.; Wright, Malcolm W.; Biswas, Abhijit

    2003-07-01

    A robust acquisition, tracking and pointing (ATP) subsystem is being developed for the 2.5 Gigabit per second (Gbps) Unmanned-Aerial-Vehicle (UAV) to ground free-space optical communications link project. The demonstration will gather HDTV images of regions of geological interest (e.g. volcanic) and then downlink those images to ground receivers at a range of 50 km while the UAV is at an altitude of 18 km. With a 200 mW downlink laser at 1550 nm for a BER of 1E-9, the pointing requirements on the flight terminal are a jitter error of 19.5 urad and a bias error of 14.5 urad with a probability of pointing induced fades of 0.1 %. In order to mitigate the effect of atmospheric fades and deal with UAV flight and vibration uncertainties (relatively new craft) the ATP subsystem requirements have been set to a stringent level in order to assure success of the communication link. The design, analysis and development of this robust ATP subsystem will be described in this paper. The key innovations that have been developed to make the ATP subsystem robust are i) the application of inertial sensors to make the acquisition and tracking functions tolerant to atmospheric fades, ii) the usage of active exposure control to provide a 16 dB dynamic range on the Focal Plane Array (FPA) tracking window, and iii) the introduction of a second ultra wide field of view camera to assure acquisition of the ground beacon.

  14. The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase

    PubMed Central

    Ding, Hao; Guo, Manhong; Vidhyasagar, Venkatasubramanian; Talwar, Tanu; Wu, Yuliang

    2015-01-01

    Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI). The Q motif, consisting of nine amino acids (GFXXPXPIQ) with an invariant glutamine (Q) residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase. PMID:26474416

  15. Calcium-induced conformational changes in the regulatory domain of the human mitochondrial ATP-Mg/Pi carrier

    PubMed Central

    Harborne, Steven P.D.; Ruprecht, Jonathan J.; Kunji, Edmund R.S.

    2015-01-01

    The mitochondrial ATP-Mg/Pi carrier imports adenine nucleotides from the cytosol into the mitochondrial matrix and exports phosphate. The carrier is regulated by the concentration of cytosolic calcium, altering the size of the adenine nucleotide pool in the mitochondrial matrix in response to energetic demands. The protein consists of three domains; (i) the N-terminal regulatory domain, which is formed of two pairs of fused calcium-binding EF-hands, (ii) the C-terminal mitochondrial carrier domain, which is involved in transport, and (iii) a linker region with an amphipathic α-helix of unknown function. The mechanism by which calcium binding to the regulatory domain modulates substrate transport in the carrier domain has not been resolved. Here, we present two new crystal structures of the regulatory domain of the human isoform 1. Careful analysis by SEC confirmed that although the regulatory domain crystallised as dimers, full-length ATP-Mg/Pi carrier is monomeric. Therefore, the ATP-Mg/Pi carrier must have a different mechanism of calcium regulation than the architecturally related aspartate/glutamate carrier, which is dimeric. The structure showed that an amphipathic α-helix is bound to the regulatory domain in a hydrophobic cleft of EF-hand 3/4. Detailed bioinformatics analyses of different EF-hand states indicate that upon release of calcium, EF-hands close, meaning that the regulatory domain would release the amphipathic α-helix. We propose a mechanism for ATP-Mg/Pi carriers in which the amphipathic α-helix becomes mobile upon release of calcium and could block the transport of substrates across the mitochondrial inner membrane. PMID:26164100

  16. ATP Interior Noise Technology and Flight Demonstration Program

    NASA Technical Reports Server (NTRS)

    Stephens, David G.; Powell, Clemans A.

    1988-01-01

    The paper provides an overview of the ATP (Advanced Turboprop Program) acoustics program with emphasis on the NASA technology program and the recent NASA/Industry demonstration programs aimed at understanding and controlling passenger cabin noise. Technology developments in propeller (source) noise, cabin noise transmission, and subjective acoustics are described. Finally, an overview of the industry demonstrator programs is presented.

  17. Detection of ATP and NADH: A Bioluminescent Experience.

    ERIC Educational Resources Information Center

    Selig, Ted C.; And Others

    1984-01-01

    Described is a bioluminescent assay for adenosine triphosphate (ATP) and reduced nicotineamide-adenine dinucleotide (NADH) that meets the requirements of an undergraduate biochemistry laboratory course. The 3-hour experiment provides students with experience in bioluminescence and analytical biochemistry yet requires limited instrumentation,…

  18. ATP-induced noncooperative thermal unfolding of hen lysozyme

    SciTech Connect

    Liu, Honglin; Yin, Peidong; He, Shengnan; Sun, Zhihu; Tao, Ye; Huang, Yan; Zhuang, Hao; Zhang, Guobin; Wei, Shiqiang

    2010-07-02

    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 noncooperative 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.

  19. Cyclodextrin-based microcapsules as bioreactors for ATP biosynthesis.

    PubMed

    Li, Jian-Hu; Wang, Yi-Fu; Ha, Wei; Liu, Yan; Ding, Li-Sheng; Li, Bang-Jing; Zhang, Sheng

    2013-09-09

    A biomimetic energy converter was fabricated via the assembly of CF0F1-ATPase on lipid-coated hollow nanocapsules composed of α-cyclodextrins/chitosan-graft-poly(ethylene glycol) methacrylate. Upon entrapped GOD into these capsules, the addition of glucose could trigger proton-motive force and then drive the rotation of ATPase to synthesize ATP.

  20. ATP Binding Turns Plant Cryptochrome Into an Efficient Natural Photoswitch

    NASA Astrophysics Data System (ADS)

    Müller, Pavel; Bouly, Jean-Pierre; Hitomi, Kenichi; Balland, Véronique; Getzoff, Elizabeth D.; Ritz, Thorsten; Brettel, Klaus

    2014-06-01

    Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH. radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD.-, from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396-. Its negative charge could trigger conformational changes necessary for signal transduction.

  1. ATP drives direct photosynthetic production of 1-butanol in cyanobacteria

    PubMed Central

    Lan, Ethan I.; Liao, James C.

    2012-01-01

    While conservation of ATP is often a desirable trait for microbial production of chemicals, we demonstrate that additional consumption of ATP may be beneficial to drive product formation in a nonnatural pathway. Although production of 1-butanol by the fermentative coenzyme A (CoA)-dependent pathway using the reversal of β-oxidation exists in nature and has been demonstrated in various organisms, the first step of the pathway, condensation of two molecules of acetyl-CoA to acetoacetyl-CoA, is thermodynamically unfavorable. Here, we show that artificially engineered ATP consumption through a pathway modification can drive this reaction forward and enables for the first time the direct photosynthetic production of 1-butanol from cyanobacteria Synechococcus elongatus PCC 7942. We further demonstrated that substitution of bifunctional aldehyde/alcohol dehydrogenase (AdhE2) with separate butyraldehyde dehydrogenase (Bldh) and NADPH-dependent alcohol dehydrogenase (YqhD) increased 1-butanol production by 4-fold. These results demonstrated the importance of ATP and cofactor driving forces as a design principle to alter metabolic flux. PMID:22474341

  2. Teacher Development Program for ATP 2000. Project Report.

    ERIC Educational Resources Information Center

    Sutphin, Dean; And Others

    Agri Tech Prep 2000 (ATP 2000) is a 4-year tech prep program linking high school and postsecondary curricula designed to prepare New York students for careers in agriculture or acceptance into a college program in agriculture. Because teacher development was designated an integral project component for fiscal year 1991-1992, a weeklong teacher…

  3. 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.

  4. Detection of ATP and NADH: A Bioluminescent Experience.

    ERIC Educational Resources Information Center

    Selig, Ted C.; And Others

    1984-01-01

    Described is a bioluminescent assay for adenosine triphosphate (ATP) and reduced nicotineamide-adenine dinucleotide (NADH) that meets the requirements of an undergraduate biochemistry laboratory course. The 3-hour experiment provides students with experience in bioluminescence and analytical biochemistry yet requires limited instrumentation,…

  5. 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)

  6. ATP-enhanced peroxidase-like activity of gold nanoparticles.

    PubMed

    Shah, Juhi; Purohit, Rahul; Singh, Ragini; Karakoti, Ajay Singh; Singh, Sanjay

    2015-10-15

    Gold nanoparticles (AuNPs) are known to possess intrinsic biological peroxidase-like activity that has applications in development of numerous biosensors. The reactivity of the Au atoms at the surface of AuNPs is critical to the performance of such biosensors, yet little is known about the effect of biomolecules and ions on the peroxidase-like activity. In this work, the effect of ATP and other biologically relevant molecules and ions over peroxidase-like activity of AuNPs are described. Contrary to the expectation that nanoparticles exposed to biomolecules may lose the catalytic property, ATP and ADP addition enhanced the peroxidase-like activity of AuNPs. The catalytic activity was unaltered by the addition of free phosphate, sulphate and carbonate anions however, addition of ascorbic acid to the reaction mixture diminished the intrinsic peroxidase-like activity of AuNPs, even in the presence of ATP and ADP. In contrast to AuNPs, ATP did not synergize and improve the peroxidase activity of the natural peroxidase enzyme, horseradish peroxidase.

  7. Animation Model to Conceptualize ATP Generation: A Mitochondrial Oxidative Phosphorylation

    ERIC Educational Resources Information Center

    Jena, Ananta Kumar

    2015-01-01

    Adenosine triphosphate (ATP) is the molecular unit of intracellular energy and it is the product of oxidative phosphorylation of cellular respiration uses in cellular processes. The study explores the growth of the misconception levels amongst the learners and evaluates the effectiveness of animation model over traditional methods. The data…

  8. ATP Binding Turns Plant Cryptochrome Into an Efficient Natural Photoswitch

    PubMed Central

    Müller, Pavel; Bouly, Jean-Pierre; Hitomi, Kenichi; Balland, Véronique; Getzoff, Elizabeth D.; Ritz, Thorsten; Brettel, Klaus

    2014-01-01

    Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH· radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD·−, from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396−. Its negative charge could trigger conformational changes necessary for signal transduction. PMID:24898692

  9. Abiogenic Photophosphorylation of ADP to ATP Sensitized by Flavoproteinoid Microspheres

    NASA Astrophysics Data System (ADS)

    Kolesnikov, Michael P.; Telegina, Taisiya A.; Lyudnikova, Tamara A.; Kritsky, Mikhail S.

    2008-06-01

    A model for abiogenic photophosphorylation of ADP by orthophosphate to yield ATP was studied. The model is based on the photochemical activity of flavoproteinoid microspheres that are formed by aggregation in an aqueous medium of products of thermal condensation of a glutamic acid, glycine and lysine mixture (8:3:1) and contain, along with amino acid polymers (proteinoids), abiogenic isoalloxazine (flavin) pigments. Irradiation of aqueous suspensions of microspheres with blue visible light or ultraviolet in the presence of ADP and orthophosphate resulted in ATP formation. The yield of ATP in aerated suspensions was 10 20% per one mol of starting ADP. Deaeration reduced the photophosphorylating activity of microspheres five to 10 times. Treatment of aerated microsphere suspensions with superoxide dismutase during irradiation partially suppressed ATP formation. Deaerated microspheres restored completely their photophosphorylating activity after addition of hydrogen peroxide to the suspension. The photophosphorylating activity of deaerated suspensions of flavoproteinoid microspheres was also recovered by introduction of Fe3+-cytochrome c, an electron acceptor alternative to oxygen. On the basis of the results obtained, a chemical mechanism of phosphorylation is proposed in which the free radical form of reduced flavin sensitizer left( {{text{FlH}}^ bullet } right) and ADP are involved.

  10. Characterization of ATP citrate lyase from Chlorobium limicola.

    PubMed Central

    Antranikian, G; Herzberg, C; Gottschalk, G

    1982-01-01

    ATP citrate lyase (EC 4.1.3.8) from Chlorobium limicola was partially purified. It was established that the consumption of substrates and the formation of products proceeded stoichiometrically and that citrate cleavage was of the si-type. ADP and oxaloacetate inhibited enzyme activity. Oxaloacetate also inhibited the growth of C. limicola. PMID:7142107

  11. IV ATP potentiates midazolam sedation as assessed by bispectral index.

    PubMed

    Sakurai, Satoru; Fukunaga, Atsuo; Ichinohe, Tatsuya; Kaneko, Yuzuru

    2014-01-01

    In this study, by measuring bispectral index (BIS), we tested the hypothesis that intravenous adenosine 5'-triphosphate (ATP) infusion would deepen the level of midazolam-induced sedation. Ten healthy volunteers underwent 2 experiments with at least 2 weeks' interval: immediately after intravenous bolus administration of midazolam (0.04 mg/kg), they received continuous infusion of either ATP infusion (100 μg/kg/min) or placebo (saline) for 40 minutes in a double-blind, randomized, crossover manner. Changes in BIS values and responsiveness to verbal command as well as cardiorespiratory variables were observed throughout the study periods. Administration of midazolam alone reduced BIS value from control: 97 ± 1 to 68 ± 18 at 25 minutes, which was accompanied by significant cardiopulmonary depressant effects, while maintaining responsiveness to verbal command (consciousness) throughout the study period. Coadministration of ATP with midazolam further reduced BIS value to 51 ± 13, associated with complete loss of consciousness without adverse effect on the cardiorespiratory systems. We conclude that the addition of ATP infusion to midazolam significantly enhances midazolam sedation without disturbing cardiorespiratory functions.

  12. ATP-association to intrabacterial nanotransportation system in Vibrio cholerae.

    PubMed

    Matsuzaki, Yuji; Wu, Hong; Nakano, Takashi; Nakahari, Takashi; Sano, Kouichi

    2015-12-01

    Vibrio cholerae colonizes the lumen of the proximal small intestine, which has an alkaline environment, and secretes cholera toxin (CT) through a type II secretion machinery. V. cholerae possesses the intrabacterial nanotransportation system (ibNoTS) for transporting CT from the inner portion toward the peripheral portion of the cytoplasm, and this system is controlled by extrabacterial pH. Association of ATP with ibNoTS has not yet been examined in detail. In this study, we demonstrated by immunoelectron microscopy that ibNoTS of V. cholerae under the extrabacterial alkaline condition was inhibited by ATP inhibitors, 2,4-dinitrophenol (DNP), a protonophore, or 8-amino-adenosine which produces inactive form of ATP. The inhibition of CT transport can be reversed by neutralization of DNP. Those inhibitions were associated with decrease of CT secretion by which ibNoTS followed. We propose that ATP closely associates with V. cholerae ibNoTS for transporting CT.

  13. ATP stimulates calcium influx in primary astrocyte cultures

    SciTech Connect

    Neary, J.T.; van Breemen, C.; Forster, E.; Norenberg, L.O.; Norenberg, M.D.

    1988-12-30

    The effect of ATP and other purines on /sup 45/Ca uptake was studied in primary cultures of rat astrocytes. Treatment of the cells with ATP for 1 to 30 min brought about an increase in cellular /sup 45/Ca. Stimulation of calcium influx by ATP was investigated using a 90 sec exposure to /sup 45/Ca and over a concentration range of 0.1 nM to 3 mM; a biphasic dose-response curve was obtained with EC50 values of 0.3 nM and 9 uM, indicating the presence of low and high affinity purinergic binding sites. Similar levels of /sup 45/Ca influx at 90 sec were observed with ATP, ADP and adenosine (all at 100 uM). Prior treatment of the cultures with LaCl3 blocked the purine-induced /sup 45/Ca influx. These findings indicate that one pathway for calcium entry in astrocytes involves purinergic receptor-operated, calcium channels.

  14. ATP drives direct photosynthetic production of 1-butanol in cyanobacteria.

    PubMed

    Lan, Ethan I; Liao, James C

    2012-04-17

    While conservation of ATP is often a desirable trait for microbial production of chemicals, we demonstrate that additional consumption of ATP may be beneficial to drive product formation in a nonnatural pathway. Although production of 1-butanol by the fermentative coenzyme A (CoA)-dependent pathway using the reversal of β-oxidation exists in nature and has been demonstrated in various organisms, the first step of the pathway, condensation of two molecules of acetyl-CoA to acetoacetyl-CoA, is thermodynamically unfavorable. Here, we show that artificially engineered ATP consumption through a pathway modification can drive this reaction forward and enables for the first time the direct photosynthetic production of 1-butanol from cyanobacteria Synechococcus elongatus PCC 7942. We further demonstrated that substitution of bifunctional aldehyde/alcohol dehydrogenase (AdhE2) with separate butyraldehyde dehydrogenase (Bldh) and NADPH-dependent alcohol dehydrogenase (YqhD) increased 1-butanol production by 4-fold. These results demonstrated the importance of ATP and cofactor driving forces as a design principle to alter metabolic flux.

  15. Photoaffinity labeling of ribulose-1,5-bisphosphate carboxylase/oxygenase activase with ATP gamma-benzophenone. Identification of the ATP gamma-phosphate binding domain.

    PubMed

    Salvucci, M E; Rajagopalan, K; Sievert, G; Haley, B E; Watt, D S

    1993-07-05

    The phosphate-binding domain of the ATP-binding site of tobacco Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activase was elucidated by photo-affinity labeling with a monoanhydride of ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([gamma-32P]ATP gamma BP). Covalent incorporation of [gamma-32P]ATP gamma BP into the 42-kDa Rubisco activase subunit was dependent upon irradiation with ultraviolet light. Photolabelling of Rubisco activase with ATP gamma BP exhibited saturation kinetics; the apparent Kd for photolabeling was 5 microM. Two lines of evidence showed that ATP gamma BP modified Rubisco activase at the ATP-binding domain. First, physiological concentrations of ATP and ADP afforded complete protection against photolabeling of Rubisco activase by ATP gamma BP. Second, photolysis of Rubisco activase in the presence of ATP gamma BP decreased both the ATPase and the Rubisco activating activities. Inactivation of enzyme activity was dependent on ATP gamma BP concentration and could be prevented by including ADP during photolabeling. The region of Rubisco activase that was modified by ATP gamma BP was identified by isolating photolabeled peptides. Sequence analysis showed that ATP gamma BP modified Rubisco activase in two distinct regions; one region, S117-A136, is adjacent to the P-loop and the other region, V223-T234, exhibits homology to a region of adenylate kinase that ligates the essential metal ion. Photolabeling of these two regions of Rubisco activase was consistent with modification of the ATP gamma-phosphate-binding domain of Rubisco activase with ATP gamma BP.

  16. A Fluorescent, Reagentless Biosensor for ATP, Based on Malonyl-Coenzyme A Synthetase

    PubMed Central

    2015-01-01

    A fluorescent reagentless biosensor for ATP has been developed, based on malonyl-coenzyme A synthetase from Rhodopseudomonas palustris as the protein scaffold and recognition element. Two 5-iodoacetamidotetramethylrhodamines were covalently bound to this protein to provide the readout. This adduct couples ATP binding to a 3.7-fold increase in fluorescence intensity with excitation at 553 nm and emission at 575 nm. It measures ATP concentrations with micromolar sensitivity and is highly selective for ATP relative to ADP. Its ability to monitor enzymatic ATP production or depletion was demonstrated in steady-state kinetic assays in which ATP is a product or substrate, respectively. PMID:26355992

  17. Online damage inspection of optics for ATP system

    NASA Astrophysics Data System (ADS)

    Chen, Jing; Jiang, Yu; Mao, Yao; Gan, Xun; Liu, Qiong

    2016-09-01

    In the Electro-Optical acquisition-tracking-pointing system (ATP), the optical components will be damaged with the several influencing factors. In this situation, the rate will increase sharply when the arrival of damage to some extent. As the complex processing techniques and long processing cycle of optical components, the damage will cause the great increase of the system development cost and cycle. Therefore, it is significant to detect the laser-induced damage in the ATP system. At present, the major research on the on-line damage detection technology of optical components is for the large optical system in the international. The relevant detection systems have complicated structures and many of components, and require enough installation space reserved, which do not apply for ATP system. To solve the problem mentioned before, This paper use a method based on machine vision to detect the damage on-line for the present ATP system. To start with, CCD and PC are used for image acquisition. Secondly, smoothing filters are used to restrain false damage points produced by noise. Then, with the shape feature included in the damage image, the OTSU Method which can define the best segmentation threshold automatically is used to achieve the goal to locate the damage regions. At last, we can supply some opinions for the lifetime of the optical components by analyzing the damage data, such as damage area, damage position. The method has the characteristics of few-detectors and simple-structures which can be installed without any changes of the original light path. With the method, experimental results show that it is stable and effective to achieve the goal of detecting the damage of optical components on-line in the ATP system.

  18. Activated sludge optimization using ATP in pulp and paper industry.

    PubMed

    Bäckman, Göran; Gytel, Ulla

    2015-01-01

    The activated sludge process is an old technology, but still the most commonly used one for treatment of wastewater. Despite the wide spread usage the technology still suffers from instability (Tandoi et al. 2006) and high operating cost. Activated sludge processes often carry a large solids inventory. Managing the total inventory without interference is the key component of the optimization process described in this paper. Use of nutrients is common in pulp and paper effluent treatment. Feeding enough nutrients to support the biomass growth is a delicate balance. Overfeeding or underfeeding of nutrients can result in higher costs. Detrimental substances and toxic components in effluents entering a biological treatment system can cause severe, long lasting disturbances (Hynninen & Ingman 1998; Bergeron & Pelletier 2004). A LumiKem test kit is used to measure biological activity with adenosine triphosphate (ATP) in a pulp and paper mill. ATP data are integrated with other standardized mill parameters. Measurements of active volatile suspended solids based on ATP can be used to quantify the living biomass in the activated sludge process and to ensure that sufficient biomass is present in order to degrade the wastewater constituents entering the process. Information about active biomass will assist in optimizing sludge inventories and feeding of nutrients allowing the living biomass to re-populate to create optimal efficiency. ATP measurements can also be used to alert operators if any components toxic to bacteria are present in wastewater. The bio stress index represents the stress level experienced by the microbiological population. This parameter is very useful in monitoring toxicity in and around bioreactors. Results from the wastewater process optimization and ATP measurements showed that treatment cost could be reduced by approximately 20-30% with fewer disturbances and sustained biological activity compared to the reference period. This was mainly achieved by

  19. Cofilin mediates ATP depletion-induced endothelial cell actin alterations.

    PubMed

    Suurna, Maria V; Ashworth, Sharon L; Hosford, Melanie; Sandoval, Ruben M; Wean, Sarah E; Shah, Bijal M; Bamburg, James R; Molitoris, Bruce A

    2006-06-01

    Ischemia and sepsis lead to endothelial cell damage, resulting in compromised microvascular flow in many organs. Much remains to be determined regarding the intracellular structural events that lead to endothelial cell dysfunction. To investigate potential actin cytoskeletal-related mechanisms, ATP depletion was induced in mouse pancreatic microvascular endothelial cells (MS1). Fluorescent imaging and biochemical studies demonstrated a rapid and progressive increase in F-actin along with a decrease in G-actin at 60 min. Confocal microscopic analysis showed ATP depletion resulted in destruction of actin stress fibers and accumulation of F-actin aggregates. We hypothesized these actin alterations were secondary to dephosphorylation/activation of actin-depolymerizing factor (ADF)/cofilin proteins. Cofilin, the predominant isoform expressed in MS1 cells, was rapidly dephosphorylated/activated during ATP depletion. To directly investigate the role of cofilin activation on the actin cytoskeleton during ischemia, MS1 cells were infected with adenoviruses containing the cDNAs for wild-type Xenopus laevis ADF/cofilin green fluorescent protein [XAC(wt)-GFP], GFP, and the constitutively active and inactive isoforms XAC(S3A)-GFP and XAC(S3E)-GFP. The rate and extent of cortical actin destruction and actin aggregate formation were increased in ATP-depleted XAC(wt)-GFP- and XAC(S3A)-GFP-expressing cells, whereas increased actin stress fibers were observed in XAC(S3E)-GFP-expressing cells. To investigate the upstream signaling pathway of ADF/cofilin, LIM kinase 1-GFP (LIMK1-GFP) was expressed in MS1 cells. Cells expressing LIMK1-GFP protein had higher levels of phosphorylated ADF/cofilin, increased stress fibers, and delayed F-actin cytoskeleton destruction during ATP depletion. These results strongly support the importance of cofilin regulation in ischemia-induced endothelial cell actin cytoskeleton alterations leading to cell damage and microvascular dysfunction.

  20. Asymmetric ring structure of Vps4 required for ESCRT-III disassembly

    PubMed Central

    Caillat, Christophe; Macheboeuf, Pauline; Wu, Yuanfei; McCarthy, Andrew A.; Boeri-Erba, Elisabetta; Effantin, Gregory; Göttlinger, Heinrich G.; Weissenhorn, Winfried; Renesto, Patricia

    2015-01-01

    The vacuolar protein sorting 4 AAA–ATPase (Vps4) recycles endosomal sorting complexes required for transport (ESCRT-III) polymers from cellular membranes. Here we present a 3.6-Å X-ray structure of ring-shaped Vps4 from Metallosphera sedula (MsVps4), seen as an asymmetric pseudohexamer. Conserved key interface residues are shown to be important for MsVps4 assembly, ATPase activity in vitro, ESCRT-III disassembly in vitro and HIV-1 budding. ADP binding leads to conformational changes within the protomer, which might propagate within the ring structure. All ATP-binding sites are accessible and the pseudohexamer binds six ATP with micromolar affinity in vitro. In contrast, ADP occupies one high-affinity and five low-affinity binding sites in vitro, consistent with conformational asymmetry induced on ATP hydrolysis. The structure represents a snapshot of an assembled Vps4 conformation and provides insight into the molecular motions the ring structure undergoes in a concerted action to couple ATP hydrolysis to ESCRT-III substrate disassembly. PMID:26632262

  1. Asymmetric ring structure of Vps4 required for ESCRT-III disassembly

    NASA Astrophysics Data System (ADS)

    Caillat, Christophe; Macheboeuf, Pauline; Wu, Yuanfei; McCarthy, Andrew A.; Boeri-Erba, Elisabetta; Effantin, Gregory; Göttlinger, Heinrich G.; Weissenhorn, Winfried; Renesto, Patricia

    2015-12-01

    The vacuolar protein sorting 4 AAA-ATPase (Vps4) recycles endosomal sorting complexes required for transport (ESCRT-III) polymers from cellular membranes. Here we present a 3.6-Å X-ray structure of ring-shaped Vps4 from Metallosphera sedula (MsVps4), seen as an asymmetric pseudohexamer. Conserved key interface residues are shown to be important for MsVps4 assembly, ATPase activity in vitro, ESCRT-III disassembly in vitro and HIV-1 budding. ADP binding leads to conformational changes within the protomer, which might propagate within the ring structure. All ATP-binding sites are accessible and the pseudohexamer binds six ATP with micromolar affinity in vitro. In contrast, ADP occupies one high-affinity and five low-affinity binding sites in vitro, consistent with conformational asymmetry induced on ATP hydrolysis. The structure represents a snapshot of an assembled Vps4 conformation and provides insight into the molecular motions the ring structure undergoes in a concerted action to couple ATP hydrolysis to ESCRT-III substrate disassembly.

  2. Phospholipid Flippase ATP10A Translocates Phosphatidylcholine and Is Involved in Plasma Membrane Dynamics*

    PubMed Central

    Naito, Tomoki; Takatsu, Hiroyuki; Miyano, Rie; Takada, Naoto; Nakayama, Kazuhisa; Shin, Hye-Won

    2015-01-01

    We showed previously that ATP11A and ATP11C have flippase activity toward aminophospholipids (phosphatidylserine (PS) and phosphatidylethanolamine (PE)) and ATP8B1 and that ATP8B2 have flippase activity toward phosphatidylcholine (PC) (Takatsu, H., Tanaka, G., Segawa, K., Suzuki, J., Nagata, S., Nakayama, K., and Shin, H. W. (2014) J. Biol. Chem. 289, 33543–33556). Here, we show that the localization of class 5 P4-ATPases to the plasma membrane (ATP10A and ATP10D) and late endosomes (ATP10B) requires an interaction with CDC50A. Moreover, exogenous expression of ATP10A, but not its ATPase-deficient mutant ATP10A(E203Q), dramatically increased PC flipping but not flipping of PS or PE. Depletion of CDC50A caused ATP10A to be retained at the endoplasmic reticulum instead of being delivered to the plasma membrane and abrogated the increased PC flipping activity observed by expression of ATP10A. These results demonstrate that ATP10A is delivered to the plasma membrane via its interaction with CDC50A and, specifically, flips PC at the plasma membrane. Importantly, expression of ATP10A, but not ATP10A(E203Q), dramatically altered the cell shape and decreased cell size. In addition, expression of ATP10A, but not ATP10A(E203Q), delayed cell adhesion and cell spreading onto the extracellular matrix. These results suggest that enhanced PC flipping activity due to exogenous ATP10A expression alters the lipid composition at the plasma membrane, which may in turn cause a delay in cell spreading and a change in cell morphology. PMID:25947375

  3. Negative feedback of extracellular ADP on ATP release in goldfish hepatocytes: a theoretical study.

    PubMed

    Chara, Osvaldo; Pafundo, Diego E; Schwarzbaum, Pablo J

    2010-06-21

    A mathematical model was built to account for the kinetic of extracellular ATP (ATPe) and extracellular ADP (ADPe) concentrations from goldfish hepatocytes exposed to hypotonicity. The model was based on previous experimental results on the time course of ATPe accumulation, ectoATPase activity, and cell viability [Pafundo et al., 2008]. The kinetic of ATPe is controlled by a lytic ATP flux, a non-lytic ATP flux, and ecto-ATPase activity, whereas ADPe kinetic is governed by a lytic ADP flux and both ecto-ATPase and ecto-ADPase activities. Non-lytic ATPe efflux was included as a diffusion equation modulated by ATPe activation (positive feedback) and ADPe inhibition (negative feedback). The model yielded physically meaningful and stable steady-state solutions, was able to fit the experimental time evolution of ATPe and simulated the concomitant kinetic of ADPe. According to the model during the first minute of hypotonicity the concentration of ATPe is mainly governed by both lytic and non-lytic ATP efflux, with almost no contribution from ecto-ATPase activity. Later on, ecto-ATPase activity becomes important in defining the time dependent decay of ATPe levels. ADPe inhibition of the non-lytic ATP efflux was strong, whereas ATPe activation was minimal. Finally, the model was able to predict the consequences of partial inhibition of ecto-ATPase activity on the ATPe kinetic, thus emulating the exposure of goldfish cells to hypotonic medium in the presence of the ATP analog AMP-PCP. The model predicts this analog to both inhibit ectoATPase activity and increase non-lytic ATP release.

  4. How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

    NASA Astrophysics Data System (ADS)

    Dudev, Todor; Grauffel, Cédric; Lim, Carmay

    2017-02-01

    Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways.

  5. Mitochondrial ATP synthase activity is impaired by suppressed O-GlcNAcylation in Alzheimer's disease

    PubMed Central

    Cha, Moon-Yong; Cho, Hyun Jin; Kim, Chaeyoung; Jung, Yang Ouk; Kang, Min Jueng; Murray, Melissa E.; Hong, Hyun Seok; Choi, Young-Joo; Choi, Heesun; Kim, Dong Kyu; Choi, Hyunjung; Kim, Jisoo; Dickson, Dennis W.; Song, Hyun Kyu; Cho, Jin Won; Yi, Eugene C.; Kim, Jungsu; Jin, Seok Min; Mook-Jung, Inhee

    2015-01-01

    Glycosylation with O-linked β-N-acetylglucosamine (O-GlcNAc) is one of the protein glycosylations affecting various intracellular events. However, the role of O-GlcNAcylation in neurodegenerative diseases such as Alzheimer's disease (AD) is poorly understood. Mitochondrial adenosine 5′-triphosphate (ATP) synthase is a multiprotein complex that synthesizes ATP from ADP and Pi. Here, we found that ATP synthase subunit α (ATP5A) was O-GlcNAcylated at Thr432 and ATP5A O-GlcNAcylation was decreased in the brains of AD patients and transgenic mouse model, as well as Aβ-treated cells. Indeed, Aβ bound to ATP synthase directly and reduced the O-GlcNAcylation of ATP5A by inhibition of direct interaction between ATP5A and mitochondrial O-GlcNAc transferase, resulting in decreased ATP production and ATPase activity. Furthermore, treatment of O-GlcNAcase inhibitor rescued the Aβ-induced impairment in ATP production and ATPase activity. These results indicate that Aβ-mediated reduction of ATP synthase activity in AD pathology results from direct binding between Aβ and ATP synthase and inhibition of O-GlcNAcylation of Thr432 residue on ATP5A. PMID:26358770

  6. Evidence for Extracellular ATP as a Stress Signal in a Single-Celled Organism

    PubMed Central

    Sivaramakrishnan, Venketesh

    2015-01-01

    ATP is omnipresent in biology and acts as an extracellular signaling molecule in mammals. Information regarding the signaling function of extracellular ATP in single-celled eukaryotes is lacking. Here, we explore the role of extracellular ATP in cell volume recovery during osmotic swelling in the amoeba Dictyostelium. Release of micromolar ATP could be detected during cell swelling and regulatory cell volume decrease (RVD) phases during hypotonic challenge. Scavenging ATP with apyrase caused profound cell swelling and loss of RVD. Apyrase-induced swelling could be rescued by 100 μM βγ-imidoATP. N-Ethylmalemide (NEM), an inhibitor of vesicular exocytosis, caused heightened cell swelling, loss of RVD, and inhibition of ATP release. Amoebas with impaired contractile vacuole (CV) fusion (drainin knockout [KO] cells) displayed increased swelling but intact ATP release. One hundred micromolar Gd3+ caused cell swelling while blocking any recovery by βγ-imidoATP. ATP release was 4-fold higher in the presence of Gd3+. Cell swelling was associated with an increase in intracellular nitric oxide (NO), with NO-scavenging agents causing cell swelling. Swelling-induced NO production was inhibited by both apyrase and Gd3+, while NO donors rescued apyrase- and Gd3+-induced swelling. These data suggest extracellular ATP released during cell swelling is an important signal that elicits RVD. Though the cell surface receptor for ATP in Dictyostelium remains elusive, we suggest ATP operates through a Gd3+-sensitive receptor that is coupled with intracellular NO production. PMID:26048010

  7. 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.

  8. 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.

  9. The Effect of ATP on the Photoconversion of Protochlorophyllide in Isolated Etioplasts of Zea mays1

    PubMed Central

    Horton, Peter; Leech, Rachel M.

    1975-01-01

    The transformation of protochlorophyllide (PChle) into chlorophyllide (Chle) has been studied in isolated etioplasts from Zea mays. ATP (1.5mm) prevented the transformation of photoconvertible PChle 650 to PChle 630 in aged etioplasts. Curve analysis indicated that the ATP effect on photoconvertibility could be entirely accounted for by changes in the proportions of PChle 630 and PChle 650 and examination of the isolated pigments revealed that only unphytylated PChle could be activated for photoconversion by ATP. In etioplasts aged for 5 hours, ATP also stimulated photoconversion of PChle 630 into Chle 670. The process was temperature-sensitive and involved PChle 650 and Chle 680 as intermediates. AMP alone had no effect, but inhibited ATP retardation of PChle loss. ADP had a similar but lesser effect than ATP. The ADP response, but not the ATP response, was considerably enhanced in the presence of an ATP-generating system (phosphoenolpyruvate/pyruvate kinase). UTP, GTP, and CTP gave 40 to 50% of the ATP response with intact etioplasts. In envelope-free etioplasts, ATP gave the greatest response but the other nucleotides were now 80% as effective as ATP. After primary photoconversion, ATP stimulated resynthesis of PChle 650. It is proposed that ATP both gives the holochrome the ability to bind to the PChle molecule and enables additional association of the pigment-protein complex to form PChle 650. PMID:16659239

  10. How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent.

    PubMed

    Dudev, Todor; Grauffel, Cédric; Lim, Carmay

    2017-02-14

    Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg(2+) interacts with the highly charged ATP triphosphate group and Li(+) can co-bind with the native Mg(2+) to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg(2+) and Li(+) (i.e. which phosphate group(s) bind Mg(2+)/Li(+)) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg(2+)/Li(+) alone and combined: Mg(2+) prefers to bind ATP tridentately to each of the three phosphate groups, but Li(+) prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li(+) binds to Mg(2+)-loaded ATP. Hence, ATP-Mg-Li, like Mg(2+)-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways.

  11. How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

    PubMed Central

    Dudev, Todor; Grauffel, Cédric; Lim, Carmay

    2017-01-01

    Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways. PMID:28195155

  12. Kinetic and hysteretic behavior of ATP hydrolysis of the highly stable dimeric ATP synthase of Polytomella sp.

    PubMed

    Villavicencio-Queijeiro, Alexa; Pardo, Juan Pablo; González-Halphen, Diego

    2015-06-01

    The F1FO-ATP synthase of the colorless alga Polytomella sp. exhibits a robust peripheral arm constituted by nine atypical subunits only present in chlorophycean algae. The isolated dimeric enzyme exhibits a latent ATP hydrolytic activity which can be activated by some detergents. To date, the kinetic behavior of the algal ATPase has not been studied. Here we show that while the soluble F1 sector exhibits Michaelis-Menten kinetics, the dimer exhibits a more complex behavior. The kinetic parameters (Vmax and Km) were obtained for both the F1 sector and the dimeric enzyme as isolated or activated by detergent, and this activation was also seen on the enzyme reconstituted in liposomes. Unlike other ATP synthases, the algal dimer hydrolyzes ATP on a wide range of pH and temperature. The enzyme was inhibited by oligomycin, DCCD and Mg-ADP, although oligomycin induced a peculiar inhibition pattern that can be attributed to structural differences in the algal subunit-c. The hydrolytic activity was temperature-dependent and exhibited activation energy of 4 kcal/mol. The enzyme also exhibited a hysteretic behavior with a lag phase strongly dependent on temperature but not on pH, that may be related to a possible regulatory role in vivo. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. HtrA2 deficiency causes mitochondrial uncoupling through the F1F0-ATP synthase and consequent ATP depletion

    PubMed Central

    Plun-Favreau, H; Burchell, V S; Holmström, K M; Yao, Z; Deas, E; Cain, K; Fedele, V; Moisoi, N; Campanella, M; Miguel Martins, L; Wood, N W; Gourine, A V; Abramov, A Y

    2012-01-01

    Loss of the mitochondrial protease HtrA2 (Omi) in mice leads to mitochondrial dysfunction, neurodegeneration and premature death, but the mechanism underlying this pathology remains unclear. Using primary cultures from wild-type and HtrA2-knockout mice, we find that HtrA2 deficiency significantly reduces mitochondrial membrane potential in a range of cell types. This depolarisation was found to result from mitochondrial uncoupling, as mitochondrial respiration was increased in HtrA2-deficient cells and respiratory control ratio was dramatically reduced. HtrA2-knockout cells exhibit increased proton translocation through the ATP synthase, in combination with decreased ATP production and truncation of the F1 α-subunit, suggesting the ATP synthase as the source of the proton leak. Uncoupling in the HtrA2-deficient mice is accompanied by altered breathing pattern and, on a cellular level, ATP depletion and vulnerability to chemical ischaemia. We propose that this vulnerability may ultimately cause the neurodegeneration observed in these mice. PMID:22739987

  14. Volume-Dependent Atp-Conductive Large-Conductance Anion Channel as a Pathway for Swelling-Induced Atp Release

    PubMed Central

    Sabirov, Ravshan Z.; Dutta, Amal K.; Okada, Yasunobu

    2001-01-01

    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 Gd3+. In on-cell patches, single-channel events appeared with a lag period of ∼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 ∼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 IC50 of 12.3 mM and an electric distance (δ) of 0.47, whereas the inward (but not outward) conductance was inhibited by intracellular ATP with an IC50 of 12.9 mM and δ of 0.40. Despite the open channel block by ATP, the channel was ATP-conductive with PATP/PCl of 0.09. The single-channel activity was sensitive to Gd3+, 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. PMID:11524456

  15. SUPERSTARS III: K-2.

    ERIC Educational Resources Information Center

    North Carolina State Dept. of Public Education, Raleigh.

    SUPERSTARS III is a K-8 program designed as an enrichment opportunity for self-directed learners in mathematics. The basic purpose of SUPERSTARS III is to provide the extra challenge that self-motivated students need in mathematics and to do so in a structured, long-term program that does not impinge on the normal classroom routine or the…

  16. Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss.

    PubMed

    Stover, E H; Borthwick, K J; Bavalia, C; Eady, N; Fritz, D M; Rungroj, N; Giersch, A B S; Morton, C C; Axon, P R; Akil, I; Al-Sabban, E A; Baguley, D M; Bianca, S; Bakkaloglu, A; Bircan, Z; Chauveau, D; Clermont, M-J; Guala, A; Hulton, S A; Kroes, H; Li Volti, G; Mir, S; Mocan, H; Nayir, A; Ozen, S; Rodriguez Soriano, J; Sanjad, S A; Tasic, V; Taylor, C M; Topaloglu, R; Smith, A N; Karet, F E

    2002-11-01

    Autosomal recessive distal renal tubular acidosis (rdRTA) is characterised by severe hyperchloraemic metabolic acidosis in childhood, hypokalaemia, decreased urinary calcium solubility, and impaired bone physiology and growth. Two types of rdRTA have been differentiated by the presence or absence of sensorineural hearing loss, but appear otherwise clinically similar. Recently, we identified mutations in genes encoding two different subunits of the renal alpha-intercalated cell's apical H(+)-ATPase that cause rdRTA. Defects in the B1 subunit gene ATP6V1B1, and the a4 subunit gene ATP6V0A4, cause rdRTA with deafness and with preserved hearing, respectively. We have investigated 26 new rdRTA kindreds, of which 23 are consanguineous. Linkage analysis of seven novel SNPs and five polymorphic markers in, and tightly linked to, ATP6V1B1 and ATP6V0A4 suggested that four families do not link to either locus, providing strong evidence for additional genetic heterogeneity. In ATP6V1B1, one novel and five previously reported mutations were found in 10 kindreds. In 12 ATP6V0A4 kindreds, seven of 10 mutations were novel. A further nine novel ATP6V0A4 mutations were found in "sporadic" cases. The previously reported association between ATP6V1B1 defects and severe hearing loss in childhood was maintained. However, several patients with ATP6V0A4 mutations have developed hearing loss, usually in young adulthood. We show here that ATP6V0A4 is expressed within the human inner ear. These findings provide further evidence for genetic heterogeneity in rdRTA, extend the spectrum of disease causing mutations in ATP6V1B1 and ATP6V0A4, and show ATP6V0A4 expression within the cochlea for the first time.

  17. Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss

    PubMed Central

    Stover, E; Borthwick, K; Bavalia, C; Eady, N; Fritz, D; Rungroj, N; Giersch, A; Morton, C; Axon, P; Akil, I; Al-Sabban, E; Baguley, D; Bianca, S; Bakkaloglu, A; Bircan, Z; Chauveau, D; Clermont, M; Guala, A; Hulton, S; Kroes, H; Li, V; Mir, S; Mocan, H; Nayir, A; Ozen, S; Rodriguez, S; Sanjad, S; Tasic, V; Taylor, C; Topaloglu, R; Smith, A; Karet, F

    2002-01-01

    Autosomal recessive distal renal tubular acidosis (rdRTA) is characterised by severe hyperchloraemic metabolic acidosis in childhood, hypokalaemia, decreased urinary calcium solubility, and impaired bone physiology and growth. Two types of rdRTA have been differentiated by the presence or absence of sensorineural hearing loss, but appear otherwise clinically similar. Recently, we identified mutations in genes encoding two different subunits of the renal α-intercalated cell's apical H+-ATPase that cause rdRTA. Defects in the B1 subunit gene ATP6V1B1, and the a4 subunit gene ATP6V0A4, cause rdRTA with deafness and with preserved hearing, respectively. We have investigated 26 new rdRTA kindreds, of which 23 are consanguineous. Linkage analysis of seven novel SNPs and five polymorphic markers in, and tightly linked to, ATP6V1B1 and ATP6V0A4 suggested that four families do not link to either locus, providing strong evidence for additional genetic heterogeneity. In ATP6V1B1, one novel and five previously reported mutations were found in 10 kindreds. In 12 ATP6V0A4 kindreds, seven of 10 mutations were novel. A further nine novel ATP6V0A4 mutations were found in "sporadic" cases. The previously reported association between ATP6V1B1 defects and severe hearing loss in childhood was maintained. However, several patients with ATP6V0A4 mutations have developed hearing loss, usually in young adulthood. We show here that ATP6V0A4 is expressed within the human inner ear. These findings provide further evidence for genetic heterogeneity in rdRTA, extend the spectrum of disease causing mutations in ATP6V1B1 and ATP6V0A4, and show ATP6V0A4 expression within the cochlea for the first time. PMID:12414817

  18. Activation of ATP binding for the autophosphorylation of DosS, a Mycobacterium tuberculosis histidine kinase lacking an ATP lid motif.

    PubMed

    Cho, Ha Yeon; Lee, Young-Hoon; Bae, Young-Seuk; Kim, Eungbin; Kang, Beom Sik

    2013-05-03

    The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg(440) in the DHp domain and Glu(537) in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg(440) and Glu(537) reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity.

  19. Resveratrol plus ethanol counteract the ethanol-induced impairment of energy metabolism: ³¹P NMR study of ATP and sn-glycerol-3-phosphate on isolated and perfused rat liver.

    PubMed

    Gallis, Jean-Louis; Serhan, Nizar; Gin, Henri; Couzigou, Patrice; Beauvieux, Marie-Christine

    2012-03-01

    The effects of trans-resveratrol (RSV) combined with ethanol (EtOH) were evaluated by (31)P NMR on total ATP and sn-glycerol-3-phosphate (sn-G3P) contents measured in real time in isolated and perfused whole liver of the rat. Mitochondrial ATP turnover was assessed by using specific inhibitors of glycolytic and mitochondrial ATP supply (iodacetate and KCN, respectively). In RSV alone, the slight decrease in ATP content (-14±5% of the initial content), sn-G3P content and ATP turnover were similar to those in the Krebs-Henseleit buffer control. Compared to control, EtOH alone (14 or 70 mmol/L) induced a decrease in ATP content (-24.95±2.95% of initial content, p<0.05) and an increase in sn-G3P (+158±22%), whereas ATP turnover tended to be increased. RSV (20 μmol/L) combined with EtOH, (i) maintained ATP content near 100%, (ii) induced a 1.6-fold increase in mitochondrial ATP turnover (p=0.049 and p=0.004 vs EtOH 14 and 70 mmol/L alone, respectively) and (iii) led to an increase in sn-G3P (+49±9% and +81±6% for 14 and 70 mmol/L EtOH, respectively). These improvements were obtained only when glycolysis was efficient at the time of addition of EtOH+RSV. Glycolysis inhibition by iodacetate (IAA) evidenced an almost 21% contribution of this pathway to ATP content. RSV alone or RSV+EtOH prevented the ATP decrease induced by IAA addition (p<0.05 vs control). This is the first demonstration of the combined effects of RSV and EtOH on liver energy metabolism. RSV increased (i) the flux of substrates through ATP producing pathways (glycolysis and phosphorylative oxidation) probably via the activation of AMPkinase, and (ii) maintained the glycolysis deviation to sn-G3P linked to NADH+H⁺ re-oxidation occurring during EtOH detoxication, thus reducing the energy cost due to the latter. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Role of the P-Type ATPases, ATP7A and ATP7B in brain copper homeostasis.

    PubMed

    Telianidis, Jonathon; Hung, Ya Hui; Materia, Stephanie; Fontaine, Sharon La

    2013-01-01

    Over the past two decades there have been significant advances in our understanding of copper homeostasis and the pathological consequences of copper dysregulation. Cumulative evidence is revealing a complex regulatory network of proteins and pathways that maintain copper homeostasis. The recognition of copper dysregulation as a key pathological feature in prominent neurodegenerative disorders such as Alzheimer's, Parkinson's, and prion diseases has led to increased research focus on the mechanisms controlling copper homeostasis in the brain. The copper-transporting P-type ATPases (copper-ATPases), ATP7A and ATP7B, are critical components of the copper regulatory network. Our understanding of the biochemistry and cell biology of these complex proteins has grown significantly since their discovery in 1993. They are large polytopic transmembrane proteins with six copper-binding motifs within the cytoplasmic N-terminal domain, eight transmembrane domains, and highly conserved catalytic domains. These proteins catalyze ATP-dependent copper transport across cell membranes for the metallation of many essential cuproenzymes, as well as for the removal of excess cellular copper to prevent copper toxicity. A key functional aspect of these copper transporters is their copper-responsive trafficking between the trans-Golgi network and the cell periphery. ATP7A- and ATP7B-deficiency, due to genetic mutation, underlie the inherited copper transport disorders, Menkes and Wilson diseases, respectively. Their importance in maintaining brain copper homeostasis is underscored by the severe neuropathological deficits in these disorders. Herein we will review and update our current knowledge of these copper transporters in the brain and the central nervous system, their distribution and regulation, their role in normal brain copper homeostasis, and how their absence or dysfunction contributes to disturbances in copper homeostasis and neurodegeneration.

  1. 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.

  2. Role of the P-Type ATPases, ATP7A and ATP7B in brain copper homeostasis

    PubMed Central

    Telianidis, Jonathon; Hung, Ya Hui; Materia, Stephanie; Fontaine, Sharon La

    2013-01-01

    Over the past two decades there have been significant advances in our understanding of copper homeostasis and the pathological consequences of copper dysregulation. Cumulative evidence is revealing a complex regulatory network of proteins and pathways that maintain copper homeostasis. The recognition of copper dysregulation as a key pathological feature in prominent neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and prion diseases has led to increased research focus on the mechanisms controlling copper homeostasis in the brain. The copper-transporting P-type ATPases (copper-ATPases), ATP7A and ATP7B, are critical components of the copper regulatory network. Our understanding of the biochemistry and cell biology of these complex proteins has grown significantly since their discovery in 1993. They are large polytopic transmembrane proteins with six copper-binding motifs within the cytoplasmic N-terminal domain, eight transmembrane domains, and highly conserved catalytic domains. These proteins catalyze ATP-dependent copper transport across cell membranes for the metallation of many essential cuproenzymes, as well as for the removal of excess cellular copper to prevent copper toxicity. A key functional aspect of these copper transporters is their copper-responsive trafficking between the trans-Golgi network and the cell periphery. ATP7A- and ATP7B-deficiency, due to genetic mutation, underlie the inherited copper transport disorders, Menkes and Wilson diseases, respectively. Their importance in maintaining brain copper homeostasis is underscored by the severe neuropathological deficits in these disorders. Herein we will review and update our current knowledge of these copper transporters in the brain and the central nervous system, their distribution and regulation, their role in normal brain copper homeostasis, and how their absence or dysfunction contributes to disturbances in copper homeostasis and neurodegeneration. PMID:23986700

  3. Identification of a mitochondrial ATP synthase-adenine nucleotide translocator complex in Leishmania.

    PubMed

    Detke, Siegfried; Elsabrouty, Rania

    2008-01-01

    The ATP synthasome is a macromolecular complex consisting of ATP synthase, adenine nucleotide translocator and phosphate carrier. To determine if this complex is evolutionary old or young, we searched for its presence in Leishmania, a mitochondria containing protozoan which evolved from the main eukaryote line soon after eukaryotes split from prokaryotes. Sucrose gradient centrifugation showed that the distribution of ANT among the fractions coincided with the distribution of ATP synthase. In addition, ATP synthase co-precipitated with FLAG tagged and wild type adenine nucleotide translocator isolated with anti FLAG and anti adenine nucleotide translocator antibodies, respectively. These data indicate that the adenine nucleotide translocator interacted with the ATP synthase to form a stable structure referred to as the ATP synthasome. The presence of the ATP synthasome in Leishmania, an organism branching off the main line of eukaryotes early in the development of eukaryotes, as well as in higher eukaryotes suggests that the ATP synthasome is a phylogenetically ancient structure.

  4. 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

  5. Regulation of Extracellular ATP in Human Erythrocytes Infected with Plasmodium falciparum

    PubMed Central

    Alvarez, Cora Lilia; Schachter, Julieta; de Sá Pinheiro, Ana Acacia; Silva, Leandro de Souza; Verstraeten, Sandra Viviana; Persechini, Pedro Muanis; Schwarzbaum, Pablo Julio

    2014-01-01

    In human erythrocytes (h-RBCs) various stimuli induce increases in [cAMP] that trigger ATP release. The resulting pattern of extracellular ATP accumulation (ATPe kinetics) depends on both ATP release and ATPe degradation by ectoATPase activity. In this study we evaluated ATPe kinetics from primary cultures of h-RBCs infected with P. falciparum at various stages of infection (ring, trophozoite and schizont stages). A “3V” mixture containing isoproterenol (β-adrenergic agonist), forskolin (adenylate kinase activator) and papaverine (phosphodiesterase inhibitor) was used to induce cAMP-dependent ATP release. ATPe kinetics of r-RBCs (ring-infected RBCs), t-RBCs (trophozoite-infected RBCs) and s-RBCs (schizont-infected RBCs) showed [ATPe] to peak acutely to a maximum value followed by a slower time dependent decrease. In all intraerythrocytic stages, values of ΔATP1 (difference between [ATPe] measured 1 min post-stimulus and basal [ATPe]) increased nonlinearly with parasitemia (from 2 to 12.5%). Under 3V exposure, t-RBCs at parasitemia 94% (t94-RBCs) showed 3.8-fold higher ΔATP1 values than in h-RBCs, indicative of upregulated ATP release. Pre-exposure to either 100 µM carbenoxolone, 100 nM mefloquine or 100 µM NPPB reduced ΔATP1 to 83–87% for h-RBCs and 63–74% for t94-RBCs. EctoATPase activity, assayed at both low nM concentrations (300–900 nM) and 500 µM exogenous ATPe concentrations increased approx. 400-fold in t94-RBCs, as compared to h-RBCs, while intracellular ATP concentrations of t94-RBCs were 65% that of h-RBCs. In t94-RBCs, production of nitric oxide (NO) was approx. 7-fold higher than in h-RBCs, and was partially inhibited by L-NAME pre-treatment. In media with L-NAME, ΔATP1 values were 2.7-times higher in h-RBCs and 4.2-times higher in t94-RBCs, than without L-NAME. Results suggest that P. falciparum infection of h-RBCs strongly activates ATP release via Pannexin 1 in these cells. Several processes partially counteracted ATPe accumulation

  6. Transport of cisplatin by the copper efflux transporter ATP7B.

    PubMed

    Safaei, Roohangiz; Otani, Shinji; Larson, Barrett J; Rasmussen, Michael L; Howell, Stephen B

    2008-02-01

    ATP7B is a P-type ATPase that mediates the efflux of copper. Recent studies have demonstrated that ATP7B regulates the cellular efflux of cisplatin (DDP) and controls sensitivity to the cytotoxic effects of this drug. To determine whether DDP is a substrate for ATP7B, DDP transport was assayed in vesicles isolated from Sf9 cells infected with a baculovirus that expressed either the wild-type ATP7B or a mutant ATP7B that was unable to transport copper as a result of conversion of the transmembrane metal binding CPC motif to CPA. Only the wild-type ATP7B-expressing vesicles exhibited copper-dependent ATPase activity, copper-induced acyl-phosphate formation, and ATP-dependent transport of copper. The amount of DDP that became bound was higher for vesicles expressing either type of ATP7B than for those not expressing either form of ATP7B, but only the vesicles expressing wild-type ATP7B mediated ATP-dependent accumulation of the drug. At pH 4.6, the vesicles expressing the wild-type ATP7B exhibited ATP-dependent accumulation of DDP with an apparent K(m) of 1.2 +/- 0.5 (S.E.M.) muM and V(max) of 0.03 +/- 0.002 (S.E.M.) nmol/mg of protein/min. DDP also induced the acyl-phosphorylation of ATP7B but at a much slower rate than copper. Copper and DDP each inhibited the ATP-dependent transport of the other. These results establish that DDP is a substrate for ATP7B but is transported at a much slower rate than copper.

  7. Arsenic Binding and Transfer by the ArsD As(III) Metallochaperone†

    PubMed Central

    Yang, Jianbo; Rawat, Swati; Stemmler, Timothy L.; Rosen, Barry P.

    2010-01-01

    ArsD is a metallochaperone that delivers trivalent metalloids [As(III) or Sb(III)] to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. Interaction with ArsD increases the affinity of ArsA for As(III), conferring resistance to environmental concentrations of arsenic. Previous genetic analysis suggested that ArsD residues Cys12, Cys13, and Cys18 are involved in the transfer of As(III) to ArsA. Here X-ray absorption spectroscopy was used to show that As(III) is coordinated with three sulfur atoms, consistent with the three cysteine residues forming the As(III) binding site. Two single-tryptophan derivatives of ArsD exhibited quenching of intrinsic protein fluorescence upon binding of As(III) or Sb(III), which allowed estimation of the rates of binding and affinities for metalloids. Substitution of Cys12, Cys13, or Cys18 decreased the affinity for As(III) more than 10-fold. Reduced glutathione greatly increased the rate of binding of As(III) to ArsD but did not affect binding of As(III) to ArsA. This suggests that in vivo cytosolic As(III) might be initially bound to GSH and transferred to ArsD and then to ArsAB, which pumps the metalloid out of the cell. The As(III) chelator dimercaptosuccinic acid did not block the transfer from ArsD to ArsA, consistent with channeling of the metalloid from one protein to the other, as opposed to release and rebinding of the metalloid. Finally, transfer of As(III) from ArsD to ArsA occurred in the presence of MgATP at 23 °C but not at 4 °C. Neither MgADP nor MgATP-γ-S could replace MgATP. These results suggest that transfer occurs with a conformation of ArsA that transiently forms during the catalytic cycle. PMID:20361763

  8. Possible role of firmly bound ATP in the energy transduction of photosynthetic membranes.

    PubMed

    Lutz, H U; Beyeler, W; Pflugshaupt, C; Bachofen, R

    1975-01-01

    Chromatophores of Rhodospirillum rubrum and spinach chloroplasts contain firmly bound ATP that is rapidly labeled along with ADP in the presence of 32Pi and endogenous nucleotides. The labeling is not entirely dependent on light. In chloroplasts three types of bound ATP can be defined methodologically by their extraction properties: buffer-soluble; acid-soluble; and SDS-soluble or firmly bound ATP. Extensive washing of the chloroplasts does reduce buffer-soluble but not acid-soluble and firmly bound ATP. Buffer-soluble [32P] ATP is almost exclusively gamma labeled while acid-soluble and firmly bound ATP are labeled in the beta and gamma position equally. CCCP, desaspidin, and phlorizin do not inhibit the labeling of firmly bound ATP, whereas the phosphorylation is almost abolished. However, EDTA and NEM pretreatments of the chloroplasts affect both reactions similarly. The postillumination [32P] ATP synthesis with chromatophores can be inhibited by adding ATP to the incubation mixture after illumination if 32Pi is included only during the dark incubation, but is without effect if 32Pi is present only during illumination. On the other hand, ADP added after illumination inhibits post-illumination [32P] ATP formation in both chromatophores and chloroplasts only if 32Pi is present during illumination. The data can be explained by a coupling factor having two sites, as proposed previously on the basis that firmly bound ATP does not transfer its phosphoryl group but seems to drive a synthesis of acid-soluble ATP which incorporates free phosphate.

  9. ATP Release from Human Airway Epithelial Cells Exposed to Staphylococcus aureus Alpha-Toxin

    PubMed Central

    Baaske, Romina; Richter, Mandy; Möller, Nils; Ziesemer, Sabine; Eiffler, Ina; Müller, Christian; Hildebrandt, Jan-Peter

    2016-01-01

    Airway epithelial cells reduce cytosolic ATP content in response to treatment with S. aureus alpha-toxin (hemolysin A, Hla). This study was undertaken to investigate whether this is due to attenuated ATP generation or to release of ATP from the cytosol and extracellular ATP degradation by ecto-enzymes. Exposure of cells to rHla did result in mitochondrial calcium uptake and a moderate decline in mitochondrial membrane potential, indicating that ATP regeneration may have been attenuated. In addition, ATP may have left the cells through transmembrane pores formed by the toxin or through endogenous release channels (e.g., pannexins) activated by cellular stress imposed on the cells by toxin exposure. Exposure of cells to an alpha-toxin mutant (H35L), which attaches to the host cell membrane but does not form transmembrane pores, did not induce ATP release from the cells. The Hla-mediated ATP-release was completely blocked by IB201, a cyclodextrin-inhibitor of the alpha-toxin pore, but was not at all affected by inhibitors of pannexin channels. These results indicate that, while exposure of cells to rHla may somewhat reduce ATP production and cellular ATP content, a portion of the remaining ATP is released to the extracellular space and degraded by ecto-enzymes. The release of ATP from the cells may occur directly through the transmembrane pores formed by alpha-toxin. PMID:27929417

  10. 76 FR 13069 - Airworthiness Directives; BAE Systems (Operations) Limited Model ATP Airplanes; BAE Systems...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-10

    ... (Operations) Limited Model ATP Airplanes; BAE Systems (Operations) Limited Model HS 748 Airplanes AGENCY... specified products. The MCAI states: Early in the life of the ATP (circa 1989), a report was received that a... by issuing SB ATP- 27-11, describing a one-time inspection of the hinge pins, which was...

  11. 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....

  12. Fullerene derived molecularly imprinted polymer for chemosensing of adenosine-5'-triphosphate (ATP).

    PubMed

    Sharma, Piyush S; Dabrowski, Marcin; Noworyta, Krzysztof; Huynh, Tan-Phat; Kc, Chandra B; Sobczak, Janusz W; Pieta, Piotr; D'Souza, Francis; Kutner, Wlodzimierz

    2014-09-24

    For molecular imprinting of oxidatively electroactive analytes by electropolymerization, we used herein reductively electroactive functional monomers. As a proof of concept, we applied C60 fullerene adducts as such for the first time. For that, we derivatized C60 to bear either an uracil or an amide, or a carboxy addend for recognition of the adenosine-5'-triphosphate (ATP) oxidizable analyte with the ATP-templated molecularly imprinted polymer (MIP-ATP). Accordingly, the ATP complex with all of the functional monomers formed in solution was potentiodynamically electropolymerized to deposit an MIP-ATP film either on an Au electrode of the quartz crystal resonator or on a Pt disk electrode for the piezoelectric microgravimetry (PM) or capacitive impedimetry (CI) determination of ATP, respectively, under the flow-injection analysis (FIA) conditions. The apparent imprinting factor for ATP was ∼4.0. After extraction of the ATP template, analytical performance of the resulting chemosensors, including detectability, sensitivity, and selectivity, was characterized. The limit of detection was 0.3 and 0.03mM ATP for the PM and CI chemosensor, respectively. The MIP-ATP film discriminated structural analogues of ATP quite well. The Langmuir, Freundlich, and Langmuir-Freundlich isotherms were fitted to the experimental data of the ATP sorption and sorption stability constants appeared to be nearly independent of the adopted sorption model.

  13. Formation of a complex containing ATP, Mg2+, and spermine. Structural evidence and biological significance.

    PubMed

    Meksuriyen, D; Fukuchi-Shimogori, T; Tomitori, H; Kashiwagi, K; Toida, T; Imanari, T; Kawai, G; Igarashi, K

    1998-11-20

    The conformation of ATP in the presence of Mg2+ and/or spermine was studied by 31P and 1H NMR, to clarify how polyamines interact with ATP. Spermine predominantly interacted with the beta- and gamma-phosphates of ATP in the presence of Mg2+. A conformational change of the beta- and gamma-phosphate of ATP with spermine could not be observed in the absence of Mg2+ by 31P NMR. It was found by 1H NMR that the conformation of adenosine moiety of ATP was not influenced significantly by spermine. The binding of Mg2+ to ATP was slightly inhibited by spermine and vice versa. The results indicate that the binding sites of Mg2+ and spermine on ATP only partially overlap. The PotA protein, an ATP-dependent enzyme, was used as a model system to study the biological role of the ATP-Mg2+-spermine complex. The ATPase activity of PotA was greatly enhanced by spermine. Double reciprocal plots at several concentrations of spermine as an activator indicate that spermine interacts with ATP, but not with PotA. The activity of protein kinase A was also stimulated about 2-fold by spermine. The results suggest that a ternary complex of ATP-Mg2+-spermine may play an important role in some ATP-dependent reactions in vivo and in the physiological effects of endogenous polyamines.

  14. Characterization of an ATP translocase identified in the plant pathogen, Candidatus Liberibacter asiaticus

    USDA-ARS?s Scientific Manuscript database

    ATP/ADP translocases allow for the transport of ATP across a lipid bilayer, which is normally impermeable to this molecule due to its size and charge. These transport proteins appear to be unique to mitochondria, plant plastids, and obligate-intracellular bacteria. Of the bacterial ATP/ADP translo...

  15. ATP6V1G1 — EDRN Public Portal

    Cancer.gov

    ATP6V1G1 is a subunit of vacuolar ATPase (V-ATPase), a multisubunit enzyme. V-ATPase is an enzyme transporter that functions to acidify intracellular compartments in eukaryotic cells. This acidification process is necessary for such intracellular processes as protein sorting, zymogen activation, receptor-mediated endocytosis, and synaptic vesicle proton gradient generation. V-ATPase is ubiquitously expressed and is present in endomembrane organelles such as vacuoles, lysosomes, endosomes, the Golgi apparatus, chromaffin granules and coated vesicles, as well as in the plasma membrane. V-ATPase is composed of a cytosolic V1 domain and a transmembrane V0 domain. The V1 domain consists of three A, three B, and two G subunits, as well as a C, D, E, F, and H subunit. The V1 domain contains the ATP catalytic site.

  16. Calcium induced ATP synthesis: Isotope effect, magnetic parameters and mechanism

    NASA Astrophysics Data System (ADS)

    Buchachenko, A. L.; Kuznetsov, D. A.; Breslavskaya, N. N.; Shchegoleva, L. N.; Arkhangelsky, S. E.

    2011-03-01

    ATP synthesis by creatine kinase with calcium ions is accompanied by 43Ca/ 40Ca isotope effect: the enzyme with 43Ca 2+ was found to be 2.0 ± 0.3 times more active than enzymes, in which Ca 2+ ions have nonmagnetic nuclei 40Ca. The effect demonstrates that primary reaction in ATP synthesis is electron transfer between reaction partners, Сa( HO)n2+ ( n ⩽ 3) and Ca 2+(ADP) 3-. It generates ion-radical pair, in which spin conversion results in the isotope effect. Magnetic parameters (g-factors and HFC constants a( 43Ca) and a( 31P)) confirm that namely terminal oxygen atom of the ADP ligand in the complex Ca 2+(ADP) 3- donates electron to the Ca( HO)n2+ ion.

  17. Bioluminescence microscopy: application to ATP measurements in single living cells

    NASA Astrophysics Data System (ADS)

    Brau, Frederic; Helle, Pierre; Bernengo, Jean C.

    1997-12-01

    Bioluminescence microscopy can be used to measure intracellular cofactors and ionic concentrations (Ca2+, K+, ATP, NADH), as an alternative to micro- spectrophotometry and micro-fluorimetry, due to the development of sensitive detectors (cooled photomultipliers tubes and CCD). The main limitation comes from the very small and brief intensity of the emitted light. Our instrumentation based on an inverted microscope, equipped with high aperture immersion lenses is presented. Light intensity measurements are carried out through a photomultiplier sorted for low dark current and cooled at -5 degree(s)C to reduce thermal noise. Our first aim is to quantify ATP on single living cells using the firefly luciferin-luciferase couple. Experimental and kinetic aspects are presented to emphasize the potentialities of the technique.

  18. Interactions of ATP, oestradiol, genistein and the anti-oestrogens, faslodex (ICI 182780) and tamoxifen, with the human erythrocyte glucose transporter, GLUT1.

    PubMed Central

    Afzal, Iram; Cunningham, Philip; Naftalin, Richard J

    2002-01-01

    17 beta-Oestradiol (ED when subscript to K) and the phytoestrogen isoflavone genistein (GEN) inhibit glucose transport in human erythrocytes and erythrocyte ghosts. The selective oestrogen receptor modulators or anti-oestrogens, faslodex (ICI 182780) (FAS) and tamoxifen (TAM), competitively antagonize oestradiol inhibition of glucose exit from erythrocytes (K(i(ED/FAS))=2.84+/-0.16 microM and K(i(ED/TAM))=100+/-2 nM). Faslodex has no significant inhibitory effect on glucose exit, but tamoxifen alone inhibits glucose exit (K(i(TAM))=300+/-100 nM). In ghosts, ATP (1-4 mM) competitively antagonizes oestradiol, genistein and cytochalasin B (CB)-dependent inhibitions of glucose exit, (K(i(ATP/ED))=2.5+/-0.23 mM, K(i(ATP/GEN))=0.99+/-0.17 mM and K(i(ATP/CB))=0.76+/-0.08 mM). Tamoxifen and faslodex reverse oestradiol-dependent inhibition of glucose exit with ATP>1 mM (K(i(ED/TAM))=130+/-5 nM and K(i(ED/FAS))=2.7+/-0.9 microM). The cytoplasmic surface of the glucose transporter (GLUT)1 contains four sequences with close homologies to sequences in the ligand-binding domain of human oestrogen receptor beta (hesr-2). One homology is adjacent to the Walker ATP-binding motif II (GLUT1, residues 225-229) in the large cytoplasmic segment linking transmembrane helices 6 and 7; another GLUT (residues 421-423) contains the Walker ATP-binding motif III. Mapping of these regions on to a three-dimensional template of GLUT indicates that a possible oestrogen-binding site lies between His(337), Arg(349) and Glu(249) at the cytoplasmic entrance to the hydrophilic pore spanning GLUT, which have a similar topology to His(475), Glu(305) and Arg(346) in hesr-2 that anchor the head and tail hydroxy groups of oestradiol and genistein, and thus are suitably placed to provide an ATP-sensitive oestrogen binding site that could modulate glucose export. PMID:12133004

  19. ATP binding/hydrolysis by and phosphorylation of peroxisomal ATP-binding cassette proteins PMP70 (ABCD3) and adrenoleukodystrophy protein (ABCD1).

    PubMed

    Tanaka, Arowu R; Tanabe, Kouichi; Morita, Masashi; Kurisu, Mikinori; Kasiwayama, Yoshinori; Matsuo, Michinori; Kioka, Noriyuki; Amachi, Teruo; Imanaka, Tsuneo; Ueda, Kazumitsu

    2002-10-18

    The 70-kDa peroxisomal membrane protein (PMP70) and adrenoleukodystrophy protein (ALDP), half-size ATP-binding cassette transporters, are involved in metabolic transport of long and very long chain fatty acids into peroxisomes. We examined the interaction of peroxisomal ATP-binding cassette transporters with ATP using rat liver peroxisomes. PMP70 was photoaffinity-labeled at similar efficiencies with 8-azido-[alpha-32P]ATP and 8-azido-[gamma-32P]ATP when peroxisomes were incubated with these nucleotides at 37 degrees C in the absence Mg2+ and exposed to UV light without removing unbound nucleotides. The photoaffinity-labeled PMP70 and ALDP were co-immunoprecipitated together with other peroxisomal proteins, which also showed tight ATP binding properties. Addition of Mg2+ reduced the photoaffinity labeling of PMP70 with 8-azido-[gamma-32P]ATP by 70%, whereas it reduced photoaffinity labeling with 8-azido-[alpha-32P]ATP by only 20%. However, two-thirds of nucleotide (probably ADP) was dissociated during removal of unbound nucleotides. These results suggest that ATP binds to PMP70 tightly in the absence of Mg2+, the bound ATP is hydrolyzed to ADP in the presence of Mg2+, and the produced ADP is dissociated from PMP70, which allows ATP hydrolysis turnover. Properties of photoaffinity labeling of ALDP were essentially similar to those of PMP70. Vanadate-induced nucleotide trapping in PMP70 and ALDP was not observed. PMP70 and ALDP were also phosphorylated at a tyrosine residue(s). ATP binding/hydrolysis by and phosphorylation of PMP70 and ALDP are involved in the regulation of fatty acid transport into peroxisomes.

  20. 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

  1. 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

  2. Persister formation in Staphylococcus aureus is associated with ATP depletion

    SciTech Connect

    Conlon, Brian P.; Rowe, Sarah E.; Gandt, Autumn Brown; Nuxoll, Austin S.; Donegan, Niles P.; Zalis, Eliza A.; Clair, Geremy; Adkins, Joshua N.; Cheung, Ambrose L.; Lewis, Kim

    2016-04-18

    Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics1. Persisters are associated with chronic bacterial infection and antibiotic treatment failure. In Escherichia coli, toxin/antitoxin (TA) modules are responsible for persister formation. The mechanism of persister formation in Gram positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting TA modules in S. aureus did not affect the level of persisters. Here we show that S. aureus persisters are produced due to a stochastic entrance to stationary phase accompanied by a drop in intracellular ATP. Cells expressing stationary state markers are present throughout the growth phase, increasing in frequency with cell density. Cell sorting revealed that expression of stationary markers was associated with a 100-1000 fold increased likelihood of survival to antibiotic challenge. We find that the antibiotic tolerance of these cells is due to a drop in intracellular ATP. The ATP level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotic treatment.

  3. Honing in on the ATP Release Channel in Taste Cells

    PubMed Central

    2015-01-01

    Studies over the last 8 years have identified 3 potential channels that appear to release ATP from Type II cells in response to taste stimuli. These studies have taken different methodological approaches but have all provided data supporting their candidate channel as the ATP release channel. These potential channels include Pannexin 1, Connexins (30 and/or 43), and most recently, the Calhm1 channel. Two papers in this issue of Chemical Senses provide compelling new evidence that Pannexin 1 is not the ATP release channel. Tordoff et al. did a thorough behavioral analysis of the Pannexin1 knock out mouse and found that these animals have the same behavioral responses as wild type mice for 7 different taste stimuli that were tested. Vandenbeuch et al. presented an equally thorough analysis of the gustatory nerve responses in the Pannexin1 knock out mouse and found no differences compared with controls. Thus when the role of Pannexin 1 is analyzed at the systems level, it is not required for normal taste perception. Further studies are needed to determine the role of this hemichannel in taste cells. PMID:26126730

  4. Light Effect on Water Viscosity: Implication for ATP Biosynthesis.

    PubMed

    Sommer, Andrei P; Haddad, Mike Kh; Fecht, Hans-Jörg

    2015-07-08

    Previous work assumed that ATP synthase, the smallest known rotary motor in nature, operates at 100% efficiency. Calculations which arrive to this result assume that the water viscosity inside mitochondria is constant and corresponds to that of bulk water. In our opinion this assumption is not satisfactory for two reasons: (1) There is evidence that the water in mitochondria prevails to 100% as interfacial water. (2) Laboratory experiments which explore the properties of interfacial water suggest viscosities which exceed those of bulk water, specifically at hydrophilic interfaces. Here, we wish to suggest a physicochemical mechanism which assumes intramitochondrial water viscosity gradients and consistently explains two cellular responses: The decrease and increase in ATP synthesis in response to reactive oxygen species and non-destructive levels of near-infrared (NIR) laser light, respectively. The mechanism is derived from the results of a new experimental method, which combines the technique of nanoindentation with the modulation of interfacial water layers by laser irradiation. Results, including the elucidation of the principle of light-induced ATP production, are expected to have broad implications in all fields of medicine.

  5. Light Effect on Water Viscosity: Implication for ATP Biosynthesis

    PubMed Central

    Sommer, Andrei P.; Haddad, Mike Kh.; Fecht, Hans-Jörg

    2015-01-01

    Previous work assumed that ATP synthase, the smallest known rotary motor in nature, operates at 100% efficiency. Calculations which arrive to this result assume that the water viscosity inside mitochondria is constant and corresponds to that of bulk water. In our opinion this assumption is not satisfactory for two reasons: (1) There is evidence that the water in mitochondria prevails to 100% as interfacial water. (2) Laboratory experiments which explore the properties of interfacial water suggest viscosities which exceed those of bulk water, specifically at hydrophilic interfaces. Here, we wish to suggest a physicochemical mechanism which assumes intramitochondrial water viscosity gradients and consistently explains two cellular responses: The decrease and increase in ATP synthesis in response to reactive oxygen species and non-destructive levels of near-infrared (NIR) laser light, respectively. The mechanism is derived from the results of a new experimental method, which combines the technique of nanoindentation with the modulation of interfacial water layers by laser irradiation. Results, including the elucidation of the principle of light-induced ATP production, are expected to have broad implications in all fields of medicine. PMID:26154113

  6. 2,3-Diphosphoglycerate and ATP dissociate erythrocyte membrane skeletons.

    PubMed

    Sheetz, M P; Casaly, J

    1980-10-25

    Since ATP and 2,3-diphosphoglycerate cause an increase in the lateral mobility of integral membrane proteins in the erythrocyte (Schindler, M., Koppel, D., and Sheetz, M. P. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 1457-1461), we have studied their effects on the membrane skeletal complex or shell (composed of spectrin, actin, and bands 4.1 (78,000 daltons) and 4.9 (50,000 daltons)) and its interaction with the erythrocyte membrane. Both phosphate compounds dissociated the delipidated shell complex, with half-maximal dissociation at 2.5 mM 2,3-diphosphoglycerate and 8 mM ATP, whereas equivalent concentrations of EDTA did not. Concomitant with complex dissociation, spectrin was solubilized but band 4.1 and actin remained in a complexed or polymeric form. When proteins which were involved in linking spectrin to the membrane were present on the shell, higher concentrations of the phosphate compounds still dissociated the complex but less spectrin was solubilized. Treatment of erythrocyte membranes with the same phosphate compounds caused membrane vesiculation but no proteins were solubilized. We suggest that ATP and 2,3-diphosphoglycerate, at concentrations which are normally present in erythrocytes, can weaken associations in the shell but will not dissociate the complex from membrane attachment sites.

  7. Overview of photo-induced therapy for ATP production

    NASA Astrophysics Data System (ADS)

    Abdalla, Mohamed; Nagy, A.; Ye, W. N.; Mussivand, T.

    2012-10-01

    The purpose of this report is to provide a review of the effects of low-power photo-induced therapy using lasers of different device parameters such as intensity, wavelength, lasing mechanism (i.e., pulsed or continuous) on the production of Adenosine triphosphate (ATP) in mammalian cells. This is a very important research topic as it is suggested in literature that there might be a relationship between the ATP levels and specific diseases. It has been shown that the ATP production was enhanced at wavelengths ranging between 600 nm and 1000 nm (also known as the optical window), in particular at 600nm, 632.8nm, 635nm, 650nm, and 904nm. However, certain experiments showed that the effectiveness of the photo-induced therapy was also dependent on the dosage and the duration of the supplied light. We present the research conclusions drawn from the experiments reported within the last decade, and provide a list of potential medical treatment(s) for patients using visible and near infrared (NIR) light.

  8. ATP-gated channels in vascular smooth muscle cells.

    PubMed

    Benham, C D

    1990-01-01

    ATP acting through P2x-purinoceptors activates cation channels with some similarities to the activation of channels gated by acetylcholine and glutamate (channels that can also act as fast excitatory transmitters). These experiments clearly demonstrate an ATP-mediated Ca2+ influx through agonist-gated channels and a consequent elevation of [Ca2+]i in these single vascular smooth muscle cells. The combination of the ability to hold these cells under voltage-clamp and to measure [Ca2+]i simultaneously has allowed us to exclude other possible explanations for the rise in [Ca2+]i under these conditions. Thus, although the major cation entering through the channels is Na+, ATP receptor activation will also generate subtle, localized increases in [Ca2+]. These increases might directly activate contractile proteins or, if insufficient to do this, might upregulate other Ca2(+)-dependent enzymes modulating the contractile process and provide an enhanced source of Ca2+ for uptake into internal Ca2+ stores. Further understanding of the physiological role of this conductance pathway may require the development of specific receptor antagonists or channel blockers.

  9. ATP6AP2 — EDRN Public Portal

    Cancer.gov

    ATP6AP2 functions as a renin and prorenin cellular receptor. It may mediate renin-dependent cellular responses by activating ERK1 and ERK2. By increasing the catalytic efficiency of renin in AGT/angiotensinogen conversion to angiotensin I, it may also play a role in the renin-angiotensin system (RAS). ATP6AP2 is expressed in brain, heart, placenta, liver, kidney and pancreas; it is barely detectable in lung and skeletal muscles. In the kidney cortex it is restricted to the mesangium of glomeruli. In the coronary and kidney artery it is expressed in the subendothelium, associated to smooth muscles where it colocalizes with REN. It is expressed in vascular structures and by syncytiotrophoblast cells in the mature fetal placenta. Defects in ATP6AP2 are a cause of mental retardation X-linked with epilepsy (MRXE). MRXE is a syndromic mental retardation. Patients manifest mild to moderate mental retardation associated with epilepsy, delays in motor milestones and speech acquisition in infancy.

  10. TCDD decreases ATP levels and increases reactive oxygen production through changes in mitochondrial F F{sub 1}-ATP synthase and ubiquinone

    SciTech Connect

    Shertzer, Howard G. . E-mail: shertzhg@ucmail.uc.edu; Genter, Mary Beth; Shen, Dongxiao; Nebert, Daniel W.; Chen, Ying; Dalton, Timothy P.

    2006-12-15

    Mitochondria generate ATP and participate in signal transduction and cellular pathology and/or cell death. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) decreases hepatic ATP levels and generates mitochondrial oxidative DNA damage, which is exacerbated by increasing mitochondrial glutathione redox state and by inner membrane hyperpolarization. This study identifies mitochondrial targets of TCDD that initiate and sustain reactive oxygen production and decreased ATP levels. One week after treating mice with TCDD, liver ubiquinone (Q) levels were significantly decreased, while rates of succinoxidase and Q-cytochrome c oxidoreductase activities were increased. However, the expected increase in Q reduction state following TCDD treatment did not occur; instead, Q was more oxidized. These results could be explained by an ATP synthase defect, a premise supported by the unusual finding that TCDD lowers ATP/O ratios without concomitant changes in respiratory control ratios. Such results suggest either a futile cycle in ATP synthesis, or hydrolysis of newly synthesized ATP prior to release. The TCDD-mediated decrease in Q, concomitant with an increase in respiration, increases complex 3 redox cycling. This acts in concert with glutathione to increase membrane potential and reactive oxygen production. The proposed defect in ATP synthase explains both the greater respiratory rates and the lower tissue ATP levels.

  11. Oxa1 Directly Interacts with Atp9 and Mediates Its Assembly into the Mitochondrial F1Fo-ATP Synthase Complex

    PubMed Central

    Jia, Lixia; Dienhart, Mary K.

    2007-01-01

    The yeast Oxa1 protein is involved in the biogenesis of the mitochondrial oxidative phosphorylation (OXPHOS) machinery. The involvement of Oxa1 in the assembly of the cytochrome oxidase (COX) complex, where it facilitates the cotranslational membrane insertion of mitochondrially encoded COX subunits, is well documented. In this study we have addressed the role of Oxa1, and its sequence-related protein Cox18/Oxa2, in the biogenesis of the F1Fo-ATP synthase complex. We demonstrate that Oxa1, but not Cox18/Oxa2, directly supports the assembly of the membrane embedded Fo-sector of the ATP synthase. Oxa1 was found to physically interact with newly synthesized mitochondrially encoded Atp9 protein in a posttranslational manner and in a manner that is not dependent on the C-terminal, matrix-localized region of Oxa1. The stable manner of the Atp9-Oxa1 interaction is in contrast to the cotranslational and transient interaction previously observed for the mitochondrially encoded COX subunits with Oxa1. In the absence of Oxa1, Atp9 was observed to assemble into an oligomeric complex containing F1-subunits, but its further assembly with subunit 6 (Atp6) of the Fo-sector was perturbed. We propose that by directly interacting with newly synthesized Atp9 in a posttranslational manner, Oxa1 is required to maintain the assembly competence of the Atp9-F1-subcomplex for its association with Atp6. PMID:17344477

  12. TCDD decreases ATP levels and increases reactive oxygen production through changes in mitochondrial F0F1-ATP synthase and ubiquinone

    PubMed Central

    Shertzer, Howard G.; Genter, Mary Beth; Shen, Dongxiao; Nebert, Daniel W.; Chen, Ying; Dalton, Timothy P.

    2007-01-01

    Mitochondria generate ATP and participate in signal transduction and cellular pathology and/or cell death. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) decreases hepatic ATP levels and generates mitochondrial oxidative DNA damage, which is exacerbated by increasing mitochondrial glutathione redox state and by inner-membrane hyperpolarization. This study identifies mitochondrial targets of TCDD that initiate and sustain reactive oxygen production and decreased ATP levels. One week after treating mice with TCDD, liver ubiquinone (Q) levels were significantly decreased, while rates of succinoxidase and Q-cytochrome c oxidoreductase activities were increased. However, the expected increase in Q reduction state following TCDD treatment did not occur; instead, Q was more oxidized. These results could be explained by an ATP synthase defect, a premise supported by the unusual finding that TCDD lowers ATP/O ratios without concomitant changes in respiratory control ratios. Such results suggest either a futile cycle in ATP synthesis, or hydrolysis of newly-synthesized ATP prior to release. The TCDD-mediated decrease in Q, concomitant with an increase in respiration, increases complex 3 redox-cycling. This acts in concert with glutathione to increase membrane potential and reactive oxygen production. The proposed defect in ATP synthase explains both the greater respiratory rates and the lower tissue ATP levels. PMID:17109908

  13. Differential proteomic profiling unveils new molecular mechanisms associated with mitochondrial complex III deficiency

    PubMed Central

    Morán, María; López-Bernardo, Elia; Cadenas, Susana; Hidalgo, Beatriz; Sánchez, Ricardo; Seneca, Sara; Arenas, Joaquín; Martín, Miguel A.; Ugalde, Cristina

    2014-01-01

    We have analyzed the cellular pathways and metabolic adaptations that take place in primary skin fibroblasts from patients with mutations in BCS1L, a major genetic cause of mitochondrial complex III enzyme deficiency. Mutant fibroblasts exhibited low oxygen consumption rates and intracellular ATP levels, indicating that the main altered molecular event probably is a limited respiration-coupled ATP production through the OXPHOS system. Two-dimensional DIGE and MALDI-TOF/TOF mass spectrometry analyses unambiguously identified 39 proteins whose expression was significantly altered in complex III-deficient fibroblasts. Extensive statistical and cluster analyses revealed a protein profile characteristic for the BCS1L mutant fibroblasts that included alterations in energy metabolism, cell signaling and gene expression regulation, cytoskeleton formation and maintenance, and intracellular stress responses. The physiological validation of the predicted functional adaptations of human cultured fibroblasts to complex III deficiency confirmed the up-regulation of glycolytic enzyme activities and the accumulation of branched-chain among other amino acids, suggesting the activation of anaerobic glycolysis and cellular catabolic states, in particular protein catabolism, together with autophagy as adaptive responses to mitochondrial respiratory chain dysfunction and ATP deficiency. Our data point to an overall metabolic and genetic reprogramming that could contribute to explain the clinical manifestations of complex III deficiency in patients. PMID:25239759

  14. Real-time imaging of inflation-induced ATP release in the ex vivo rat lung.

    PubMed

    Furuya, Kishio; Tan, Ju Jing; Boudreault, Francis; Sokabe, Masahiro; Berthiaume, Yves; Grygorczyk, Ryszard

    2016-11-01

    Extracellular ATP and other nucleotides are important autocrine/paracrine mediators that regulate diverse processes critical for lung function, including mucociliary clearance, surfactant secretion, and local blood flow. Cellular ATP release is mechanosensitive; however, the impact of physical stimuli on ATP release during breathing has never been tested in intact lungs in real time and remains elusive. In this pilot study, we investigated inflation-induced ATP release in rat lungs ex vivo by real-time luciferin-luciferase (LL) bioluminescence imaging coupled with simultaneous infrared tissue imaging to identify ATP-releasing sites. With LL solution introduced into air spaces, brief inflation of such edematous lung (1 s, ∼20 cmH2O) induced transient (<30 s) ATP release in a limited number of air-inflated alveolar sacs during their recruitment/opening. Released ATP reached concentrations of ∼10(-6) M, relevant for autocrine/paracrine signaling, but it remained spatially restricted to single alveolar sacs or their clusters. ATP release was stimulus dependent: prolonged (100 s) inflation evoked long-lasting ATP release that terminated upon alveoli deflation/derecruitment while cyclic inflation/suction produced cyclic ATP release. With LL introduced into blood vessels, inflation induced transient ATP release in many small patchlike areas the size of alveolar sacs. Findings suggest that inflation induces ATP release in both alveoli and the surrounding blood capillary network; the functional units of ATP release presumably consist of alveolar sacs or their clusters. Our study demonstrates the feasibility of real-time ATP release imaging in ex vivo lungs and provides the first direct evidence of inflation-induced ATP release in lung air spaces and in pulmonary blood capillaries, highlighting the importance of purinergic signaling in lung function.

  15. Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1

    PubMed Central

    Althoff, Thorsten; Mills, Deryck J; Popot, Jean-Luc; Kühlbrandt, Werner

    2011-01-01

    The respiratory chain in the inner mitochondrial membrane contains three large multi-enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19-Å 3D map of the 1.7-MDa amphipol-solubilized supercomplex I1III2IV1 from bovine heart obtained by single-particle electron cryo-microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X-ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol-binding sites of complexes I and III, and of 10–11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred. PMID:21909073

  16. Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1.

    PubMed

    Althoff, Thorsten; Mills, Deryck J; Popot, Jean-Luc; Kühlbrandt, Werner

    2011-09-09

    The respiratory chain in the inner mitochondrial membrane contains three large multi-enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19-Å 3D map of the 1.7-MDa amphipol-solubilized supercomplex I(1)III(2)IV(1) from bovine heart obtained by single-particle electron cryo-microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X-ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol-binding sites of complexes I and III, and of 10-11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred.

  17. Antithrombin III blood test

    MedlinePlus

    ... AT III) is a protein that helps control blood clotting. A blood test can determine the amount of ... may mean you have an increased risk of blood clotting. This can occur when there is not enough ...

  18. Phylogenetic relationships of Salmonella based on DNA sequence comparison of atpD encoding the beta subunit of ATP synthase.

    PubMed

    Christensen, H; Olsen, J E

    1998-04-01

    DNA sequences covering 57% of atpD encoding the beta subunit of ATP synthase were determined for 16 strains of Salmonella enterica, two strains of S. bongori, and one strain each of Citrobacter freundii and Yersinia enterocolitica, and comparison was made with the published Escherichia coli and Enterobacter aerogenes sequences. The phylogenetic tree based on maximum-likelihood analysis showed separation of the subspecies of S. enterica except for two serotypes of subspecies II which were unsupported by a common node. The two serotypes of S. bongori were separated from S. enterica and related to the serotypes of subspecies II. A tight relationship was found between S. enterica subspecies IIIa consisting of monophasic serotypes and subspecies IIIb consisting of diphasic serotypes. This is in conflict with results obtained for most other housekeeping genes and the 23S rRNA gene separating mono- from diphasic subspecies.

  19. Mammalian copper-transporting P-Type ATPases, ATP7A and ATP7B: Emerging roles

    PubMed Central

    La Fontaine, Sharon; Ackland, M. Leigh; Mercer, Julian F.B.

    2010-01-01

    Copper (Cu) has a role in a diverse and increasing number of pathways, physiological and disease processes. These roles are testament to the fundamental importance of Cu in biology and the need to understand the mechanisms that regulate Cu homeostasis. The mammalian Cu-transporting P-type ATPases ATP7A and ATP7B are two key proteins that regulate the Cu status of the body. They transport Cu across cellular membranes for biosynthetic and protective functions, enabling Cu to fulfill its role as a structural cofactor for many essential enzymes, and to prevent a toxic build-up of Cu inside cells. A variety of regulatory mechanisms operate at transcriptional and post-translational levels to ensure adequate Cu supplies for both physiological and pathophysiological processes. This review summarizes the recent literature that is revealing the emerging roles of the Cu-ATPases in health and disease. PMID:19922814

  20. Protons, the thylakoid membrane, and the chloroplast ATP synthase.

    PubMed

    Junge, W

    1989-01-01

    According to the chemiosmotic theory, proton pumps and ATP synthases are coupled by lateral proton flow through aqueous phases. Three long-standing challenges to this concept, all of which have been loosely subsumed under 'localized coupling' in the literature, were examined in the light of experiments carried out with thylakoids: (1) Nearest neighbor interaction between pumps and ATP synthases. Considering the large distances between photosystem II and CFoCF1, in stacked thylakoids this is a priori absent. (2) Enhanced proton diffusion along the surface of the membrane. This could not be substantiated for the outer side of the thylakoid membrane. Even for the interface between pure lipid and water, two laboratories have reported the absence of enhanced diffusion. (3) Localized proton ducts in the membrane. Intramembrane domains that can transiently trap protons do exist in thylakoid membranes, but because of their limited storage capacity for protons, they probably do not matter for photophosphorylation under continuous light. Seemingly in favor of localized proton ducts is the failure of a supposedly permeant buffer to enhance the onset lag of photophosphorylation. However, it was found that failure of some buffers and the ability of others in this respect were correlated with their failure/ability to quench pH transients in the thylakoid lumen, as predicted by the chemiosmotic theory. It was shown that the chemiosmotic concept is a fair approximation, even for narrow aqueous phases, as in stacked thylakoids. These are approximately isopotential, and protons are taken in by the ATP synthase straight from the lumen. The molecular mechanism by which F0F1 ATPases couple proton flow to ATP synthesis is still unknown. The threefold structural symmetry of the headpiece that, probably, finds a corollary in the channel portion of these enzymes appeals to the common wisdom that structural symmetry causes functional symmetry. "Rotation catalysis" has been proposed. It is

  1. Oligomycin Inhibition of Phosphate Uptake and ATP Labeling in Excised Maize Roots 1

    PubMed Central

    Bledsoe, Carolyn; Cole, C. V.; Ross, Cleon

    1969-01-01

    ATP labeling by newly absorbed 32P in excised maize roots was reduced 34% by the presence of oligomycin during a 4-min uptake period with no reduction in rate of phosphorus absorption. Longer exposure to oligomycin, during pretreatment periods or longer uptake periods, reduced phosphorus absorption and further reduced ATP synthesis. In these tissues it appears that oligomycin inhibits ATP production at the mitochondria, that ATP is the energy source for phosphorus uptake at the plasmalemma, and that a depletion in the ATP supply causes a reduced rate of uptake. Images PMID:16657154

  2. Alternating hemiplegia of childhood with a de novo mutation in ATP1A3 and changes in SLC2A1 responsive to a ketogenic diet.

    PubMed

    Ulate-Campos, Adriana; Fons, Carmen; Artuch, Rafael; Castejón, Esperanza; Martorell, Loreto; Ozelius, Laurie; Pascual, Juan; Campistol, Jaume

    2014-04-01

    Alternating hemiplegia of childhood (AHC) is a rare condition characterized by an early onset of hemiplegic episodes and other paroxysmal or permanent neurological dysfunctions. Recently, mutations in the ATP1A3 gene have been identified as the causal mechanism of AHC. Regarding the differential diagnosis of AHC, glucose transporter 1 deficiency syndrome may be considered because these two disorders share some paroxystic and nonparoxystic features. We report a typical case of AHC harboring a de novo mutation in the ATP1A3 gene, together with a duplication and insertion in the SLC2A1 gene who exhibited marked clinical improvement following ketogenic diet. Because the contribution of the SLC2A1 mutation to the clinical phenotype cannot be definitely demonstrated, the remarkable clinical response after ketogenic diet led us to the hypothesis that ketogenic diet might be effective in AHC as it provides an alternative energy source for the brain. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Imaging cytochrome C oxidase and FoF1-ATP synthase in mitochondrial cristae of living human cells by FLIM and superresolution microscopy

    NASA Astrophysics Data System (ADS)

    Foertsch, Franziska; Ilchenko, Mykhailo; Heitkamp, Thomas; Noßmann, Silke; Hoffmann, Birgit; Starke, Ilka; Mrowka, Ralf; Biskup, Christoph; Börsch, Michael

    2017-02-01

    Cytochrome C oxidase and FoF1-ATP synthase constitute complex IV and V, respectively, of the five membrane-bound enzymes in mitochondria comprising the respiratory chain. These enzymes are located in the inner mitochondrial membrane (IMM), which exhibits large invaginations called cristae. According to recent electron cryotomography, FoF1-ATP synthases are located predominantly at the rim of the cristae, while cytochrome C oxidases are likely distributed in planar membrane areas of the cristae. Previous FLIM measurements (K. Busch and coworkers) of complex II and III unravelled differences in the local environment of the membrane enzymes in the cristae. Here, we tagged complex IV and V with mNeonGreen and investigated their mitochondrial nano-environment by FLIM and superresolution microscopy in living human cells. Different lifetimes and anisotropy values were found and will be discussed.

  4. Hemolysis is a primary ATP-release mechanism in human erythrocytes

    PubMed Central

    Sikora, Jacek; Orlov, Sergei N.; Furuya, Kishio

    2014-01-01

    The hypothesis that regulated ATP release from red blood cells (RBCs) contributes to nitric oxide-dependent control of local blood flow has sparked much interest in underlying release mechanisms. Several stimuli, including shear stress and hypoxia, have been found to induce significant RBC ATP release attributed to activation of ATP-conducting channels. In the present study, we first evaluated different experimental approaches investigating stimulated RBC ATP release and quantifying hemolysis. We then measured ATP and free hemoglobin in each and every RBC supernatant sample to directly assess the contribution of hemolysis to ATP release. Hypotonic shock, shear stress, and hypoxia, but not cyclic adenosine monophosphate agonists, significantly enhanced ATP release. It tightly correlated, however, with free hemoglobin in RBC supernatants, indicating that lysis was responsible for most, if not all, ATP release. Luminescence ATP imaging combined with simultaneous infrared cell imaging showed that ATP was released exclusively from lysing cells with no contribution from intact cells. In summary, with all stimuli tested, we found no evidence of regulated ATP release from intact RBCs other than by cell lysis. Such a release mechanism might be physiologically relevant in vivo, eg, during exercise and hypoxia where intravascular hemolysis, predominantly of senescent cells, is augmented. PMID:25097178

  5. ATP transport through a single mitochondrial channel, VDAC, studied by current fluctuation analysis.

    PubMed Central

    Rostovtseva, T K; Bezrukov, S M

    1998-01-01

    The "molecular Coulter counter" concept has been used to study transport of ATP molecules through the nanometer-scale aqueous pore of the voltage-dependent mitochondrial ion channel, VDAC. We examine the ATP-induced current fluctuations and the change in average current through a single fully open channel reconstituted into a planar lipid bilayer. At high salt concentration (1 M NaCl), the addition of ATP reduces both solution conductivity and channel conductance, but the effect on the channel is several times stronger and shows saturation behavior even at 50 mM ATP concentration. These results and simple steric considerations indicate pronounced attraction of ATP molecules to VDAC's aqueous pore and permit us to evaluate the effect of a single ATP molecule on channel conductance. ATP addition also generates an excess noise in the ionic current through the channel. Analysis of this excess noise shows that its spectrum is flat in the accessible frequency interval up to several kilohertz. ATP exchange between the pore and the bulk is fast enough not to display any dispersion at these frequencies. By relating the low-frequency spectral density of the noise to the equilibrium diffusion of ATP molecules in the aqueous pore, we calculate a diffusion coefficient D = (1.6-3.3)10(-11) m2/s. This is one order of magnitude smaller than the ATP diffusion coefficient in the bulk, but it agrees with recent results on ATP flux measurements in multichannel membranes using the luciferin/luciferase method. PMID:9591663

  6. Spike-independent release of ATP from Xenopus spinal neurons evoked by activation of glutamate receptors

    PubMed Central

    Brown, Paul; Dale, Nicholas

    2002-01-01

    As the release of ATP from neurons has only been directly studied in a few cases, we have used patch sniffing to examine ATP release from Xenopus spinal neurons. ATP release was detected following intracellular current injection to evoke spikes. However, spiking was not essential as both glutamate and NMDA could evoke release of ATP in the presence of TTX. Neither acetylcholine nor high K+ was effective at inducing ATP release in the presence of TTX. Although Cd2+ blocked glutamate-evoked release of ATP suggesting a dependence on Ca2+ entry, neither ω-conotoxin-GVIA nor nifedipine prevented ATP release. N-type and L-type channels are thus not essential for glutamate-evoked ATP release. That glutamate receptors can elicit release in the absence of spiking suggests a close physical relationship between these receptors, the Ca2+ channels and release sites. As the dependence of ATP release on the influx of Ca2+ through Ca2+ channel subtypes differs from that of synaptic transmitter release, ATP may be released from sites that are distinct from those of the principal transmitter. In addition to its role as a fast transmitter, ATP may thus be released as a consequence of the activation of excitatory glutamatergic synapses and act to signal information about activity patterns in the nervous system. PMID:11986374

  7. 77 FR 39626 - Further Definition of “Swap Dealer,” “Security-Based Swap Dealer,” “Major Swap Participant...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-05

    ... the third column, correct paragraph (hhh)(6)(iii)(B)(2) to read as follows: Sec. 1.3 Definitions. * * * * * (hhh) * * * (6) * * * (iii) * * * (B) * * * (2) The sum of the amount calculated under paragraph (hhh...

  8. Toward a multiscale description of microvascular flow regulation: o(2)-dependent release of ATP from human erythrocytes and the distribution of ATP in capillary networks.

    PubMed

    Goldman, Daniel; Fraser, Graham M; Ellis, Christopher G; Sprague, Randy S; Ellsworth, Mary L; Stephenson, Alan H

    2012-01-01

    Integration of the numerous mechanisms that have been suggested to contribute to optimization of O(2) supply to meet O(2) need in skeletal muscle requires a systems biology approach which permits quantification of these physiological processes over a wide range of length scales. Here we describe two individual computational models based on in vivo and in vitro studies which, when incorporated into a single robust multiscale model, will provide information on the role of erythrocyte-released ATP in perfusion distribution in skeletal muscle under both physiological and pathophysiological conditions. Healthy human erythrocytes exposed to low O(2) tension release ATP via a well characterized signaling pathway requiring activation of the G-protein, Gi, and adenylyl cyclase leading to increases in cAMP. This cAMP then activates PKA and subsequently CFTR culminating in ATP release via pannexin 1. A critical control point in this pathway is the level of cAMP which is regulated by pathway-specific phosphodiesterases. Using time constants (~100 ms) that are consistent with measured erythrocyte ATP release, we have constructed a dynamic model of this pathway. The model predicts levels of ATP release consistent with measurements obtained over a wide range of hemoglobin O(2) saturations (sO(2)). The model further predicts how insulin, at concentrations found in pre-diabetes, enhances the activity of PDE3 and reduces intracellular cAMP levels leading to decreased low O(2)-induced ATP release from erythrocytes. The second model, which couples O(2) and ATP transport in capillary networks, shows how intravascular ATP and the resulting conducted vasodilation are affected by local sO(2), convection and ATP degradation. This model also predicts network-level effects of decreased ATP release resulting from elevated insulin levels. Taken together, these models lay the groundwork for investigating the systems biology of the regulation of microvascular perfusion distribution by

  9. Extracellular ATP activates an Arabidopsis plasma membrane Ca2+-permeable conductance

    PubMed Central

    Shang, Zhonglin; Laohavisit, Anuphon

    2009-01-01

    Extracellular ATP has been found to elevate cytosolic free Ca2+ in Arabidopsis thaliana and trigger gene transcription, suggesting that it acts as a plant cell regulator. Recent findings place extracellular ATP upstream of Arabidopsis thaliana NADPH oxidase activity and plasma membrane Ca2+-permeable channels in the root epidermis. Here we show that increasing extracellular ATP concentration evokes a larger but more irregular Ca2+ influx conductance in root epidermal protoplasts. This may help modulate changes in cytosolic free Ca2+ as a second messenger and help explain the dose-dependent effects of extracellular ATP on cell function. The receptors for ATP and the downstream plasma membrane Ca2+ channels remain unknown at the protein or gene level. No equivalents of animal ATP receptors have been identified in higher plant genomes. We propose here that annexins could perceive extracellular ATP and participate in Ca2+ influx. PMID:19826233

  10. Coassembly of Photosystem II and ATPase as Artificial Chloroplast for Light-Driven ATP Synthesis.

    PubMed

    Feng, Xiyun; Jia, Yi; Cai, Peng; Fei, Jinbo; Li, Junbai

    2016-01-26

    Adenosine triphosphate (ATP) is one of the most important energy sources in living cells, which can drive serial key biochemical processes. However, generation of a proton gradient for ATP production in an artificial way poses a great challenge. In nature, photophosphorylation occurring in chloroplasts is an ideal prototype of ATP production. In this paper we imitate the light-to-ATP conversion process occurring in the thylakoid membrane by construction of FoF1-ATPase proteoliposome-coated PSII-based microspheres with well-defined core@shell structures using molecular assembly. Under light illumination, PSII can split water into protons, oxygen, and electrons and can generate a proton gradient for ATPase to produce ATP. Thus, an artificially designed chloroplast for PSII-driven ATP synthesis is realized. This biomimetic system will help to understand the photophosphorylation process and may facilitate the development of ATP-driven devices by remote light control.

  11. ATP oscillations mediate inductive action of FGF and Shh signalling on prechondrogenic condensation.

    PubMed

    Kwon, Hyuck Joon

    2013-01-01

    Skeletal patterns are prefigured by prechondrogenic condensation. Morphogens such as fibroblast growth factor (FGF) and sonic hedgehog (Shh) specify the skeletal patterns in limb development. However, how morphogens regulate prechondrogenic condensation has remained unclear. Recently, it was demonstrated that synchronized Adenosine triphosphate (ATP) oscillations play a critical role in prechondrogenic condensation. Thus, the present study has focused on whether ATP oscillations mediate the actions of major developmental morphogens such as FGF and Shh on prechondrogenic condensation. It has been shown that both FGF and Shh signalling promoted cellular condensation but not chondrogenic differentiation and also induced ATP oscillations. In addition, blockage of FGF and Shh signalling prevented both ATP oscillations and prechondrogenic condensation. Furthermore, it was found that inhibition of ATP oscillations suppressed FGF/Shh-induced prechondrogenic condensation. These results indicate that ATP oscillations mediate the actions of FGF and Shh signalling on prechondrogenic condensation. This study proposes that morphogens organize skeletal patterns via ATP oscillations.

  12. Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay.

    PubMed

    Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji

    2008-08-01

    We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87(N). Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.

  13. Isolation of ATP from a yeast fermentation broth using a cryogel column at high flow velocities.

    PubMed

    Yan, Chen; Shen, Shaochuan; Yun, Junxian; Wang, Lianghua; Yao, Kejian; Yao, Shan-Jing

    2008-12-01

    This communication presents an effective method for isolating adenosine triphosphate (ATP) from a yeast fermentation broth using an anion-exchange supermacroporous cryogel column at high flow velocities. The breakthrough and elution behaviors of pure ATP in the cryogel bed were investigated at flow velocities of 2, 5, and 10 cm/min and the ATP binding capacities were determined. Then the ATP-containing yeast fermentation broth was employed as the test feedstock and various chromatographic runs were conducted to isolate ATP by the cryogel at different high flow velocities. The ATP samples obtained were analyzed quantitatively by HPLC. The results showed that even at a flow velocity of 5 or 10 cm/min, a product purity of 97.4 or 98.0% can be achieved, illustrating the potential of the present method for separation of high-purity ATP directly from fermentation feedstock at high flow velocities.

  14. ATP protects, by way of receptor-mediated mechanisms, against hypoxia-induced injury in renal proximal tubules.

    PubMed

    Kribben, Andreas; Feldkamp, Thorsten; Horbelt, Markus; Lange, Bettina; Pietruck, Frank; Herget-Rosenthal, Stefan; Heemann, Uwe; Philipp, Thomas

    2003-01-01

    We examined the effect of ATP on hypoxia-induced injury in freshly isolated rat renal proximal tubules and compared it with the effects of stable ATP analogues and ATP degradation products. Extracellular ATP significantly reduced hypoxia-induced structural cell damage (lactate dehydrogenase release). P(2)-receptor agonistic ATP analogues, including 2'-methylthio-ATP (2-Me-S-ATP), were also protective. In contrast, the P(1)-agonistic degradation products AMP and adenosine were not protective. Hypoxia-induced functional cell damage (loss of cellular potassium) was not changed by ATP or 2-Me-S-ATP. We therefore conclude that the protective property of ATP is not based on an effect of the degradation products or on a direct effect on cellular energy metabolism. The data indicate that the protective effect of ATP is mediated by P(2) receptors.

  15. [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.

  16. Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase

    PubMed Central

    He, Jiuya; Ford, Holly C.; Carroll, Joe; Ding, Shujing; Fearnley, Ian M.

    2017-01-01

    The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme’s rotor. The c-subunit is produced from three nuclear genes, ATP5G1, ATP5G2, and ATP5G3, encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1, ATP5G2, and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F1-catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP. PMID:28289229

  17. Developmental changes in the expression of ATP7A during a critical period in postnatal neurodevelopment.

    PubMed

    Niciu, M J; Ma, X-M; El Meskini, R; Ronnett, G V; Mains, R E; Eipper, B A

    2006-01-01

    ATP7A is a P-type ATPase that transports copper from cytosol into the secretory pathway for loading onto cuproproteins or efflux. Mutations in Atp7a cause Menkes disease, a copper-deficiency disorder fatal in the postnatal period due to severe neurodegeneration. Early postnatal copper injections are known to diminish degenerative changes in some human patients and mice bearing mutations in Atp7a. In situ hybridization studies previously demonstrated that ATP7A transcripts are expressed widely in the brain. ATP7A-specific antibody was used to study the neurodevelopmental expression and localization of ATP7A protein in the mouse brain. Based on immunoblot analyses, ATP7A expression is most abundant in the early postnatal period, reaching peak levels at P4 in neocortex and cerebellum. In the developing and adult brain, ATP7A levels are greatest in the choroid plexus/ependymal cells of the lateral and third ventricles. ATP7A expression decreases in most neuronal subpopulations from birth to adulthood. In contrast, ATP7A expression increases in CA2 hippocampal pyramidal and cerebellar Purkinje neurons. ATP7A is expressed in a subset of astrocytes, microglia, oligodendrocytes, tanycytes and endothelial cells. ATP7A is largely localized to the trans-Golgi network, adopting the cell-specific and developmentally-regulated morphology of this organelle. The presence of ATP7A in the axons of postnatal, but not adult, optic nerve suggests stage-specific roles for this enzyme. In sum, the precisely-regulated neurodevelopmental expression of ATP7A correlates well with the limited therapeutic window for effective treatment of Menkes disease.

  18. ATP facilitates spontaneous glycinergic IPSC frequency at dissociated rat dorsal horn interneuron synapses

    PubMed Central

    Rhee, Jeong Seop; Wang, Zhi Ming; Nabekura, Junichi; Inoue, Kazuhide; Akaike, Norio

    2000-01-01

    The ATP action on spontaneous miniature glycinergic inhibitory postsynaptic currents (mIPSCs) was investigated in rat substantia gelatinosa (SG) neurons mechanically dissociated from the 2nd layer of the dorsal horn in which their presynaptic glycinergic nerve terminals remained intact.ATP reversibly facilitated the frequency of the mIPSCs in a concentration-dependent manner without affecting their amplitude distribution. The ATP agonist, 2-methylthioATP (2MeSATP), mimicked the ATP action, while another ATP receptor agonist, αβ-methylene-ATP (α,β-meATP), had no effect on mIPSCs.The ATP receptor antagonists, suramin (1 × 10−6 M) and pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (1 × 10−5 M), completely blocked the facilitatory effect of ATP on glycine release (102·0 ± 11·2 % and 99·3 ± 16·2 %, n = 6, respectively) without altering the current amplitude distributions.N-Ethylmaleimide (NEM), a sulphydryl alkylating agent, suppressed the inhibitory effect of adenosine on mIPSC frequency (111·2 ± 13·3 %, n = 4) without altering the current amplitude distribution. However, ATP still facilitated the mIPSC frequency (693·3 ± 245·2 %, n = 4) even in the presence of NEM.The facilitatory effect of ATP (1 × 10−5 M) on mIPSC frequency was not affected by adding 1 × 10−4 M Cd2+ to normal external solution but was eliminated in a Ca2+-free external solution.These results suggest that ATP enhances glycine release from nerve terminals, presumably resulting in the inhibition of SG neurons which conduct nociceptive signals to the CNS. This presynaptic P2X-type ATP receptor may function to prevent excess excitability in SG neurons, thus preventing an excessive pain signal and/or SG cell death. PMID:10766927

  19. The ATP requirements of adenovirus type 5 DNA replication and cellular DNA replication.

    PubMed

    De Jong, P J; Kwant, M M; van Driel, W; Jansz, H S; van der Vliet, P C

    1983-01-15

    Several in vitro DNA replication systems were employed to characterize the ATP dependency of adenovirus type 5 (Ad5) DNA replication. Ad5 DNA synthesis in isolated nuclei, representing the elongation of nascent DNA chains, was slightly ATP dependent. Reduction of the ATP concentration from the optimum (8 mM) to the endogenous value (0.16 microM) reduced Ad5 DNA replication only to 70%. No change in the pattern of replication was observed as indicated by the analysis of replicative intermediates using agarose gel electrophoresis. ATP could be replaced by dATP, but not by GTP or other nucleoside triphosphates. By contrast, cellular DNA replication in isolated nuclei from HeLa cells was reduced to 12% by the omission of ATP. These differences could not be explained by different ATP pools or by effects of ATP on dNTP pools. Cellular DNA replication in contrast to viral DNA replication was sensitive to low concentrations of adenosine 5'-O-(3-thiotriphosphate). Inhibition by this ATP analog was competitive with ATP (Ki = 0.4 mM). Adenovirus DNA replication by DNA-free nuclear extracts, representing initiation plus elongation (Challberg and Kelly, Proc. Nat. Acad. Sci. USA 76, 655-659, 1979), exhibited a nearly absolute requirement for ATP. ATP could be substituted not only by dATP, but also by GTP and dGTP and to a lesser extent by pyrimidine triphosphates. Similar results were found when the formation of a covalent complex between dCTP and the precursor terminal protein was studied. This reaction is essential for the initiation of Ad5 DNA replication. The results indicate that different ATP-requiring functions are employed during the initiation and elongation stages of adenovirus DNA replication.

  20. ATP/ADP Turnover and Import of Glycolytic ATP into Mitochondria in Cancer Cells Is Independent of the Adenine Nucleotide Translocator.

    PubMed

    Maldonado, Eduardo N; DeHart, David N; Patnaik, Jyoti; Klatt, Sandra C; Gooz, Monika Beck; Lemasters, John J

    2016-09-09

    Non-proliferating cells oxidize respiratory substrates in mitochondria to generate a protonmotive force (Δp) that drives ATP synthesis. The mitochondrial membrane potential (ΔΨ), a component of Δp, drives release of mitochondrial ATP(4-) in exchange for cytosolic ADP(3-) via the electrogenic adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane, which leads to a high cytosolic ATP/ADP ratio up to >100-fold greater than matrix ATP/ADP. In rat hepatocytes, ANT inhibitors, bongkrekic acid (BA), and carboxyatractyloside (CAT), and the F1FO-ATP synthase inhibitor, oligomycin (OLIG), inhibited ureagenesis-induced respiration. However, in several cancer cell lines, OLIG but not BA and CAT inhibited respiration. In hepatocytes, respiratory inhibition did not collapse ΔΨ until OLIG, BA, or CAT was added. Similarly, in cancer cells OLIG and 2-deoxyglucose, a glycolytic inhibitor, depolarized mitochondria after respiratory inhibition, which showed that mitochondrial hydrolysis of glycolytic ATP maintained ΔΨ in the absence of respiration in all cell types studied. However in cancer cells, BA, CAT, and knockdown of the major ANT isoforms, ANT2 and ANT3, did not collapse ΔΨ after respiratory inhibition. These findings indicated that ANT did mediate mitochondrial ATP/ADP exchange in cancer cells. We propose that suppression of ANT contributes to low cytosolic ATP/ADP, activation of glycolysis, and a Warburg metabolic phenotype in proliferating cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Time course of the initial [Ca2+]i response to extracellular ATP in smooth muscle depends on [Ca2+]e and ATP concentration.

    PubMed Central

    Mahoney, M G; Slakey, L L; Benham, C D; Gross, D J

    1998-01-01

    In response to extracellular application of 50 microM ATP, all individual porcine aortic smooth muscle cells respond with rapid rises from basal [Ca2+]i to peak [Ca2+]i within 5 s. The time from stimulus to the peak of the [Ca2+]i response increases with decreasing concentration of ATP. At ATP concentrations of 0.5 microM and below, the time to the [Ca2+]i peak varies more significantly from cell to cell than at higher concentrations, and each cell shows complicated initiation and decay kinetics. For any individual cell, the lag phase before a response decreases with increasing concentration of ATP. An increase in lag time with decreasing ATP concentration is also observed in the absence of extracellular Ca2+, but the lag phase is more pronounced, especially at concentrations of ATP below 0.5 microM. Whole-cell patch-clamp electrophysiology shows that in porcine aortic smooth muscle cells, ATP stimulates an inward current carried mainly by Cl- ion efflux with a time course similar to the [Ca2+]i changes and no detectable current from an ATP-gated cation channel. A simple signal cascade initiation kinetics model, starting with nucleotide receptor activation leading to IP3-mediated Ca2+ release from IP3-sensitive internal stores, fits the data and suggests that the kinetics of the Ca2+ response are dominated by upstream signal cascade components. PMID:9746547

  2. 12 CFR 221.2 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... holding company of which a bank is a subsidiary within the meaning of the Bank Holding Company Act of 1956, as amended (12 U.S.C. 1841(d)); (ii) Any other subsidiary of such bank holding company; and (iii) Any... 12 Banks and Banking 3 2012-01-01 2012-01-01 false Definitions. 221.2 Section 221.2 Banks and...

  3. Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases.

    PubMed

    Günther Sillero, María A; de Diego, Anabel; Pérez-Zúñiga, Francisco J; Sillero, Antonio

    2008-05-15

    T4 DNA ligase and the ubiquitin activating enzyme (E1), catalyze the synthesis of ATP beta,gamma-bisphosphonate derivatives. Concerning T4 DNA ligase: (i) etidronate (pC(OH)(CH(3))p) displaced the AMP moiety of the complex E-AMP in a concentration dependent manner; (ii) the K(m) values and the rate of synthesis k(cat) (s(-1)), determined for the following compounds were, respectively: etidronate, 0.73+/-0.09 mM and (70+/-10)x10(-3) s(-1); clodronate (pCCl(2)p), 0.08+/-0.01 mM and (4.1+/-0.3)x10(-3) s(-1); methylenebisphosphonate (pCH(2)p), 0.024+/-0.001 mM and (0.6+/-0.1)x10(-3) s(-1); tripolyphosphate (P(3)) (in the synthesis of adenosine 5'-tetraphosphate, p(4)A), 1.30+/-0.30 mM and (6.2+/-1.1)x10(-3) s(-1); (iii) in the presence of GTP and ATP, inhibition of the synthesis of Ap(4)G was observed with clodronate but not with pamidronate (pC(OH)(CH(2)-CH(2)-NH(3))p). Concerning the ubiquitin activating enzyme (E1): methylenebisphosphonate was the only bisphosphonate, out of the ones tested, that served as substrate for the synthesis of an ATP derivative (K(m)=0.36+/-0.09 mM and k(cat)=0.15+/-0.02 s(-1)). None of the above bisphosphonates were substrates of the reaction catalyzed by luciferase or by acyl-CoA synthetase. The ability of acetyl-CoA synthetase to use methylenebisphosphonate as substrate depended on the commercial source of the enzyme. In our view this report widens our knowledge of the enzymes able to metabolize bisphosphonates, a therapeutic tool widely used in the treatment of osteoporosis.

  4. Type III burst pair

    NASA Astrophysics Data System (ADS)

    Ning, Zongjun; Fu, Qijun; Lu, Quankang

    2000-05-01

    We present a special solar radio burst detected on 5 January 1994 using the multi-channel (50) spectrometer (1.0-2.0 GHz) of the Beijing Astronomical Observatory (BAO). Sadly, the whole event could not be recorded since it had a broader bandwidth than the limit range of the instrument. The important part was obtained, however. The event is composed of a normal drift type III burst on the lower frequency side and a reverse drift type III burst appearing almost simultaneously on the high side. We call the burst type III a burst pair. It is a typical characteristic of two type III bursts that they are morphologically symmetric about some frequency from 1.64 GHz to 1.78 GHz on the dynamic spectra records, which indicates that there are two different electron beams from the same acceleration region travelling simultaneously in opposite directions (upward and downward). A magnetic reconnection mode is a nice interpretation of type III burst pair since the plasma beta β~=0.01 is much less than 1 and the beams have velocity of about 1.07×10^8 cm s^-1 after leaving the reconnection region if we assume that the ambient magnetic field strength is about 100 G.

  5. Type III burst pair.

    NASA Astrophysics Data System (ADS)

    Zongjun, Ning; Fu, Qijun; Quankang, Lu

    2000-05-01

    Presents a special solar radio burst detected on 5 January 1994 using the multi-channel (50) spectrometer (1.0 - 2.0 GHz) of the Beijing Astronomical Observatory. Sadly, the whole event could not be recorded since it had a broader bandwidth than the limit range of the instrument. The important part was obtained, however. The event is composed of a normal drift type III burst on the lower frequency side and a reverse drift type III burst appearing almost simultaneously on the high side. The authors call the burst type III a burst pair. It is a typical characteristic of two type III bursts that they are morphologically symmetric about some frequency from 1.64 GHz to 1.78 GHz on the dynamic spectra records, which indicates that there are two different electron beams from the same acceleration region travelling simultaneously in opposite directions (upward and downward). A magnetic reconnection mode is an interpretation of type III burst pair.

  6. Bifidobacterium lactis DSM 10140: Identification of the atp (atpBEFHAGDC) Operon and Analysis of Its Genetic Structure, Characteristics, and Phylogeny

    PubMed Central

    Ventura, Marco; Canchaya, Carlos; van Sinderen, Douwe; Fitzgerald, Gerald F.; Zink, Ralf

    2004-01-01

    The atp operon is highly conserved among eubacteria, and it has been considered a molecular marker as an alternative to the 16S rRNA gene. PCR primers were designed from the consensus sequences of the atpD gene to amplify partial atpD sequences from 12 Bifidobacterium species and nine Lactobacillus species. All PCR products were sequenced and aligned with other atpD sequences retrieved from public databases. Genes encoding the subunits of the F1F0-ATPase of Bifidobacterium lactis DSM 10140 (atpBEFHAGDC) were cloned and sequenced. The deduced amino acid sequences of these subunits showed significant homology with the sequences of other organisms. We identified specific sequence signatures for the genus Bifidobacterium and for the closely related taxa Bifidobacterium lactis and Bifidobacterium animalis and Lactobacillus gasseri and Lactobacillus johnsonii, which could provide an alternative to current methods for identification of lactic acid bacterial species. Northern blot analysis showed that there was a transcript at approximately 7.3 kb, which corresponded to the size of the atp operon, and a transcript at 4.5 kb, which corresponded to the atpC, atpD, atpG, and atpA genes. The transcription initiation sites of these two mRNAs were mapped by primer extension, and the results revealed no consensus promoter sequences. Phylogenetic analysis of the atpD genes demonstrated that the Lactobacillus atpD gene clustered with the genera Listeria, Lactococcus, Streptococcus, and Enterococcus and that the higher G+C content and highly biased codon usage with respect to the genome average support the hypothesis that there was probably horizontal gene transfer. The acid inducibility of the atp operon of B. lactis DSM 10140 was verified by slot blot hybridization by using RNA isolated from acid-treated cultures of B. lactis DSM 10140. The rapid increase in the level of atp operon transcripts upon exposure to low pH suggested that the ATPase complex of B. lactis DSM 10140 was

  7. 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

  8. ATP depletion inhibits the endocytosis of ClC-2.

    PubMed

    Dhani, Sonja U; Kim Chiaw, Patrick; Huan, Ling-Jun; Bear, Christine E

    2008-01-01

    The chloride channel, ClC-2 is expressed ubiquitously and participates in multiple physiological processes. In particular, ClC-2 has been implicated in the regulation of neuronal chloride ion homeostasis and mutations in ClC-2 are associated with idiopathic generalized epilepsy. Despite the physiological and pathophysiological significance of this channel, its regulation remains incompletely understood. The functional expression of ClC-2 at the cell surface has been shown to be enhanced by depletion of cellular ATP, implicating its possible role in cellular energy sensing. In the present study, biochemical assays of cell surface expression suggest that this gain of function reflects, in part, an increase in channel number due to the reduction in ClC-2 internalization by endocytosis. Cell surface expression of the disease-causing mutant: G715E, thought to lack wild-type nucleotide binding affinity, is similarly affected, suggesting that ATP-depletion modifies the function of proteins in the endocytic pathway rather than ClC-2 directly. Using a combination of immunofluorescence and biochemical studies, we confirmed that ClC-2 is internalized via dynamin-dependent endocytosis and that the change in surface expression evoked by ATP depletion is partially mimicked by inhibition of dynamin function using a dynamin dominant-negative mutant (DynK44A). Furthermore, trafficking via the early endosomal compartment occurs in part through rab5-associated vesicles and recycling of ClC-2 to the cell surface occurs through a rab11 dependent pathway. In summary, we have determined that the internalization of ClC-2 by endocytosis is inhibited by metabolic stress, highlighting the importance for understanding the molecular mechanisms mediating the endosomal trafficking of this channel. (c) 2007 Wiley-Liss, Inc.

  9. Glutamate and ATP signalling in white matter pathology

    PubMed Central

    Matute, Carlos

    2011-01-01

    Excessive signalling by excitatory neurotransmitters like glutamate and ATP can be deleterious to neurons and oligodendroglia, and cause disease. In particular, sustained activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-d-aspartate (NMDA) receptors damages oligodendrocytes, a feature that depends entirely on Ca2+ overload of the cytoplasm and that can be initiated by disruption of glutamate homeostasis. Thus, inhibition of glutamate uptake by activated microglia can compromise glutamate homeostasis and induce oligodendrocyte excitotoxicity. Moreover, non-lethal, brief activation of kainate receptors in oligodendrocytes rapidly sensitizes these cells to complement attack as a consequence of oxidative stress. In addition to glutamate, ATP signalling can directly trigger oligodendrocyte excitotoxicity via activation of Ca2+-permeable P2X7 purinergic receptors, which mediates ischaemic damage to white matter (WM) and causes lesions that are reminiscent of multiple sclerosis (MS) plaques. Conversely, blockade of P2X7 receptors attenuates post-ischaemic injury to WM and ameliorates chronic experimental autoimmune encephalomyelitis, a model of MS. Importantly, P2X7 expression is elevated in normal-appearing WM in patients with MS, suggesting that signalling through this receptor in oligodendrocytes may be enhanced in this disease. Altogether, these observations reveal novel mechanisms by which altered glutamate and ATP homeostasis can trigger oligodendrocyte death. This review aims at summarizing current knowledge about the mechanisms leading to WM damage as a consequence of altered neurotransmitter signalling, and their relevance to disease. This knowledge will generate new therapeutic avenues to treat more efficiently acute and chronic WM pathology. PMID:21250988

  10. WAIS-III and WMS-III performance in chronic Lyme disease.

    PubMed

    Keilp, John G; Corbera, Kathy; Slavov, Iordan; Taylor, Michael J; Sackeim, Harold A; Fallon, Brian A

    2006-01-01

    There is controversy regarding the nature and degree of intellectual and memory deficits in chronic Lyme disease. In this study, 81 participants with rigorously diagnosed chronic Lyme disease were administered the newest revisions of the Wechsler Adult Intelligence Scale (WAIS-III) and Wechsler Memory Scale (WMS-III), and compared to 39 nonpatients. On the WAIS-III, Lyme disease participants had poorer Full Scale and Performance IQ's. At the subtest level, differences were restricted to Information and the Processing Speed subtests. On the WMS-III, Lyme disease participants performed more poorly on Auditory Immediate, Immediate, Auditory Delayed, Auditory Recognition Delayed, and General Memory indices. Among WMS-III subtests, however, differences were restricted to Logical Memory (immediate and delayed) and Family Pictures (delayed only), a Visual Memory subtest. Discriminant analyses suggest deficits in chronic Lyme are best characterized as a combination of memory difficulty and diminished processing speed. Deficits were modest, between one-third and two-thirds of a standard deviation, consistent with earlier studies. Depression severity had a weak relationship to processing speed, but little other association to test performance. Deficits in chronic Lyme disease are consistent with a subtle neuropathological process affecting multiple performance tasks, although further work is needed to definitively rule out nonspecific illness effects.

  11. Nucleotide sequence of the Rhodospirillum rubrum atp operon.

    PubMed Central

    Falk, G; Hampe, A; Walker, J E

    1985-01-01

    The nucleotide sequence was determined of a 8775-base-pair region of DNA cloned from the photosynthetic non-sulphur bacterium Rhodospirillum rubrum. It contains a cluster of five genes encoding F1-ATPase subunits. The genes are arranged in the same order as F1 genes in the Escherichia coli unc operon. However, as in the related organism Rhodopseudomonas blastica, neither genes for components of F0, the membrane sector of ATP synthase, nor a homologue of the E. coli uncI gene are associated with this locus, as they are in E. coli. Images Fig. 2. PMID:2861810

  12. ATP-Induced Helicase Slippage Reveals Highly Coordinated Subunits

    PubMed Central

    Sun, Bo; Johnson, Daniel S.; Patel, Gayatri; Smith, Benjamin Y.; Pandey, Manjula; Patel, Smita S.; Wang, Michelle D.

    2011-01-01

    Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair, and recombination1,2. Bacteriophage T7 gene product 4 is a model hexameric helicase which has been observed to utilize dTTP, but not ATP, to unwind dsDNA as it translocates from 5′ to 3′ along ssDNA2–6. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate1,7. Here we address this question using a single molecule approach to monitor helicase unwinding. We discovered that T7 helicase does in fact unwind dsDNA in the presence of ATP and the unwinding rate is even faster than that with dTTP. However unwinding traces showed a remarkable sawtooth pattern where processive unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behavior was not observed with dTTP alone and was greatly reduced when ATP solution was supplemented with a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. We found T7 helicase binds and hydrolyzes ATP and dTTP by competitive kinetics such that the unwinding rate is dictated simply by their respective Vmax, KM, and concentrations. In contrast, processivity does not follow a simple competitive behavior and shows a cooperative dependence on nucleotide concentrations. This does not agree with an uncoordinated mechanism where each subunit functions independently, but supports a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Our data indicate that only one subunit at a time can accept a nucleotide while other subunits are nucleotide-ligated and thus interact with the DNA to ensure processivity. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding

  13. Detection and quantification of ATP in human blood serum.

    PubMed

    Akdeniz, Ali; Caglayan, Mehmet Gokhan; Polivina, Irina; Anzenbacher, Pavel

    2016-08-21

    Two fluorometric sensors based on the tri-serine tri-lactone scaffold and thiourea or sulfonamide moieties serving as hydrogen bond donors allowing for anion binding are described. The sensor utilizing thiourea as a recognition moiety shows fluorescence enhancement while the sensor with sulfonamide shows quenching upon addition of phosphates. Sensor arrays composed of two sensors are able to discriminate structurally similar organic phosphates in the presence of interferents in human blood serum. The quantitative analysis of ATP in human blood serum shows high accuracy (the root mean square error of prediction, 1.65%) without requiring any sample pretreatment.

  14. Chemical synthesis of yeast mitochondrial ATP synthase membranous subunit 8.

    PubMed

    Goetz, M; Schmitter, J M; Geoffre, S; Dufourc, E J

    1999-06-01

    Chemical synthesis of highly hydrophobic peptides and proteins remains a challenging problem. Strong interchain associations within the peptide-resin matrix have to be overcome. A synthetic strategy for solid phase peptide synthesis is proposed, mainly based on prolonged coupling time using aprotic polar solvent mixtures. A tailored chromatographic purification was required to obtain a sample sufficiently pure for structural analysis. In this work, the total chemical synthesis of the membrane-embedded yeast mitochondrial ATP synthase subunit 8 is described. The quality of the synthetic protein was checked by electrospray mass spectrometry, its tendency to adopt alpha-helical secondary structure is evidenced by circular dichroism spectroscopy.

  15. Detection of ATP hydrolysis through motion of nanoconfined DNA

    NASA Astrophysics Data System (ADS)

    Roushan, Maedeh; Livshits, Gideon; Azad, Zubair; Wang, Hong; Riehn, Robert

    Confinement of DNA to nanochannels with a cross-section of 100 ×100 nm2 and hundreds of micrometer long has previously been used to investigate the equilibrium binding properties of proteins to DNA. Here we report on the observation that a range of proteins which catalyze a modification of DNA, and that do so by hydrolyzing ATP, cause a net directed motion of nanochannel-confined DNA. We present a model for this observation that does not require any motor-like action of the protein and that is purely dependent on the catalytic properties.

  16. Terrestrial evolution of polymerization of amino acids - Heat to ATP

    NASA Technical Reports Server (NTRS)

    Fox, S. W.; Nakashima, T.

    1981-01-01

    Sets of amino acids containing sufficient trifunctional monomer are thermally polymerized at temperatures such as 65 deg; the amino acids order themselves. Various polymers have diverse catalytic activities. The polymers aggregate, in aqueous solution, to cell-like structures having those activities plus emergent properties, e.g. proliferatability. Polyamino acids containing sufficient lysine catalyze conversion of free amino acids, by ATP, to small peptides and a high molecular weight fraction. The lysine-rich proteinoid is active in solution, within suspensions of cell-like particles, or in other particles composed of lysine-rich proteinoid and homopolyribonucleotide. Selectivities are observed. An archaic polyamino acid prelude to coded protein synthesis is indicated.

  17. ATP11B Mediates Platinum Resistance in Ovarian Cancer

    DTIC Science & Technology

    2013-05-01

    rather than cations) from the outer and inner leaf- let of membrane bilayers (13, 17–23). Although the putative role of ATP11B, based on its protein... membrane protein 4 is implicated in trans-Golgi network vesicle traf- ficking. Mol Biol Cell. 1999;10(6):1957–1972. 29. Ohmichi M, Hayakawa J, Tasaka K...isolation and character- ization of cholesterol-rich membrane domains from trans-Golgi network vesicles . J Lipid Res. 2011; 52(3):582–589.

  18. The relationship between constitutive ATP release and its extracellular metabolism in isolated rat kidney glomeruli.

    PubMed

    Karczewska, J; Martyniec, L; Dzierzko, G; Stepiński, J; Angielski, S

    2007-06-01

    ATP and adenosine are important extracellular regulators of glomerular functions. In this study, ATP release from glomeruli suspension and its extracellular metabolism were investigated. Basal extraglomerular ATP concentration (1nM) increased several fold during inhibition of ecto-ATPase activity, reflecting the basal ATP release rate. Mechanical perturbation increased the amounts of ATP released from glomeruli. ATP added to glomeruli was almost completely degraded within 20 minutes. In that time, AMP was the main product of extracellular ATP metabolism. Significant accumulation of AMP was observed after 5 min (194 +/-16 microM) and 20 min (271 +/-11 microM), whereas at the same time concentration of adenosine was only 10 muM. A competitive inhibitor of ecto-5-nucleotidase alpha-beta-methylene-ADP (AOPCP), decreased extraglomerular ATP and adenosine concentration by 80% and 50%, respectively. Similarly, AMP (100 microM) also markedly reduced extraglomerular ATP accumulation, whereas IMP, its deamination product, was not effective. P1, P5-diadenosine pentaphosphate (Ap5A) - an inhibitor of ecto-adenylate kinase prevented significantly the disappearance of ATP from extraglomerular media caused by AMP. These findings demonstrate that the decrease in extracellular ATP concentration observed after addition of AOPCP or AMP is caused by the presence of ecto-adenylate kinase activity in the glomeruli. The enzyme catalyses reversible reaction 2ADP<->ATP+AMP, and a rise in the AMP concentration can lead to fall in ATP level. The present study provides evidence