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Sample records for alkaline unwinding assay

  1. Alkaline unwinding flow cytometry assay to measure nucleotide excision repair.

    PubMed

    Thyagarajan, Bharat; Anderson, Kristin E; Lessard, Christopher J; Veltri, Gregory; Jacobs, David R; Folsom, Aaron R; Gross, Myron D

    2007-03-01

    Nucleotide excision repair (NER), one of the DNA repair pathways, is the primary mechanism for repair of bulky adducts caused by physical and chemical agents, such as UV radiation, cisplatin and 4-nitroquinolones. Variations in DNA repair may be a significant risk factor for several cancers, but its measurement in epidemiological studies has been hindered by the high variability, complexity and laborious nature of currently available assays. An alkaline unwinding flow cytometric assay using UV-C radiation as a DNA-damaging agent was adapted for measurement of NER-mediated breaks. This assay was based on the principle of alkaline unwinding of strand breaks in double-stranded DNA to yield single-stranded DNA with the number of strand breaks being proportional to the amount of DNA damage. This assay measured 50,000 events per sample with several samples being analyzed per specimen, thereby providing very reliable measurements, which can be performed on a large-scale basis. Using area under the curve (AUC) to quantitate amount of NER-mediated breaks, this assay was able to detect increased NER-mediated breaks with increasing doses of UV-C radiation. The assay detected NER-mediated breaks in lymphocytes from normal donors and not in xeroderma pigmentosum lymphoblastoid cell lines indicating specificity for the detection of NER-mediated breaks. The assay measured NER-mediated breaks within G(1), S and G(2)/M phases of the cell cycle; thereby decreasing variability in measurements of NER-mediated breaks due to differences in cell cycle phases. Intraindividual variability for AUC after 120 min of repair was 15% with interindividual variability being approximately 43% for cells in the G(1) phase, indicating substantial between-subject variation and relatively low within-subject variation. Thus, the alkaline unwinding flow cytometry-based assay provides a high-throughput method for the specific measurement of NER-mediated breaks in lymphocytes.

  2. Genotoxicity of cadmium chloride in the marine gastropod Nerita chamaeleon using comet assay and alkaline unwinding assay.

    PubMed

    Sarkar, Anupam; Bhagat, Jacky; Ingole, Baban S; Rao, Durga P; Markad, Vijaykumar L

    2015-02-01

    This paper presents an evaluation of the genotoxic effects of cadmium chloride (CdCl2 ) on marine gastropod, Nerita chamaeleon following the technique of comet assay and the DNA alkaline unwinding assay (DAUA). In this study, the extent of DNA damage in gill cells of N. chamaeleon was measured after in vivo exposure to four different concentrations (10, 25, 50, and 75 µg/L) of CdCl2 . In vitro exposure of hydrogen peroxide (H2 O2 ; 1, 10, 25, and 50 µM) of the gill cells showed a significant increase in the percentage tail DNA, Olive tail moment, and tail length (TL). Significant changes in percentage tail DNA by CdCl2 exposure were observed in all exposed groups of snails with respect to those in control. Exposure to 75 µg/L of CdCl2 produced significant decrease in DNA integrity as measured by DAUA at all duration with respect to control. In vivo exposure to different concentrations of CdCl2 (10, 25, 50, and 75 µg/L) to N. chamaeleon showed considerable increase in DNA damage as observed by both alkaline comet assay and the DAUA. The extent of DNA damage in marine gastropods determined by the application of alkaline comet assay and DAUA clearly indicated the genotoxic responses of marine gastropod, N. chamaeleon to a wide range of cadmium concentration in the marine environment.

  3. Using the DNA alkaline unwinding assay to detect DNA damage in laboratory and environmentally exposed cells and tissues

    SciTech Connect

    Nacci, D.; Jackim, E.

    1990-01-01

    The DNA alkaline unwinding assay is being evaluated for use in the detection of DNA damage in marine animals exposed to environmental pollutants. In preliminary work, DNA unwinding methods were used with in vitro cell systems to demonstrate DNA strand breaks. Cultured mammalian fibroblasts and sperm from marine fish and invertebrates (Pseudopleuronectes, Arbacia and Mytilus) showed concentration-dependent increases in DNA strand breaks after brief exposures to alkylating agents. DNA unwinding methods were also used on DNA extracted from marine animals injected with genotoxicants and from animals exposed in situ at an estuarine site. Gills from blue mussels caged at the New Bedford Harbor Superfund Site (MA, USA) highly contaminated with many organic (e.g. PCBs) and inorganic contaminants, were also examined. A significant increase in DNA strand breaks was seen in gill tissues of animals held in the contaminated site for as little as 3 days. Although not as severe, an increase in strand breaks was also seen in animals held at the control site for 28 days.

  4. Shortening of alkaline DNA unwinding time does not interfere with detecting DNA damage to mouse and human spermatozoa in the comet assay.

    PubMed

    Kusakabe, Hirokazu; Tateno, Hiroyuki

    2011-01-01

    The comet assay was performed on mouse and human spermatozoa to examine the effect of alkaline DNA unwinding time. The spermatozoa were treated in vitro with the DNA-damaging agents, methyl methanesulfonate (MMS) or hydrogen peroxide (H₂O₂), and then embedded in agarose gel on glass slides. The slides were immersed in alkaline solution (> pH 13) for 1, 5, 10 and 20 min, and then subjected to the electrophoresis under neutral conditions. In mouse spermatozoa, comet tails seen in solvent controls became brighter and longer as the alkaline DNA unwinding time increased. However, in the MMS-treated mouse spermatozoa, a smaller difference in the damage from that in the solvent control was seen with time within a dose. DNA damage induced by H₂O₂ could also be detected accurately after alkali treatment for 1-20 min. In human spermatozoa, DNA damage induced by MMS and H₂O₂ could be detected in a dose-dependent manner after alkali treatment for 1 min. The ability of the comet assay to detect DNA damage was not adversely affected by the short period (1 min) of the alkaline DNA unwinding time.

  5. Towards a more reliable comet assay: optimising agarose concentration, unwinding time and electrophoresis conditions.

    PubMed

    Azqueta, Amaya; Gutzkow, Kristine B; Brunborg, Gunnar; Collins, Andrew R

    2011-09-18

    The comet assay is now the method of choice for measuring most kinds of DNA damage in cells. However, due to the lack of a standardised protocol inter-laboratory comparisons are of limited value. The aim of this paper is to demonstrate how small changes in comet-assay variables may significantly affect the results. We examined the effect of varying agarose concentrations, alkaline unwinding time, electrophoresis time, voltage and current, by use of two cell types, viz. human peripheral blood lymphocytes and the lymphoblastoid cell line TK-6. All these variables have marked effects on assay performance and, therefore, on the determination of DNA damage. Here we identify factors of particular importance.

  6. Real-time fluorescence assays to monitor duplex unwinding and ATPase activities of helicases.

    PubMed

    Özeş, Ali R; Feoktistova, Kateryna; Avanzino, Brian C; Baldwin, Enoch P; Fraser, Christopher S

    2014-07-01

    Many physiological functions of helicases are dependent on their ability to unwind nucleic acid duplexes in an ATP-dependent fashion. Determining the kinetic frameworks of these processes is crucial to understanding how these proteins function. We recently developed a fluorescence assay to monitor RNA duplex unwinding by DEAD-box helicases in real time. In this assay, two fluorescently modified short reporter oligonucleotides are annealed to an unmodified RNA loading strand of any length so that the fluorescent moieties of the two reporters find themselves in close proximity to each other and fluorescence is quenched. One reporter is modified with cyanine 3 (Cy3), whereas the other is modified with a spectrally paired black-hole quencher (BHQ). As the helicase unwinds the loading strand, the enzyme displaces the Cy3-modified reporter, which will bind to a capture or competitor DNA strand, permanently separating it from the BHQ-modified reporter. Complete separation of the Cy3-modified reporter strand is thus detected as an increase in total fluorescence. This assay is compatible with reagentless biosensors to monitor ATPase activity so that the coupling between ATP hydrolysis and duplex unwinding can be determined. With the protocol described, obtaining data and analyzing results of unwinding and ATPase assays takes ∼4 h.

  7. Binding mode and affinity studies of DNA-binding agents using topoisomerase I DNA unwinding assay.

    PubMed

    McKnight, Ruel E; Gleason, Aaron B; Keyes, James A; Sahabi, Sadia

    2007-02-15

    A topoisomerase I DNA unwinding assay has been used to determine the relative DNA-binding affinities of a model pair of homologous naphthalene diimides. Binding affinity data were corroborated using calorimetric (ITC) and spectrophotometric (titration and T(m)) studies, with substituent size playing a significant role in binding. The assay was also used to investigate the mode of binding adopted by several known DNA-binding agents, including SYBR Green and PicoGreen. Some of the compounds exhibited unexpected binding modes.

  8. The further development of a mammalian DNA alkaline unwinding bioassay with potential application to hazard identification for contaminants from environmental samples

    SciTech Connect

    Daniel, F.B.; Chang, L.W.; Schenck, K.M.; DeAngelo, A.B.; Skelly, M.F. )

    1989-10-01

    Recently, we have detailed a DNA alkaline unwinding assay (DAUA) that can be used to rapidly measure chemically induced strand breaks in mammalian cells. In this paper we present further development of this assay, including: (1) studies on the relationship between DNA adducts and DNA strand breaks; (2) evaluation of the role of cytotoxicity in DNA strand breaks; and (3) application of the DAUA to cell preparations from the liver of mice dosed with methylating agents. The level of DNA adducts produced in human CCRF-CEM cells by treatment with benzo(a)pyrene diol-epoxide (BPDE), N-acetoxy-2-acetyl aminofluorene (AAAF), and various methylating agents was linear with concentration over several orders of magnitude. Likewise, the level of strand breaks increased with the concentration over the same dose range. The strand breaks/adduct ratio ranged from 0.05 for the methyl adducts to 0.001 for the BPDE adducts. Using these values and the inherent sensitivity of the DAUA (circa 100 to 1000 breaks/cell) the ability of the assay to detect DNA damage induced by various classes of chemical carcinogens can be calculated. The DAUA appears to be useful for assessing the relative potency of various environmental genotoxic effects on mammalian cells. In addition, it can be conducted on cells isolated from target organs of whole animals.

  9. Single-strand breaks in DNA of various organs of mice induced by methyl methanesulfonate and dimethylsulfoxide determined by the alkaline unwinding technique

    SciTech Connect

    Solveig Walles, S.A.; Erixon, K.

    1984-03-01

    The method for determination of single-strand breaks (SSB) in DNA by the technique of alkaline unwinding and hydroxylapatite chromatography has been applied for cell nuclei from organs of mice. Male mice were given methyl methane-sulfonate (MMS) and dimethylsulfoxide (DMSO) by i.p. administration. Cell nuclei were prepared from various organs and then lysed in alkali. The amount of DNA was determined by fluorometry using 4',6-diamidino-2-phenylindole.2HCl. The relative level of SSB in DNA was determined in various organs (liver, kidney, lung, spleen, testis and brain) 1-24 h after administration of the agent. After MMS-treatment the number of SSB in DNA increased to about the same extent in all organs 1 h post-treatment but then decreased by time. The SSB persisted for the longest time in brain- and lung-DNA. DMSO induced SSB only in DNA of kidney.

  10. Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

    PubMed

    Pu, Xinzhu; Wang, Zemin; Klaunig, James E

    2015-08-06

    Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies.

  11. Acellular comet assay: a tool for assessing variables influencing the alkaline comet assay.

    PubMed

    Kennedy, Erin K; McNamee, James P; Prud'homme Lalonde, Louise; Jones, Trevor; Wilkinson, Diana

    2012-01-01

    In this study, an acellular modification to the alkaline comet assay to further evaluate key variables within the assay that may influence the outcome of genotoxicity studies is described. This acellular comet assay can detect differences of 0.2 Gy of (60)Co gamma-ray radiation between 0 and 1 Gy and differences of 1 Gy between 0 and 8 Gy; thus, this assay is applicable for a wide range of DNA damage levels. It is also shown that DNA damage from different radiation energies was not significantly different from (60)Co gamma-ray. This assay displayed a statistical increase in DNA damage due to uncontrolled exposure to natural light; however, the slope of the dose-response curve for light-exposed samples was similar to that for samples protected from light. A comparison of the alkaline comet assay with the acellular comet assay allowed for the intrinsic repair capacity of the alkaline comet assay to be quantified.

  12. Unwinding inflation

    SciTech Connect

    D'Amico, Guido; Gobbetti, Roberto; Kleban, Matthew; Schillo, Marjorie E-mail: rg1509@nyu.edu E-mail: mls604@nyu.edu

    2013-03-01

    Higher-form flux that extends in all 3+1 dimensions of spacetime is a source of positive vacuum energy that can drive meta-stable eternal inflation. If the flux also threads compact extra dimensions, the spontaneous nucleation of a bubble of brane charged under the flux can trigger a classical cascade that steadily unwinds many units of flux, gradually decreasing the vacuum energy while inflating the bubble, until the cascade ends in the self-annihilation of the brane into radiation. With an initial number of flux quanta Q{sub 0}∼>N, this can result in N efolds of inflationary expansion while producing a scale-invariant spectrum of adiabatic density perturbations with amplitude and tilt consistent with observation. The power spectrum has an oscillatory component that does not decay away during inflation, relatively large tensor power, and interesting non-Gaussianities. Unwinding inflation fits naturally into the string landscape, and our preliminary conclusion is that consistency with observation can be attained without fine-tuning the string parameters. The initial conditions necessary for the unwinding phase are produced automatically by bubble formation, so long as the critical radius of the bubble is smaller than at least one of the compact dimensions threaded by flux.

  13. Genotoxicity of environmental agents assessed by the alkaline comet assay.

    PubMed

    Møller, Peter

    2005-01-01

    Generation of DNA damage is considered to be an important initial event in carcinogenesis. A considerable battery of assays exists for the detection of different genotoxic effects of compounds in experimental systems, or for investigations of exposure to genotoxic agents in environmental or occupational settings. Some of the tests may have limited use because of complicated technical setup or because they only are applicable to a few cell types. The single cell gel electrophoresis (comet) assay is technically simple, relatively fast, cheap, and DNA damage can be investigated in virtually all mammalian cell types without requirement for cell culture. The aim of this thesis was to evaluate the comet assay as a genotoxicity test in genetic toxicology of environmental agents, encompassing both experimental animal models and biomonitoring. The comet assay detects strand breaks (SB). The cells are embedded in agarose and lysed, generating nucleus-like structures in the gel (referred to as nucleoids). Following alkaline electrophoresis, the DNA strands migrate toward the anode, and the extent of migration depends on the number of SB in the nucleoid. The migration is visualized and scored in a fluorescence microscope after staining. Broad classes of oxidative DNA damage can be detected as additional SB if nucleoids are incubated with bacterial DNA glycosylase/endonuclease enzymes. Oxidized pyrimidines and purines can be detected by incubation with endonuclease III and formamidopyrimidine DNA glycosylase, respectively. The animal experimental studies indicated that the comet assay was able to detect genotoxic effects of diesel exhaust particles in lung tissue, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced DNA damage in colon epithelial cells and liver tissue, and benzene-induced damage in bone marrow and liver cells. The strength of the comet assay was further outlined by application of repair enzymes, indicating no oxidative DNA base damage following IQ treatment

  14. Dynamic look at DNA unwinding by a replicative helicase.

    PubMed

    Lee, Seung-Jae; Syed, Salman; Enemark, Eric J; Schuck, Stephen; Stenlund, Arne; Ha, Taekjip; Joshua-Tor, Leemor

    2014-03-04

    A prerequisite for DNA replication is the unwinding of duplex DNA catalyzed by a replicative hexameric helicase. Despite a growing body of research, key elements of helicase mechanism remain under substantial debate. In particular, the number of DNA strands encircled by the helicase ring during unwinding and the ring orientation at the replication fork completely contrast in contemporary mechanistic models. Here we use single-molecule and ensemble assays to address these questions for the papillomavirus E1 helicase. We find that E1 unwinds DNA with a strand-exclusion mechanism, with the N-terminal side of the helicase ring facing the replication fork. We show that E1 generates strikingly heterogeneous unwinding patterns stemming from varying degrees of repetitive movements, which is modulated by the DNA-binding domain. Together, our studies reveal previously unrecognized dynamic facets of replicative helicase unwinding mechanisms.

  15. Alkaline Phosphatase Assay for Freshwater Sediments: Application to Perturbed Sediment Systems

    PubMed Central

    Sayler, Gary S.; Puziss, Marla; Silver, Martin

    1979-01-01

    The p-nitrophenyl phosphate hydrolysis-phosphatase assay was modified for use in freshwater sediment. Laboratory studies indicated that the recovery of purified alkaline phosphatase activity was 100% efficient in sterile freshwater sediments when optimized incubation and sonication conditions were used. Field studies of diverse freshwater sediments demonstrated the potential use of this assay for determining stream perturbation. Significant correlations between phosphatase and total viable cell counts, as well as adenosine triphosphate biomass, suggested that alkaline phosphatase activity has utility as an indicator of microbial population density and biomass in freshwater sediments. PMID:16345464

  16. Repetitive RNA unwinding by RNA helicase A facilitates RNA annealing.

    PubMed

    Koh, Hye Ran; Xing, Li; Kleiman, Lawrence; Myong, Sua

    2014-07-01

    Helicases contribute to diverse biological processes including replication, transcription and translation. Recent reports suggest that unwinding of some helicases display repetitive activity, yet the functional role of the repetitiveness requires further investigation. Using single-molecule fluorescence assays, we elucidated a unique unwinding mechanism of RNA helicase A (RHA) that entails discrete substeps consisting of binding, activation, unwinding, stalling and reactivation stages. This multi-step process is repeated many times by a single RHA molecule without dissociation, resulting in repetitive unwinding/rewinding cycles. Our kinetic and mutational analysis indicates that the two double stand RNA binding domains at the N-terminus of RHA are responsible for such repetitive unwinding behavior in addition to providing an increased binding affinity to RNA. Further, the repetitive unwinding induces an efficient annealing of a complementary RNA by making the unwound strand more accessible. The complex and unusual mechanism displayed by RHA may help in explaining how the repetitive unwinding of helicases contributes to their biological functions.

  17. Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay.

    PubMed

    Ribas-Maynou, J; García-Peiró, A; Fernández-Encinas, A; Abad, C; Amengual, M J; Prada, E; Navarro, J; Benet, J

    2013-09-01

    Sperm DNA fragmentation (SDF) is becoming an important test to assess male infertility. Several different tests are available, but no consensus has yet been reached as to which tests are most predictive of infertility. Few publications have reported a comprehensive analysis comparing these methods within the same population. The objective of this study was to analyze the differences between the five most common methodologies, to study their correlations and to establish their cut-off values, sensitivity and specificity in predicting male infertility. We found differences in SDF between fertile donors and infertile patients in TUNEL, SCSA, SCD and alkaline Comet assays, but none with the neutral Comet assay. The alkaline COMET assay was the best in predicting male infertility followed by TUNEL, SCD and SCSA, whereas the neutral COMET assay had no predictive power. For our patient population, threshold values for infertility were 20.05% for TUNEL assay, 18.90% for SCSA, 22.75% for the SCD test, 45.37% for alkaline Comet and 34.37% for neutral Comet. This work establishes in a comprehensive study that the all techniques except neutral Comet are useful to distinguish fertile and infertile men.

  18. Online assay of bone specific alkaline phosphatase with a flow injection-bead injection system.

    PubMed

    Hartwell, Supaporn Kradtap; Somprayoon, Duangporn; Kongtawelert, Prachya; Ongchai, Siriwan; Arppornchayanon, Olarn; Ganranoo, Lucksagoon; Lapanantnoppakhun, Somchai; Grudpan, Kate

    2007-09-26

    Alkaline phosphatase (ALP) has been used as one of the biomarkers for bone resorption and liver diseases. Normally, total alkaline phosphatase is quantified along with other symptoms to determine the releasing source of the alkaline phosphatase. A semi-automated flow injection-bead injection system was proposed to conveniently and selectively assay bone alkaline phosphatase (BALP) based on its specific binding to wheat germ coated beads. Amount of BALP in serum was determined from the intensity of the yellow product produced from bound BALP on the retained beads and its substrate pNPP. The used beads were discarded and the fresh ones were introduced for the next analysis. The reaction cell was designed to be opened and closed using a computer controlled solenoid valve for a precise incubation time. The performance of the proposed system was evaluated by using it to assay BALP in human serum. The results were compared to those obtained by using a commercial ELISA kit. The system is proposed to be an easy and cost effective system for quantification of BALP as an alternative to batch wise wheat germ specific binding technique.

  19. Bio-nanocapsules for signal enhancement of alkaline phosphatase-linked immunosorbent assays.

    PubMed

    Iijima, Masumi; Yamamoto, Mikako; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi

    2013-01-01

    The bio-nanocapsules displaying about 240 molecules of immunoglobulin G Fc-binding Z domains (ZZ-BNCs) enhanced the signals of enzyme-linked immunosorbent assay by tethering the Fc regions of secondary antibodies (Abs), which were eliminated using high-molecular mass enzymes (e.g., alkaline phosphatase). By way of optimizing the distance between enzymes and Abs, ZZ-BNCs improved sensitivity independently of enzymes.

  20. A modified alkaline Comet assay for in vivo detection of oxidative DNA damage in Drosophila melanogaster.

    PubMed

    Shukla, A K; Pragya, P; Chowdhuri, D Kar

    2011-12-24

    Modifications to the alkaline Comet assay by using lesion-specific endonucleases, such as formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (ENDOIII, also known as Nth), can detect DNA bases with oxidative damage. This modified assay can be used to assess the genotoxic/carcinogenic potential of environmental chemicals. The goal of this study was to validate the ability of this modified assay to detect oxidative stress-induced genotoxicity in Drosophila melanogaster (Oregon R(+)). In this study, we used three well known chemical oxidative stress inducers: hydrogen peroxide (H(2)O(2)), cadmium chloride (CdCl(2)) and copper sulfate (CuSO(4)). Third instar larvae of D. melanogaster were fed various concentrations of the test chemicals (50-200μM) mixed with a standard Drosophila food for 24h. Alkaline Comet assays with and without the FPG and ENDOIII enzymes were performed with midgut cells that were isolated from the control and treated larvae. Our results show a concentration-dependent increase (p<0.05-0.001) in the migration of DNA from the treated larvae. ENDOIII treatment detected more oxidative DNA damage (specifically pyrimidine damage) in the H(2)O(2) exposed larvae compared to FPG or no enzyme treatment (buffer only). In contrast, FPG treatment detected more oxidative DNA damage (specifically purine damage) in CuSO(4) exposed larvae compared to ENDOIII. Although previously reported to be a potent genotoxic agent, CdCl(2) did not induce more oxidative DNA damage than the other test chemicals. Our results show that the modified alkaline Comet assay can be used to detect oxidative stress-induced DNA damage in D. melanogaster and thus may be applicable for in vivo genotoxic assessments of environmental chemicals.

  1. Cytogenetic status of healthy children assessed with the alkaline comet assay and the cytokinesis-block micronucleus cytome assay.

    PubMed

    Gajski, Goran; Gerić, Marko; Oreščanin, Višnja; Garaj-Vrhovac, Vera

    2013-01-20

    In the present study the alkaline comet assay and the cytokinesis-block micronucleus cytome (CBMN Cyt) assay were used to evaluate the baseline frequency of cytogenetic damage in peripheral blood lymphocytes (PBLs) of 50 healthy children from the general population in Croatia (age, 11.62±1.81 years). Mean values of tail length, tail intensity and tail moment, as comet assay parameters, were 12.92±0.10, 0.73±0.06 and 0.08±0.01, respectively. The mean frequency of micronuclei (MN) for all subjects was 2.32±0.28 per 1000 bi-nucleated cells, while the mean frequency of nucleoplasmic bridges (NPBs) was 1.72±0.24 and of nuclear buds (NBUDs) 1.44±0.19. The mean nuclear division index (NDI) was 1.70±0.05. When comet-assay parameters were considered, higher mean values for all three were found for the female population. According to the Mann-Whitney U test applied on the results of the comet assay, the only statistically significant difference between the male and female populations was found for tail length. Similar to the results obtained by the comet assay, girls showed higher mean values of all three measured parameters of the CBMN Cyt assay. This difference was statistically significant for total number of NPBs only. In the case of the NDI, a higher mean value was also obtained in girls, but this difference was not statistically significant. The results obtained present background data that could be considered as normal values for healthy children living in urban areas, and can later on serve as baseline values for further toxicological monitoring. Additionally, the usefulness of both techniques in measuring cytogenetic damage during bio-monitoring of children is confirmed.

  2. In vitro osteogenesis assays: influence of the primary cell source on alkaline phosphatase activity and mineralization.

    PubMed

    Hoemann, C D; El-Gabalawy, H; McKee, M D

    2009-06-01

    In trabecular bone fracture repair in vivo, osteogenesis occurs through endochondral ossification under hypoxic conditions, or through woven bone deposition in the vicinity of blood vessels. In vitro osteogenesis assays are routinely used to test osteoblastic responses to drugs, hormones, and biomaterials for bone and cartilage repair applications. These cell culture models recapitulate events that occur in woven bone synthesis, and are carried out using primary osteoblasts, osteoblast precursors such as bone marrow-derived mesenchymal stromal cells (BMSCs), or various osteoblast cell lines. With time in culture, cell differentiation is typically assessed by examining levels of alkaline phosphatase activity (an early osteoblast marker) and by evaluating the assembly of a collagen (type I)-containing fibrillar extracellular matrix that mineralizes. In this review, we have made a comparative analysis of published osteogenic assays using calvarial cells, calvaria-derived cell lines, and bone marrow stromal cells. In all of these cell types, alkaline phosphatase activity shows similar progression over time using a variety of osteogenic and mineralizing media conditions; however, levels of alkaline phosphatase activity are not proportional to observed mineralization levels.

  3. Cryopreservation of human blood for alkaline and Fpg-modified comet assay.

    PubMed

    Pu, Xinzhu; Wang, Zemin; Klaunig, James E

    2016-01-01

    The Comet assay is a reproducible and sensitive assay for the detection of DNA damage in eukaryotic cells and tissues. Incorporation of lesion specific, oxidative DNA damage repair enzymes (for example, Fpg, OGG1 and EndoIII) in the standard alkaline Comet assay procedure allows for the detection and measurement of oxidative DNA damage. The Comet assay using white blood cells (WBC) has proven useful in monitoring DNA damage from environmental agents in humans. However, it is often impractical to performance Comet assay immediately after blood sampling. Thus, storage of blood sample is required. In this study, we developed and tested a simple storage method for very small amount of whole blood for standard and Fpg-modified modified Comet assay. Whole blood was stored in RPMI 1640 media containing 10% FBS, 10% DMSO and 1 mM deferoxamine at a sample to media ratio of 1:50. Samples were stored at -20 °C and -80 °C for 1, 7, 14 and 28 days. Isolated lymphocytes from the same subjects were also stored under the same conditions for comparison. Direct DNA strand breakage and oxidative DNA damage in WBC and lymphocytes were analyzed using standard and Fpg-modified alkaline Comet assay and compared with freshly analyzed samples. No significant changes in either direct DNA strand breakage or oxidative DNA damage was seen in WBC and lymphocytes stored at -20 °C for 1 and 7 days compared to fresh samples. However, significant increases in both direct and oxidative DNA damage were seen in samples stored at -20 °C for 14 and 28 days. No changes in direct and oxidative DNA damage were observed in WBC and lymphocytes stored at -80 °C for up to 28 days. These results identified the proper storage conditions for storing whole blood or isolated lymphocytes to evaluate direct and oxidative DNA damage using standard and Fpg-modified alkaline Comet assay.

  4. Assessment of DNA interstrand crosslinks using the modified alkaline comet assay.

    PubMed

    Wu, Jian Hong; Jones, Nigel J

    2012-01-01

    The single cell gel electrophoresis (SCGE) assay, more commonly known as the comet assay, due to the "comet-like" appearance of the cells, was originally developed as a technique to measure the presence of DNA single-strand breaks. The assay is performed on single cells embedded in agar and placed in an electrical field at alkaline pH, so that fragments of negatively charged single-stranded DNA move through the gel toward the positively charged anode. Undamaged DNA moves relatively slowly, forming the head of the comet, while DNA fragmented due to the presence of single-strand breaks, moves more quickly giving the appearance of the tail. The extent of DNA migration is a measure of the DNA damage present. Since it was first developed, the comet assay has been adapted for measuring other types of DNA damage. The neutral comet assay has been employed for DNA double-strand breaks, while techniques using DNA repair enzymes to cleave specific adducts, UvrABC for ultraviolet radiation induced adducts, for example, have also been described. Here, we describe a modified version of the comet assay for the measurement of interstrand crosslinks (ICLs). Interstrand crosslinking agents include the chemotherapeutic agents mitomycin C and cis-platin, psoralen plus UVA light (PUVA) used to treat hyperproliferative skin disorders and diepoxybutane, a metabolite of 1,3-butadiene used in industrial processes and an environmental pollutant. ICLs are a potent and cytotoxic form of DNA damage as they prevent DNA strand separation, thereby preventing DNA replication. Their removal requires several different DNA repair processes including translesion synthesis and homologous recombination. As ICLs prevent separation of the DNA strands, their presence results in less DNA migration in the comet assay. To successfully measure ICLs, it is necessary to incorporate a step that induces single-strand breaks (using a defined dose of ionizing radiation) that allows the crosslinked DNA to migrate.

  5. A rapid, quantitative assay for measuring alkaline phosphatase activity in osteoblastic cells in vitro.

    PubMed

    Sabokbar, A; Millett, P J; Myer, B; Rushton, N

    1994-10-01

    Alkaline phosphatase (ALP) is the most widely recognized biochemical marker for osteoblast activity. Although its precise function is poorly understood, it is believed to play a role in skeletal mineralization. The aim of this study was to develop an assay suitable for measuring the activity of this enzyme in microtiter plate format. Using the well-characterized osteoblast-like cell line Saos-2, this paper describes an optimized biochemical assay suitable for measuring ALP activity in tissue culture samples. We have determined that a p-nitrophenyl phosphate substrate concentration of 9 mM provides highest enzyme activities. We have found that cell concentration, and hence enzyme concentration, affects both the kinetics and precision of the assay. We also tested several methods of enzyme solubilization and found that freeze-thawing the membrane fractions twice at -70 degrees C/37 degrees C or freeze-thawing once with sonication yielded highest enzyme activities. The activity of the enzyme decreased by 10% after 7 days storage. This assay provides a sensitive and reproducible method that is ideally suited for measuring ALP activity in isolated osteoblastic cells, although sample preparation and storage can influence results.

  6. Robotic implementation of assays: tissue-nonspecific alkaline phosphatase (TNAP) case study.

    PubMed

    Chung, Thomas D Y

    2013-01-01

    Laboratory automation and robotics have "industrialized" the execution and completion of large-scale, enabling high-capacity and high-throughput (100 K-1 MM/day) screening (HTS) campaigns of large "libraries" of compounds (>200 K-2 MM) to complete in a few days or weeks. Critical to the success these HTS campaigns is the ability of a competent assay development team to convert a validated research-grade laboratory "benchtop" assay suitable for manual or semi-automated operations on a few hundreds of compounds into a robust miniaturized (384- or 1,536-well format), well-engineered, scalable, industrialized assay that can be seamlessly implemented on a fully automated, fully integrated robotic screening platform for cost-effective screening of hundreds of thousands of compounds. Here, we provide a review of the theoretical guiding principles and practical considerations necessary to reduce often complex research biology into a "lean manufacturing" engineering endeavor comprising adaption, automation, and implementation of HTS. Furthermore we provide a detailed example specifically for a cell-free in vitro biochemical, enzymatic phosphatase assay for tissue-nonspecific alkaline phosphatase that illustrates these principles and considerations.

  7. In vivo genotoxicity assessment of sertraline by using alkaline comet assay and the cytokinesis-block micronucleus assay.

    PubMed

    Battal, Dilek; Aktas, Ayca; Sungur, Mehmet Ali; Kadioglu, Ela; Eker, Ebru Derici; Sahin, Nefise Ozlen; Saygi, Sahan

    2013-11-01

    Sertraline, a leading antidepressant in the selective serotonin reuptake inhibitor (SSRI) group of medicine, is the most frequently prescribed drug. In this study, the alkaline comet assay and the cytokinesis-block micronucleus (CBMN) assay were used to investigate genotoxicity potential of sertraline in the peripheral blood lymphocytes (PBLs) of acute and chronic sertraline-treated Wistar albino rats. Male Wistar albino rats (n = 48) were administered low, medium and high doses of sertraline (10, 40, 80 mg/kg) for acute and chronic treatment by employing the gavage method to investigate genotoxicity of the administered drug. The data (tail length, tail intensity and tail moment) were analysed and indicated that there was no statistically significant difference between sertraline-treated groups and the negative control group with respect to DNA damage (p > 0.05). However, it was observed that acute sertraline administration had caused much more DNA damage in comparison with chronic treatment (p < 0.05). According to the data obtained from the CBMN test, an increase in the micronucleus (MN) frequency was detected at chronic and high-dose acute sertraline treatment. Based on the outcome of comet assay, detection of statistically insignificant DNA damage may be due to the fact that sertraline did not cause damage on DNA. Also, increase in frequency of MN in chronic sertraline treatment suggests that chronic sertraline administration might influence some mechanisms of cell division. Therefore, dose adjustment in depressed patients seems significant as it may help prevent further prognosis of the diseases.

  8. Spectrophotometric total protein assay with copper(II)-neocuproine reagent in alkaline medium.

    PubMed

    Sözgen, Kevser; Cekic, Sema Demirci; Tütem, Esma; Apak, Resat

    2006-02-28

    Total protein assay was made using copper(II)-neocuproine (Nc) reagent in alkaline medium (with the help of a hydroxide-carbonate-tartarate solution) after 30min incubation at 40 degrees C. The absorbance of the reduction product, Cu(I)-Nc complex, was recorded at 450nm against a reagent blank. The absorptivity of the developed method for bovine serum albumin (BSA) was 0.023lmg(-1)cm(-1), greater than that of Lowry assay (0.0098), and much greater than that of Cu(II)-bicinchoninic acid (BCA) assay (0.00077). The linear range of the developed method (8-100mgl(-1) BSA) was as wide as that of Lowry, and much wider than that of BCA (200-1000mgl(-1) BSA) assay. The sensitivity of the method was greater than those of Cu-based assays (biuret, Lowry, and BCA) with a LOD of 1mgl(-1) BSA. The within-run and between-run precisions as RSD were 0.73 and 1.01%, respectively. The selectivity of the proposed method for protein was much higher than those of dye-binding and Lowry assays: Most common interferents to other protein assays such as tris, ethanolamine, deoxycholate, CsCl, citrate, and triton X-100 were tolerated at 100-fold concentrations in the analysis of 10mgl(-1) BSA, while the tolerance limits for other interferents, e.g., (NH(4))(2)SO(4) and acetylsalicylic acid (50-fold), SDS (25-fold), and glycerol (20-fold) were at acceptable levels. The redox reaction of Cu(II)-Nc as an outer-sphere electron transfer agent with the peptide bond and with four amino acid residues (cystine, cysteine, tryptophan, and tyrosine) was kinetically more favourable than that of Cu(II) alone in the biuret assay. Since the reduction product of Cu(II) with protein, i.e., Cu(I), was coordinatively saturated with Nc in the stable Cu(Nc)(2)(+) chelate, re-oxidation of the formed Cu(I) with Fenton-like reactions was not possible, thereby preventing a loss of chromophore. After conventional protein extraction, precipitation, and redissolution procedures, the protein contents of the minced meat

  9. Does the duration of lysis affect the sensitivity of the in vitro alkaline comet assay?

    PubMed

    Enciso, José Manuel; Sánchez, Oscar; López de Cerain, Adela; Azqueta, Amaya

    2015-01-01

    The alkaline comet assay is now the method of choice for measuring different kinds of DNA damage in cells. Several attempts have been made to identify and evaluate the critical points affecting the comet assay outcome, highlighting the requirement of arriving at a standardised protocol in order to be able to compare the results obtained in different laboratories. However, reports on the effect of modifying the time of lysis are lacking. Here we tested different times of lysis (from no lysis to 1 week) in control HeLa cells and HeLa cells treated with different concentrations of methyl methanesulfonate (MMS) or H2O2. We also tested different times of lysis in the comet assay combined with formamidopyrimidine DNA glycosylase (FPG) in untreated and Ro 19-8022 plus light-treated HeLa cells. The same DNA damage levels were detected in the absence of lysis or after 1h of lysis when the standard comet assay was used to detect the MMS- and H2O2-induced lesions; the response increased when longer lysis was used, up to at least 1 week. When FPG was used, a minimum lysis period of 5 min was necessary to allow the enzyme to reach the DNA; the same DNA damage levels were detected after 5 min or 1h of lysis and the response increased up to 24h. In conclusion, the time of lysis can be varied depending on the sensitivity needed in both versions of the assay, and a constant time of lysis should be used if results from different experiments or laboratories are to be compared.

  10. Conjugated polyelectrolyte-based real-time fluorescence assay for alkaline phosphatase with pyrophosphate as substrate.

    PubMed

    Liu, Yan; Schanze, Kirk S

    2008-11-15

    The fluorescence of the anionic, carboxylate-substituted poly(phenylene ethynylene) polymer PPECO2 is quenched very efficiently via the addition of 1 equiv of Cu(2+). Addition of pyrophosphate (PPi) into the weakly fluorescent solution of PPECO2 and Cu(2+) induces recovery of the polymer's fluorescence; the recovery occurs because PPi complexes with Cu(2+), effectively sequestering the ion so it cannot bind to the carboxylate groups of the polymer. A calibration curve was developed that relates the extent of fluorescence recovery to [PPi], making the PPECO2-Cu(2+) system a sensitive and selective turn-on sensor for PPi. Using the PPECO2-Cu(2+) system as the signal transducer, a real-time fluorescence turn-off assay for the enzyme alkaline phosphatase (ALP) using PPi as the substrate is developed. The assay operates with [PPi] in the micromolar range, and it offers a straightforward and rapid detection of ALP activity with the enzyme present in the nanomolar concentration range, operating either in an end point or real-time format. Kinetic and product inhibition parameters are derived by converting time-dependent fluorescence intensity into PPi (substrate) concentration, thus allowing calculation of the initial reaction rates (v(o)). Weak, nonspecific fluorescence responses are observed concomitant to addition of other proteins to the assay solution; however, the signal response to ALP is demonstrated to arise from the ALP catalyzed hydrolysis of PPi to phosphate (Pi).

  11. Alkaline single-cell gel (comet) assay and genotoxicity monitoring using two species of tadpoles.

    PubMed

    Ralph, S; Petras, M; Pandrangi, R; Vrzoc, M

    1996-01-01

    Small bodies of water (e.g., creeks, ponds, and drainage ditches) have received very little attention in genotoxicity studies, yet these areas are important because they are often the first to be affected by industrial effluents, sewage contaminants, accidental spills, internal combustion engine emissions, landfill runoffs, and pesticide uses. To address this deficiency, we examined erythrocytes in two species of tadpoles, Rana clamitans and Bufo americanus, using the alkaline single-cell gel (SCG) ("comet") assay. This approach involves detection, under alkaline conditions, of cell DNA fragments, which on electrophoresis migrate from the nuclear core, resulting in a "comet-with-tail" formation. Exposure of R. clamitans todpoles to a range of concentrations of methyl methanesulfonate (MMS) produced a linear increase in DNA length to DNA core width ratios. This is consistent with findings in a number of other species. Time-dose experiments using MMS suggest that the peak level of DNA damage in R. clamitans todpoles occurred 42 hr after exposure. B. americanus tadpoles exposed to 6.25 mg/l of MMS for 12 hours had a significant increase in DNA damage over that seen in the controls. Freshly caught R. clamitans tadpoles from Highgate and B. americanus tadpoles from Duart, both on the north shore of Lake Erie, gave ratios of 2.78 and 2.07, respectively. This region of Ontario is a prime agricultural area and pesticide use is extensive. Tadpoles from Highgate and Duart, maintained in the laboratory for 4 months and 6 weeks, respectively, gave ratios of 1.29 and 1.44. The results of the SCG procedure in tadpoles indicate that this assay is extremely sensitive and suitable for detecting genotoxicity in the environment.

  12. Validation of a high-throughput in vitro alkaline elution/rat hepatocyte assay for DNA damage.

    PubMed

    Gealy, Robert; Wright-Bourque, Jennifer L; Kraynak, Andrew R; McKelvey, Troy W; Barnum, John E; Storer, Richard D

    2007-04-20

    In vitro alkaline elution is a sensitive and specific short term assay which measures DNA strand breakage in a mammalian test system (primary rat hepatocytes). This lab has previously demonstrated the performance of the assay with known genotoxic and non-genotoxic compounds. The methodology employed has relatively low sample throughput and is labor-intensive, requiring a great deal of manual processing of samples in a format that is not amenable to automation. Here, we present an automated version of the assay. This high-throughput alkaline elution assay (HT-AE) was made possible through 3 key developments: (1) DNA quantitation using PicoGreen and OliGreen fluorescent DNA binding dyes; (2) design and implementation of a custom automation system; and (3) reducing the assay to a 96-well plate format. The assay can now be run with 5-50mg of test compound. HT-AE was validated in a similar manner as the original assay, including assessment of non-genotoxic and non-carcinogenic compounds and evaluation of cytotoxicity to avoid confounding effects of toxicity-associated DNA degradation. The validation test results from compounds of known genotoxic potential were used to set appropriate criteria to classify alkaline elution results for genotoxicity.

  13. Sperm DNA damage output parameters measured by the alkaline Comet assay and their importance.

    PubMed

    Simon, L; Aston, K I; Emery, B R; Hotaling, J; Carrell, D T

    2017-03-01

    The alkaline Comet assay has shown high diagnostic value to determine male reproductive health and prognostic ability to predict ART success. Here, spermatozoon was analysed in 47 fertile donors and 238 patients, including 132 couples undergoing ART [semen was collected: Group I - within 3 months of their treatment (n = 79); and Group II - 3 months prior to their treatment (n = 53)]. We introduce four Comet distribution plots (A, B1, B2 and C) by plotting the level of DNA damage (x-axis) and percentage of comets (y-axis). Fertile donors had low mean DNA damage, olive tail moment and per cent of spermatozoa with damage and increased type A plots. Comet parameters were associated with clinical pregnancies in Group I. About 66% of couples with type A distribution plot were successful after ART, whereas couples with type B1, B2 and C distribution plots achieved 56%, 44% and 33% pregnancies respectively. The efficiency of the Comet assay was due to complete decondensation process, where the compact sperm nuclear DNA (28.2 ± 0.2 μm(3) ) is decondensed to ~63 μm(3) (before lysis) and ~1018 μm(3) (after lysis). A combinational analysis of all the Comet output parameters may provide a comprehensive evaluation of patient's reproductive health as these parameters measure different aspects of DNA damage within the spermatozoa.

  14. Sensitive and selective colorimetric assay of alkaline phosphatase activity with Cu(II)-phenanthroline complex.

    PubMed

    Hu, Qiong; He, Minhui; Mei, Yaqi; Feng, Wenjie; Jing, Su; Kong, Jinming; Zhang, Xueji

    2017-01-15

    Alkaline phosphatase (ALP) plays a vital role in dephosphorylation- and phosphorylation-related cellular regulation and signaling processes. Accordingly, the development of efficient methods for ALP activity assay is of significant importance in clinical diagnosis. In this work, a simple and practical method is reported for the first time for the sensitive and selective colorimetric assay of ALP activity by exploiting a water-soluble Cu(II)-phenanthroline complex as the probe, on the basis of the distinctive metal-to-ligand charge-transfer (MLCT) properties. This method is simply built on a two-step chromogenic reaction: the enzymatic hydrolysis of the substrate ascorbic acid 2-phosphate to ascorbic acid (AA), followed by the reduction of the colorimetric probe Cu(BPDS)2(2-) (BPDS=bathophenanthroline disulfonate) by AA to its cuprous form. The latter process triggers a turn-on spectral absorption at 424nm and a striking color change of the solution from colorless to blackish-green. Needless of complicated protocols and instrumentation, this method allows a sensitive readout of ALP activity within a wide linear range of 0-200mUmL(-)(1), with a detection limit down to 1.25mUmL(-1). Results also reveal that it is highly selective and holds great potential in ALP inhibitor efficiency evaluation. In addition, quantitative analysis of ALP activity in spiked serum samples has been realized successfully in the linear range of 0-200mUmL(-1), with a detection limit of 1.75mUmL(-1). Advantages of simplicity, wide linear range, high sensitivity and selectivity, low cost, and little background interference render this method great potential in practical applications.

  15. Visualizing helicases unwinding DNA at the single molecule level.

    PubMed

    Fili, Natali; Mashanov, Gregory I; Toseland, Christopher P; Batters, Christopher; Wallace, Mark I; Yeeles, Joseph T P; Dillingham, Mark S; Webb, Martin R; Molloy, Justin E

    2010-07-01

    DNA helicases are motor proteins that catalyze the unwinding of double-stranded DNA into single-stranded DNA using the free energy from ATP hydrolysis. Single molecule approaches enable us to address detailed mechanistic questions about how such enzymes move processively along DNA. Here, an optical method has been developed to follow the unwinding of multiple DNA molecules simultaneously in real time. This was achieved by measuring the accumulation of fluorescent single-stranded DNA-binding protein on the single-stranded DNA product of the helicase, using total internal reflection fluorescence microscopy. By immobilizing either the DNA or helicase, localized increase in fluorescence provides information about the rate of unwinding and the processivity of individual enzymes. In addition, it reveals details of the unwinding process, such as pauses and bursts of activity. The generic and versatile nature of the assay makes it applicable to a variety of DNA helicases and DNA templates. The method is an important addition to the single-molecule toolbox available for studying DNA processing enzymes.

  16. Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase

    NASA Astrophysics Data System (ADS)

    Ha, Taekjip; Rasnik, Ivan; Cheng, Wei; Babcock, Hazen P.; Gauss, George H.; Lohman, Timothy M.; Chu, Steven

    2002-10-01

    Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid, and are involved in several human diseases. Some function as hexameric rings, but the functional form of non-hexameric helicases has been debated. Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays-which do not interfere with biological activity-to probe DNA unwinding by the Escherichia coli Rep helicase. Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding. DNA unwinding initiates only if a functional helicase is formed via additional protein binding. Partial dissociation of the functional complex during unwinding results in interruptions (`stalls') that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re-formation of the functional helicase. These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex. We expect that these techniques will be useful for dynamic studies of other helicases and protein-DNA interactions.

  17. Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase.

    PubMed

    Ha, Taekjip; Rasnik, Ivan; Cheng, Wei; Babcock, Hazen P; Gauss, George H; Lohman, Timothy M; Chu, Steven

    2002-10-10

    Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid, and are involved in several human diseases. Some function as hexameric rings, but the functional form of non-hexameric helicases has been debated. Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays--which do not interfere with biological activity--to probe DNA unwinding by the Escherichia coli Rep helicase. Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding. DNA unwinding initiates only if a functional helicase is formed via additional protein binding. Partial dissociation of the functional complex during unwinding results in interruptions ('stalls') that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re-formation of the functional helicase. These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex. We expect that these techniques will be useful for dynamic studies of other helicases and protein-DNA interactions.

  18. In vitro assessment of genotoxic effects of electric arc furnace dust on human lymphocytes using the alkaline comet assay.

    PubMed

    Garaj-Vrhovac, Vera; Orescanin, Visnja; Ruk, Damir; Gajski, Goran

    2009-02-15

    In vitro genotoxic effects of leachates of electric arc furnace dust (EAFD) on human peripheral lymphocytes, assessed prior and following the treatment with a strong alkaline solution were investigated using the alkaline comet assay. Prior and following the treatment, lymphocytes were incubated with leachate of EAFD for 6 and 24 hours at 37 degrees C. Negative controls were also included. Mean values of the tail lengths established in the samples treated with the leachate stemming from the original dust for 6 and 24 hours, were 15.70 microm and 16.78 microm, respectively, as compared to 12.33 microm found in the control sample. Slight, but significant increase in the tail length was also found with the dust treated with a strong alkaline solution (13.37 microm and 13.60 microm). In case of high heavy metal concentrations (the extract of the original furnace dust), the incubation period was revealed to be of significance as well. The obtained results lead to the conclusion that alkaline comet assay could be used as a rapid, sensitive and low-cost tool when assessing genotoxicity of various waste materials, such as leachates of the electric arc furnace dust.

  19. Facile colorimetric assay of alkaline phosphatase activity using Fe(II)-phenanthroline reporter.

    PubMed

    Hu, Qiong; Zhou, Baojing; Dang, Pengyun; Li, Lianzhi; Kong, Jinming; Zhang, Xueji

    2017-01-15

    We report a versatile approach for the colorimetric assay of alkaline phosphatase (ALP) activity based on the distinctive metal-to-ligand charge-transfer (MLCT) absorption properties of Fe(II)-phenanthroline reporter. In the presence of ALP, the applied substrate ascorbic acid 2-phosphate is enzymatically hydrolyzed to produce ascorbic acid, which then reduces Fe(3+) to Fe(2+). The complexation of Fe(2+) with the bathophenanthroline disulfonate (BPS) ligand generates a blood-red Fe(BPS)3(4-) reporter, which is characterized by an intense MLCT absorption band at 535 nm in the visible range. Under optimal conditions, the spectral output exhibits a good quantitative relationship with ALP activity over the range of 0-220 mU mL(-1) with a detection limit of 0.94 mU mL(-1). Moreover, the activity of ALP can also be conveniently judged through naked-eye observations. Results indicate that it is highly selective and can be applied to the screening of ALP inhibitors. In addition, it has been successfully employed to detect the endogenous ALP level of undiluted human serum samples, with a detection limit of 1.05 mU mL(-1) being achieved. This approach avoids any elaborately designed substrates and holds considerable simplicity and flexibility for reporter design. This study broadens the horizon of the applications of phenanthroline-based transition metal complexes. Furthermore, an efficient and practical method like this has the potential to be widely used in clinical applications and in the point-of-care testing.

  20. Determination of genotoxic effects of methidathion alkaline hydrolysis in human lymphocytes using the micronucleus assay and square-wave voltammetry.

    PubMed

    Stivaktakis, Polychronis D; Giannakopoulos, Evangelos; Vlastos, Dimitris; Matthopoulos, Demetrios P

    2017-02-01

    The interaction of pesticides with environmental factors, such as pH, may result in alterations of their physicochemical properties and should be taken into consideration in regard to their classification. This study investigates the genotoxicity of methidathion and its alkaline hydrolysis by-products in cultured human lymphocytes, using the square-wave voltammetry (square wave-adsorptive cathodic stripping voltammetry (SW-AdCSV) technique) and the cytokinesis block micronucleus assay (CBMN assay). According to the SW-AdCSV data the alkaline hydrolysis of methidathion results in two new molecules, one non-electro-active and a second electro-active which is more genotoxic than methidathion itself in cultured human lymphocytes, inducing higher micronuclei frequencies. The present study confirms the SW-AdCSV technique as a voltammetric method which can successfully simulates the electrodynamics of the cellular membrane.

  1. DNA breakage detection-fish (DBD-FISH): effect of unwinding time.

    PubMed

    Vázquez-Gundín, F; Gosálvez, J; de la Torre, J; Fernández, J L

    2000-09-20

    DBD-FISH is a new procedure that allows detection and quantification of DNA breakage in situ within specific DNA target sites. Cells embedded in an agarose matrix on a slide are treated in an alkaline unwinding solution to transform DNA breaks into single-stranded DNA (ssDNA). After removal of proteins, DNA probes are hybridized and detected. DNA breaks increase the ssDNA and relax supercoiling of DNA loops, so more probe hybridizes, thereby increasing the surface area and fluorescence intensity of the FISH signal. The probe selects the chromatin area to be analysed. In order to restrict the extension of unwound ssDNA to a region closer to the origin of the DNA break, human leukocytes were processed for DBD-FISH with a whole genome probe, after a 10 Gy dose of X-rays, for various unwinding times: 5, 2 min and 30s. Two cell populations were detected after 30s, but not with the 5 or 2 min unwinding times. One cell group had small to medium haloes corresponding to the relaxation of DNA supercoiling after DAPI staining, and strong DBD-FISH labelling of induced DNA breaks, whereas the other cell group showed big haloes of DNA loop unfolding and an absence of DBD-FISH labelling. The latter group was similar to cells processed by DBD-FISH without the unwinding step. Thus, they should correspond to cells unaffected by the alkaline unwinding solution, possibly because very brief unwinding times do not allow the diffusion of the alkali into the cells deep within the gel, thus biasing the results. Taking this into account, 2 min seems to be the minimum unwinding time required for an accurate detection of a signal by DBD-FISH.

  2. A single-molecule approach to visualize the unwinding activity of DNA helicases.

    PubMed

    Fili, Natalia; Toseland, Christopher P; Dillingham, Mark S; Webb, Martin R; Molloy, Justin E

    2011-01-01

    Almost all aspects of DNA metabolism involve separation of double-stranded DNA catalyzed by helicases. Observation and measurement of the dynamics of these events at the single-molecule level provide important mechanistic details of helicase activity and give the opportunity to probe aspects that are not revealed in bulk solution measurements. The assay, presented here, provides information about helicase unwinding rates and processivity. Visualization is achieved by using a fluorescent single-stranded DNA-binding protein (SSB), which allows the time course of individual DNA unwinding events to be observed using total internal reflection fluorescence microscopy. Observation of a prototypical helicase, Bacillus subtilis AddAB, shows that the unwinding process consists of bursts of unwinding activity, interspersed with periods of pausing.

  3. Duplex unwinding with DEAD-box proteins.

    PubMed

    Jankowsky, Eckhard; Putnam, Andrea

    2010-01-01

    DEAD-box proteins, which comprise the largest helicase family, are involved in virtually all aspects of RNA metabolism. DEAD-box proteins catalyze diverse ATP-driven functions including the unwinding of RNA secondary structures. In contrast to many well-studied DNA and viral RNA helicases, DEAD-box proteins do not rely on translocation on one of the nucleic acid strands for duplex unwinding, but directly load onto helical regions and then locally pry the strands apart in an ATP-dependent fashion. In this chapter, we outline substrate design and unwinding protocols for DEAD-box proteins and focus on the quantitative evaluation of their unwinding activity.

  4. Monitoring helicase-catalyzed DNA unwinding by fluorescence anisotropy and fluorescence cross-correlation spectroscopy.

    PubMed

    Xi, Xu Guang; Deprez, Eric

    2010-07-01

    In order to elucidate molecular mechanism of helicases, we have developed two new rapid and sensitive fluorescence assays to measure helicase-mediated DNA unwinding. The fluorescence anisotropy (FA) assay takes the advantage of the substantial change in fluorescence polarization upon helicase binding to DNA and DNA unwinding. The extent of depolarization depends on the rate of tumbling of the fluorescently labeled DNA molecule, which decreases with increasing size. This assay therefore can simultaneously monitor the DNA binding of helicase and the subsequent helicase-catalyzed DNA unwinding in real-time. For size limitation reasons, the FA approach is more suitable for single-turnover kinetic studies. A fluorescence cross-correlation spectroscopy method (FCCS) is also described for measuring DNA unwinding. This assay is based on the degree of concomitant diffusion of the two complementary DNA strands in a small excitation volume, each labeled by a different color. The decrease in the amplitude of the cross-correlation signal is then directly related to the unwinding activity. By contrast with FA, the FCCS-based assay can be used to measure the unwinding activity under both single- and multiple-turnover conditions, with no limitation related to the size of the DNA strands constituting the DNA substrate. These methods used together have proven to be useful for studying molecular mechanism underlying efficient motor function of helicases. Here, we describe the theoretical basis and framework of FA and FCCS and some practical implications for measuring DNA binding and unwinding. We discuss sample preparation and potential troubleshooting. Special attention is paid to instrumentation, data acquisition and analysis.

  5. A single-molecule digital enzyme assay using alkaline phosphatase with a cumarin-based fluorogenic substrate.

    PubMed

    Obayashi, Yusuke; Iino, Ryota; Noji, Hiroyuki

    2015-08-07

    Digitalization of fluorogenic enzymatic assays through the use of femtoliter chamber array technology is an emerging approach to realizing highly quantitative bioassays with single-molecule sensitivity. However, only a few digital fluorogenic enzyme assays have been reported, and the variations of the digital enzyme assays are basically limited to fluorescein- and resorufin-based fluorogenic assays. This limitation hampers the realization of a multiplex digital enzyme assay such as a digital enzyme-linked immunosorbent assay (ELISA). In this study, after optimization of buffer conditions, we achieved a single-molecule digital enzyme alkaline phosphatase (ALP) assay with a cumarin-based fluorogenic substrate, 4-methylunbelliferyl phosphate (4-MUP). When ALP molecules were encapsulated in a 44-femtoliter chamber array at a low ratio of less than 1 molecule per chamber, each chamber showed a discrete fluorescence signal in an all-or-none manner, allowing the digital counting of the number of active enzyme molecules. The fraction of fluorescent chambers linearly decreased with the enzyme concentration, obeying the Poisson distribution as expected. We also demonstrated a dual-color digital enzyme assay with a ALP/4-MUP and β-galactosidase (β-gal)/resorufin-β-d-galactopyranoside combination. The activities of single ALP and β-gal molecules were clearly detected simultaneously. The method developed in this study will enable us to carry out a parallelized, multiplex digital ELISA.

  6. Alkaline comet assay in liver and stomach, and micronucleus assay in bone marrow, from rats treated with 2-acetylaminofluorene, azidothymidine, cisplatin, or isobutyraldehyde.

    PubMed

    Kraynak, A R; Barnum, J E; Cunningham, C L; Ng, A; Ykoruk, B A; Bennet, B; Stoffregen, D; Merschman, M; Freeland, E; Galloway, S M

    2015-07-01

    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM) initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined the ability of the assay to determine the genotoxicity of 2-acetylaminofluorene (AAF), azidothymidine (AZT), cisplatin (CPN), and isobutyraldehyde (IBA) in liver and glandular stomach of male Sprague-Dawley rats. Rats were given oral doses of test compound or control once daily for three days. High dose levels were approximately maximum tolerated doses and were based on preliminary range-finding studies. Tissues were harvested 3h after the final dose (48h after the initial dose). A bone marrow micronucleus assay (MN) was also conducted on the rats treated with AZT, CPN, and IBA. Acute toxic effects of treatment were determined primarily through histomorphologic analysis of liver and stomach but also by body weight and serum liver enzyme changes. The comet assay was conducted on fresh tissue preparations but frozen samples from two studies were also assayed. Statistically significant dose-related differences in comet % DNA in tail were found in liver and stomach for the genotoxin AZT and in liver for the genotoxin CPN, but not in liver or stomach for the non-genotoxin IBA. Statistically significant differences in % DNA in tail were measured in liver for the low and mid dose of the genotoxin AAF, but not the high dose. The comet assays of frozen liver suspensions from CPN- and AAF-treated rats yielded comparable results to the assays of fresh preparations. There were no indications of significant toxicity induced by any treatment. The micronucleus assay was positive for CPN and AZT and negative for IBA. In conclusion, the in vivo comet assay is capable of detecting genotoxic effects of a variety of chemicals and may fill an important role in the genotoxicity test battery.

  7. Vortex unwinding in a turbulent boundary layer

    NASA Technical Reports Server (NTRS)

    Mcginley, Catherine B.; Beeler, George B.

    1987-01-01

    The vortex unwinding method is used as a tool in performing vortex cancellation in a turbulent boundary layer. Sufficient reduction in the isotach variation was achieved to verify the usefulness of this technique, for the cases of both wall turbulence control and horseshoe vortex alleviation. More detailed measurements of vortex strength and position improve the optimization process and increase the amount of vortex unwinding.

  8. Unwinding in Hopfion vortex bunches

    SciTech Connect

    Jaeykkae, Juha; Hietarinta, Jarmo

    2009-06-15

    We investigate the behavior of parallel Faddeev-Hopf vortices under energy minimization in a system with physically relevant, but unusual boundary conditions. The homotopy classification is no longer provided by the Hopf invariant, but rather by the set of integer homotopy invariants proposed by Pontrjagin. The nature of these invariants depends on the boundary conditions. A set of tightly wound parallel vortices of the usual Hopfion structure is observed to form a bunch of intertwined vortices or unwind completely, depending on the boundary conditions.

  9. Preliminary study of genotoxicity evaluation of orthodontic miniscrews on mucosa oral cells by the alkaline comet assay.

    PubMed

    Martín-Cameán, Ana; Puerto, María; Jos, Ángeles; Azqueta, Amaya; Iglesias-Linares, Alejandro; Solano, Enrique; Cameán, Ana M

    2015-01-01

    Miniscrew implants are widely used nowadays in orthodontic treatments due to their good results in clinical practice. However, data regarding the biocompatibility of commercially available orthodontic miniscrews and temporary devices are very scarce, and their role as genotoxicity inducers has been not previously evaluated with the alkaline comet assay. The aim of this study was to investigate the DNA damage in buccal cells of patients subjected to orthodontic treatments. The alkaline comet assay has been applied in oral mucosa cells from patients treated with conventional orthodontic treatment in comparison to patients treated additionally with miniscrews, non-treated volunteers (control) and smoking volunteers (positive control). The application of orthodontic appliances and miniscrews induced significant and similar (2-fold) increases of %DNA in tail in comparison to control group. Females experienced a significant increase in %DNA in all the treatments in comparison to the control group, whereas males showed significant damage only with the combined orthodontic and miniscrew treatment. In conclusion, conventional orthodontic appliances induced genotoxicity, and the incorporation of miniscrews assayed did not imply any additional increase of DNA damage.

  10. Do DEAD-box proteins promote group II intron splicing without unwinding RNA?

    PubMed

    Del Campo, Mark; Tijerina, Pilar; Bhaskaran, Hari; Mohr, Sabine; Yang, Quansheng; Jankowsky, Eckhard; Russell, Rick; Lambowitz, Alan M

    2007-10-12

    The DEAD-box protein Mss116p promotes group II intron splicing in vivo and in vitro. Here we explore two hypotheses for how Mss116p promotes group II intron splicing: by using its RNA unwinding activity to act as an RNA chaperone or by stabilizing RNA folding intermediates. We show that an Mss116p mutant in helicase motif III (SAT/AAA), which was reported to stimulate splicing without unwinding RNA, retains ATP-dependent unwinding activity and promotes unfolding of a structured RNA. Its unwinding activity increases sharply with decreasing duplex length and correlates with group II intron splicing activity in quantitative assays. Additionally, we show that Mss116p can promote ATP-independent RNA unwinding, presumably via single-strand capture, also potentially contributing to DEAD-box protein RNA chaperone activity. Our findings favor the hypothesis that DEAD-box proteins function in group II intron splicing as in other processes by using their unwinding activity to act as RNA chaperones.

  11. Assessment of the serum levels of bone alkaline phosphatase with a new immunoradiometric assay in patients with metabolic bone disease

    SciTech Connect

    Garnero, P.; Delmas, P.D.

    1993-10-01

    The authors measured serum bone alkaline phosphatase (B-ALP) with a new immunoradiometric assay (IRMA) in a large sample of healthy controls comprising 173 women and 180 men, 20-88 yr of age, and in patients with metabolic bone disease. Using serum samples from patients with liver disease and patients with Paget's disease with elevated total alkaline phosphatase (T-ALP) as a source of, respectively, liver and bone isoenyzmes, they determined a liver cross-reactivity of the IRMA of 16% that was confirmed by electrophoresis of the circulating alkaline phosphatase isoenzymes. The IRMA was linear for serial sample dilutions, the recovery ranged from 89-110%, and the intra- and interassay variations were below 7% and 9%, respectively. B-ALP increased linearly with age in both sexes, and the mean B-ALP serum levels were not significantly different for women and men (11.3 [+-] 4.8 ng/mL for women; 11.0 [+-] 4.0 ng/mL for men). The increase in B-ALP after the menopause was significantly higher than that in T-ALP (+77% vs. +24%; P<0.001). When the values of postmenopausal women were expressed as the SD from the mean of premenopausal women, the mean Z scores were 2.2[+-] 1.8 for B-ALP and 0.9 [+-] 1.3 for T-ALP (P<0.001 between the two).

  12. Carbon quantum dots-based recyclable real-time fluorescence assay for alkaline phosphatase with adenosine triphosphate as substrate.

    PubMed

    Qian, Zhaosheng; Chai, Lujing; Tang, Cong; Huang, Yuanyuan; Chen, Jianrong; Feng, Hui

    2015-03-03

    A convenient, reliable, and highly sensitive real-time assay for alkaline phosphatase (ALP) activity in the continuous and recyclable way is established on the basis of aggregation and disaggregation of carbon quantum dots (CQDs) through the competitive assay approach. CQDs and adenosine triphosphate (ATP) were used as the fluorescent indicator and substrate for ALP activity assessment, respectively. Richness of carboxyl groups on the surface of CQDs enables their severe aggregation triggered by cerium ions, which results in effective fluorescence quenching. Under the catalytic hydrolysis of ALP, ATP can be rapidly transformed to phosphate ions. Stronger affinity of phosphate ions to cerium ions than carboxyl groups is taken advantage of to achieve fluorescence recovery induced by redispersion of CQDs in the presence of ALP and ATP. Quantitative evaluation of ALP activity in a broad range from 4.6 to 383.3 U/L with the detection limit of 1.4 U/L can be realized in this way, which endows the assay with high enough sensitivity for practical detection in human serum. The assay can be used in a recyclable way for more than three times since the generated product CePO4 as a precipitate can be easily removed from the standard assay system. This strategy broadens the sensing application of fluorescent CQDs with excellent biocompatibility and provides an example based on disaggregation in optical probe development.

  13. Flow screen-printed amperometric detection of p-nitrophenol in alkaline phosphatase-based assays.

    PubMed

    Fanjul-Bolado, Pablo; González-García, María Begoña; Costa-García, Agustín

    2006-08-01

    p-Nitrophenyl phosphate is one of the most widely used substrates for alkaline phosphatase in ELISAs because its yellow, water-soluble product, p-nitrophenol, absorbs strongly at 405 nm. p-Nitrophenol is also electroactive; an oxidative peak at 0.97 V (vs. an Ag pseudoreference electrode) is obtained when a bare screen-printed carbon electrode is used. When an amperometric detector was coupled to a flow-injection analysis system the detection limit achieved for p-nitrophenol was 2x10(-8) mol L(-1), almost two orders of magnitude lower than that obtained by measuring the absorbance of the compound. By use of this electrochemical detection method, measurement of 7x10(-14) mol L(-1) alkaline phosphatase was achieved after incubation for 20 min. The feasibility of coupling immunoassay to screen-printed carbon electrode amperometric detection has been demonstrated by performing an ELISA for detection of pneumolysin, a toxin produced by Streptococcus pneumoniae, which causes respiratory infections. The method is simple, reproducible, and much more sensitive than traditional spectrophotometry.

  14. A sensitive enzyme-catalytic nanogold-resonance scattering spectral assay for alkaline phosphate.

    PubMed

    Jiang, Zhiliang; Wu, Meng; Liu, Gaosan; Liang, Aihui

    2012-06-01

    In pH 8.9 Tris-HCl buffer solutions, alkaline phosphatase (ALP) catalyzed the hydrolysis of ascorbic acid 2-phosphate (AAP) substrate to form ascorbic acid. Then H(3)PO(4) was added to stop the enzymatic reaction and HAuCl(4) was used to react with ascorbic acid to generate gold nanoparticles that exhibited a resonance scattering (RS) peak at 600 nm. Under the selected conditions, when the activity of ALP increased, the formed ascorbic acid and gold nanoparticles also increased. Thus, the RS intensity at 600 nm enhanced linearly. The linear range was 0.06-22 U/L, with a detection limit of 0.03 U/L. The ALP in serum was analyzed, and the results were in agreement with those of the fluorescence method.

  15. Real-time fluorescence assays of alkaline phosphatase and ATP sulfurylase activities based on a novel PPi fluorescent probe.

    PubMed

    Wang, Xiaobo; Zhang, Zhiyang; Ma, Xiaoyan; Wen, Jinghan; Geng, Zhirong; Wang, Zhilin

    2015-05-01

    An anthracene-armed tetraaza macrocyclic fluorescent probe 3-(9-anthrylmethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene(l) for detecting Zn(2+) in aqueous medium was synthesized. L-Zn(2+) complex, showed selectivity toward pyrophosphate ion (PPi) by quenching the fluorescence in aqueous HEPES buffer (pH 7.4). Furthermore, L-Zn(2+) was also used to set up a real-time fluorescence assay for monitoring enzyme activities of alkaline phosphatase (ALP) and adenosine triphosphate sulfurylase (ATPS). In the presence of ALP inhibitor Na3VO4 and ATPS inhibitor chlorate, two enzymes activities decreased obviously, respectively.

  16. Screening for Novel Endogenous Inflammatory Stimuli Using the Secreted Embryonic Alkaline Phosphatase NF-κB Reporter Assay

    PubMed Central

    Zuliani-Alvarez, Lorena; Piccinini, Anna M.; Midwood, Kim S

    2017-01-01

    An immune response can be activated by pathogenic stimuli, as well as endogenous danger signals, triggering the activation of pattern recognition receptors and initiating signalling cascades that lead to inflammation. This method uses THP1-Blue™ cells, a human monocytic cell line which contains an embryonic alkaline phosphatase reporter gene allowing the detection of NF-κB-induced transcriptional activation. We validated this protocol by assessing NF-κB activation after stimulation of toll-like receptor 4 (TLR4) by two different agonists: lipopolysaccharide (LPS), derived from the cell wall of Gram negative bacteria, and tenascin-C, an extracellular matrix protein whose expression is induced upon tissue injury. We then used this protocol to screen for potential new endogenous TLR4 agonists, but this method can also be used as a quick, economical and reliable means to assay the activity of other inflammatory stimuli resulting in TLR-dependent NF-κB activation.

  17. Real-time ratiometric fluorescent assay for alkaline phosphatase activity with stimulus responsive infinite coordination polymer nanoparticles.

    PubMed

    Deng, Jingjing; Yu, Ping; Wang, Yuexiang; Mao, Lanqun

    2015-03-03

    This study demonstrates a novel ratiometric fluorescent method for real-time alkaline phosphatase (ALP) activity assay with stimulus responsive infinite coordination polymer (ICP) nanoparticles as the probe. The ICP nanoparticles used in this study are composed of two components; one is the supramolecular ICP network formed with guanine monophosphate (GMP) as the ligand and Tb(3+) as the central metal ion, and the other is a fluorescent dye, i.e., 7-amino-4-methyl coumarin (coumarin) encapsulated into the ICP network. Upon being excited at 315 nm, the ICP network itself emits green fluorescence at 552 nm. Coumarin dye encapsulated in the ICP network emits weak fluorescence at 450 nm upon excitation at the same wavelength (315 nm), and this fluorescence emission becomes strong when the encapsulated dye is released from the network into the solution phase. Hence, we develop a ratiometric fluorescent assay based on the ALP-induced destruction of the supramolecular ICP network and the release of coumarin. This mechanism can be used for real-time ratiometric fluorescent monitoring of ALP activity by continuously measuring the ratio of fluorescent intensity at the wavelength of 552 nm (F552) to that at 450 nm (F450) (F552/F450) in the time-dependent fluorescent spectra of the coumarin@Tb-GMP suspension containing ALP with different activities. Under the experimental conditions employed here, the F552/F450 value is linear with the ALP activity within a range from 0.025 U/mL to 0.2 U/mL. The detection limit is down to 0.010 U/mL (S/N = 3). Moreover, the assay developed here is employed for ALP inhibitor evaluation. This study offers a simple yet sensitive method for real-time ALP activity assay.

  18. ANALYSIS OF DNA DAMAGE AND REPAIR IN SKIN FIBROBLASTS OF INFANT AND OLDER CHILDREN USING THE IN VITRO ALKALINE COMET ASSAY

    EPA Science Inventory

    ANALYSIS OF DNA DAMAGE AND REPAIR IN SKIN FIBROBLASTS OF INFANT AND OLDER CHILDREN USING THE IN VITRO ALKALINE COMET ASSAY, Alan H. Tennant1, Geremy W. Knapp1 and Andrew D. Kligerman1, 1Environmental Carcinogenesis Division, National Health and Environmental Effects Research Lab...

  19. Use of agar diffusion assay to measure bactericidal activity of alkaline salts of fatty acids against bacteria associated with poultry processing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The agar diffusion assay was used to examine antibacterial activity of alkaline salts of caproic, caprylic, capric, lauric, and myristic acids. A 0.5M concentration of each fatty acid was dissolved in 1.0 M potassium hydroxide (KOH), and pH of the mixtures was adjusted to 10.5 with citric acid. Solu...

  20. Use of agar diffusion assay to evaluate bactericidal activity of formulations of alkaline salts of fatty acids against bacteria associated with poultry processing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The agar diffusion assay was used to examine antibacterial activity of alkaline salts of fatty acids (FA). Wells in agar media seeded with bacteria were filled with FA-potassium hydroxide (KOH) solutions, plates were incubated, and zones of inhibition were measured. The relationship between bacteric...

  1. AN EVALUATION OF THE RELATIVE GENOTOXICITY OF ARSENITE, ARSENATE, AND FOUR METHYLATED METABOLITES IN VITRO USING THE ALKALINE SINGLE CELL GEL ASSAY

    EPA Science Inventory

    An Evaluation of the Relative Genotoxicity of Arsenite, Arsenate, and Four Methylated
    Metabolites In Vitro Using the Alkaline Single Cell Gel Assay (ASCG).

    Arsenic ( As) is a genotoxic and carcinogenic metal found in many drinking water systems throughout the world. ...

  2. Genotoxic effects of boric acid and borax in zebrafish, Danio rerio using alkaline comet assay

    PubMed Central

    Gülsoy, Nagihan; Yavas, Cüneyd; Mutlu, Özal

    2015-01-01

    The present study is conducted to determine the potential mechanisms of Boron compounds, boric acid (BA) and borax (BX), on genotoxicity of zebrafish Danio rerio for 24, 48, 72 and 96-hours acute exposure (level:1, 4, 16, 64 mg/l BA and BX) in semi-static bioassay experiment. For that purpose, peripheral erythrocytes were drawn from caudal vein and Comet assay was applied to assess genotoxicity. Acute (96 hours) exposure and high concentrations of boric acid and borax increases % tail DNA and Olive tail moment. Genotoxicity was found for BA as concentration-dependent and BX as concentration and time dependent manner. In general, significant effects (P < 0,05) on both concentrations and exposure times were observed in experimental groups. DNA damage was highest at 96 h and 24 h for all BX and BA concentrations, respectively in peripheral blood of D. rerio. For the first time, our study demonstrates the effect of waterborne BA and BX exposure on genotoxicity at the molecular level, which may contribute to understanding the mechanism of boric acid and borax-induced genotoxicity in fish. PMID:26862320

  3. Genotoxic effects of boric acid and borax in zebrafish, Danio rerio using alkaline comet assay.

    PubMed

    Gülsoy, Nagihan; Yavas, Cüneyd; Mutlu, Özal

    2015-01-01

    The present study is conducted to determine the potential mechanisms of Boron compounds, boric acid (BA) and borax (BX), on genotoxicity of zebrafish Danio rerio for 24, 48, 72 and 96-hours acute exposure (level:1, 4, 16, 64 mg/l BA and BX) in semi-static bioassay experiment. For that purpose, peripheral erythrocytes were drawn from caudal vein and Comet assay was applied to assess genotoxicity. Acute (96 hours) exposure and high concentrations of boric acid and borax increases % tail DNA and Olive tail moment. Genotoxicity was found for BA as concentration-dependent and BX as concentration and time dependent manner. In general, significant effects (P < 0,05) on both concentrations and exposure times were observed in experimental groups. DNA damage was highest at 96 h and 24 h for all BX and BA concentrations, respectively in peripheral blood of D. rerio. For the first time, our study demonstrates the effect of waterborne BA and BX exposure on genotoxicity at the molecular level, which may contribute to understanding the mechanism of boric acid and borax-induced genotoxicity in fish.

  4. The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments.

    PubMed

    Ersson, Clara; Möller, Lennart

    2011-11-01

    The single cell gel electrophoresis (comet assay) is a popular method for measuring DNA migration as an estimate of DNA damage. No standardised comet assay protocol exists, which make comparisons between studies complicated. In a previous inter-laboratory validation study of the comet assay, we identified important parameters in the protocol that might affect DNA migration. The aim of this study was to assess how different comet assay protocols affect DNA migration. The results in this study suggest that (i) there is a significant linear dose-response relationship between the agarose gel's density and DNA migration and that damaged cells are more sensitive to the agarose gel's density; (ii) incubation with formamidopyrimidine DNA glycosylase for 10 min is inadequate, whereas 30 min is sufficient; (iii) the typically used 20 min of alkaline treatment might be to short when analysing samples that contain particular alkali-labile sites (ALS) and (iv) the duration of electrophoresis as well as the strength of the electric field applied affects the DNA migration. By using protocol-specific calibration curves, it is possible to reduce the variation in DNA migration caused by differences in comet assay protocols. This does, however, not completely remove the impact of the durations of alkaline treatment and electrophoresis when analysing cells containing ALS that are relatively resistant to high alkaline treatment.

  5. Genotoxicity of chlorpyrifos in freshwater fish Labeo rohita using Alkaline Single-cell Gel Electrophoresis (Comet) assay.

    PubMed

    Ismail, Muhammad; Khan, Qaiser Mahmood; Ali, Rahat; Ali, Tayyaba; Mobeen, Ameena

    2014-10-01

    Chlorpyrifos is a widely used insecticide of organophosphate group, which causes severe toxicological effects in non target aquatic organisms especially in fish. In the present study the genotoxic effects of sublethal concentrations of chlorpyrifos were observed in the erythrocytes and gill cells of Labeo rohita (commonly known as rohu) using the Alkaline Single-Cell Gel Electrophoresis (Comet) assay. Effects of chlorpyrifos on the behavior of the fish were also investigated. The 96 h LC50 value of chlorpyrifos, estimated by Trimmed Spearman-Karber (TSK) in static bioassay, was found to be 442.8 µg/L. On the basis of LC50 value, the fish were exposed to three sublethal concentrations of chlorpyrifos (SL-I ∼221.4 µg/L, SL- II ∼110.7 µg/L and SL-III ∼73.8 µg/L) for 96 h. Blood and gill samples were collected at every 24 h and were subjected to the Comet assay. The observed DNA damage was concentration dependent and time dependent and those levels of DNA damage in between the tested concentrations and times were significantly different (p < 0.01). It was also found that the gill cells are more sensitive to chlorpyrifos, though; it revealed more DNA damage as compared to the erythrocytes of fish. Fish exposed to different concentrations of chlorpyrifos showed different neurotoxic behavioral responses. It was concluded that chlorpyrifos is a genotoxic and neurotoxic insecticide causing DNA damage and neurotoxic effects in Labeo rohita.

  6. Assessment of the colorimetric and fluorometric assays for alkaline phosphatase activity in cow's, goat's, and sheep's milk.

    PubMed

    Klotz, V; Hill, Art; Warriner, K; Griffiths, M; Odumeru, J

    2008-09-01

    Raw milk is a well-established vehicle for the carriage of human pathogens, and many regulatory bodies have consequently mandated compulsory pasteurization as a food safety intervention. The residual activity of alkaline phosphatase (ALP) has historically been used to verify the adequacy of pasteurization of cow's milk. However, there is uncertainty on how the current ALP standards and methods of analysis can be applied to sheep's and goat's milk, which naturally contain different levels of the enzyme than that found in cow's milk. The official ALP methods applied in Canada (colorimetric assay; MFO-3) and in the United States (Fluorophos) were assessed for their ability to detect enzyme activity in raw and pasteurized milk derived from cows, sheep, and goats. The detection limit and the limit of quantitation were 0.8 and 2.02 microg/ml phenol, respectively, for the MFO-3 method and 43 and 85 mU/liter, respectively, for the Fluorophos method. The average ALP levels in raw goat's, cow's, and sheep's milk were 165, 1,562, and 3,512 microg/ml phenol, respectively. Raw milk detection limits, which correspond to raw milk phosphatase levels, were 0.051, 0.485, and 0.023% in cow's, goat's, and sheep's milk, respectively, for the MFO-3 method and 0.007, 0.070, and 0.004%, respectively, for the Fluorophos method. Although both methods can be used for ALP determination in cow's, goat's, and sheep's milk, the Fluorophos assay was superior to the colorimetric MFO-3 method based on sensitivity and time required to complete the analysis.

  7. Bioconjugation of alkaline phosphatase to mechanically processed, aqueous suspendible electrospun polymer nanofibers for use in chemiluminescent detection assays.

    PubMed

    Mark, Sonny S; Stolper, Samuel I; Baratti, Carla; Park, Jason Y; Taku, Maria A; Santiago-Avilés, Jorge J; Kricka, Larry J

    2008-06-11

    Aqueous suspendible polymer nanostructures were prepared by simple microtome processing of electrospun nylon 6 nanofibers and were used to immobilize calf intestinal alkaline phosphatase (ALP) by either covalent or noncovalent bioconjugation chemistries. It was found that noncovalent immobilization of ALP to the mechanically cut nanofibers (mean length approximately 4 microm; mean diameter approximately 80 nm) using a multi-stacked, layer-by-layer (LBL) approach with the cationic polymer Sapphire II resulted in the highest enzyme loading (48.1 +/- 0.4 microg . mg(-1) nanofiber) when compared to other covalent immobilization methods based on glutaraldehyde crosslinking. The biofunctionalized nanofibers were also characterized for their chemiluminescent activity with the dioxetane substrate, CSPD. The results indicate that the kinetic parameters, K(m) and V(max), for the catalytic activity of the nanostructure-bound ALP enzyme were influenced by the particular types of immobilization methods employed. In terms of the overall catalytic performance of the various immobilized ALP systems, a single-stacked LBL assembly approach resulted in the highest level of enzymatic activity per unit mass of nanofiber support. To the best of our knowledge, this study represents the first report examining the preparation of mechanically shortened, aqueous dispersed electrospun polymer nanofibers for potential application as enzyme scaffolds in chemiluminescent-based assay systems.

  8. A direct antigen-binding assay for detection of antibodies against native epitopes using alkaline phosphatase-tagged proteins.

    PubMed

    Baranov, Konstantin; Volkova, Olga; Chikaev, Nikolai; Mechetina, Ludmila; Laktionov, Pavel; Najakshin, Alexander; Taranin, Alexander

    2008-03-20

    We describe a simple and efficient method to detect antibodies against native epitopes following immunization with denatured proteins and peptides. With this method, soluble antigens genetically fused with placental alkaline phosphatase (AP) are used as probes to detect antibodies immobilized on nitrocellulose membranes. The AP-tagged proteins can be produced in sufficient amounts using transient transfection of eukaryotic cells with an appropriate cDNA fragment in a commercial AP-tag vector. The intrinsic thermo-stable phosphatase activity of a tagged protein obviates the need for its purification. To evaluate the method, three recently identified proteins of the FcR family, FCRLA, FCRL1, and FCRL4, were fused with AP and tested in a reaction with various polyclonal and monoclonal antibodies raised by immunization with bacterially produced antigens and peptide conjugates. All the three probes demonstrated high specificity in analysis of immune sera and hybridoma supernatants. Sensitivity of the assay varied depending on antibody tested and, in some cases, was in the subnanogram range. The results obtained show that AP-tagged proteins are useful tools for discrimination of antibodies against native epitopes when production of antigen in its native conformation is laborious and expensive.

  9. Unwinding forward and sliding back: an intermittent unwinding mode of the BLM helicase.

    PubMed

    Wang, Shuang; Qin, Wei; Li, Jing-Hua; Lu, Ying; Lu, Ke-Yu; Nong, Da-Guan; Dou, Shuo-Xing; Xu, Chun-Hua; Xi, Xu-Guang; Li, Ming

    2015-04-20

    There are lines of evidence that the Bloom syndrome helicase, BLM, catalyzes regression of stalled replication forks and disrupts displacement loops (D-loops) formed during homologous recombination (HR). Here we constructed a forked DNA with a 3' single-stranded gap and a 5' double-stranded handle to partly mimic a stalled DNA fork and used magnetic tweezers to study BLM-catalyzed unwinding of the forked DNA. We have directly observed that the BLM helicase may slide on the opposite strand for some distance after duplex unwinding at different forces. For DNA construct with a long hairpin, progressive unwinding of the hairpin is frequently interrupted by strand switching and backward sliding of the enzyme. Quantitative study of the uninterrupted unwinding length (time) has revealed a two-state-transition mechanism for strand-switching during the unwinding process. Mutational studies revealed that the RQC domain plays an important role in stabilizing the helicase/DNA interaction during both DNA unwinding and backward sliding of BLM. Especially, Lys1125 in the RQC domain, a highly conserved amino acid among RecQ helicases, may be involved in the backward sliding activity. We have also directly observed the in vitro pathway that BLM disrupts the mimic stalled replication fork. These results may shed new light on the mechanisms for BLM in DNA repair and homologous recombination.

  10. Unwinding forward and sliding back: an intermittent unwinding mode of the BLM helicase

    PubMed Central

    Wang, Shuang; Qin, Wei; Li, Jing-Hua; Lu, Ying; Lu, Ke-Yu; Nong, Da-Guan; Dou, Shuo-Xing; Xu, Chun-Hua; Xi, Xu-Guang; Li, Ming

    2015-01-01

    There are lines of evidence that the Bloom syndrome helicase, BLM, catalyzes regression of stalled replication forks and disrupts displacement loops (D-loops) formed during homologous recombination (HR). Here we constructed a forked DNA with a 3′ single-stranded gap and a 5′ double-stranded handle to partly mimic a stalled DNA fork and used magnetic tweezers to study BLM-catalyzed unwinding of the forked DNA. We have directly observed that the BLM helicase may slide on the opposite strand for some distance after duplex unwinding at different forces. For DNA construct with a long hairpin, progressive unwinding of the hairpin is frequently interrupted by strand switching and backward sliding of the enzyme. Quantitative study of the uninterrupted unwinding length (time) has revealed a two-state-transition mechanism for strand-switching during the unwinding process. Mutational studies revealed that the RQC domain plays an important role in stabilizing the helicase/DNA interaction during both DNA unwinding and backward sliding of BLM. Especially, Lys1125 in the RQC domain, a highly conserved amino acid among RecQ helicases, may be involved in the backward sliding activity. We have also directly observed the in vitro pathway that BLM disrupts the mimic stalled replication fork. These results may shed new light on the mechanisms for BLM in DNA repair and homologous recombination. PMID:25765643

  11. Is inflation from unwinding fluxes IIB?

    NASA Astrophysics Data System (ADS)

    Gautason, Fridrik Freyr; Schillo, Marjorie; Van Riet, Thomas

    2017-03-01

    In this paper we argue that the mechanism of unwinding inflation is naturally present in warped compactifications of type IIB string theory with local throats. The unwinding of flux is caused by its annihilation against branes. The resulting inflaton potential is linear with periodic modulations. We initiate an analysis of the inflationary dynamics and cosmological observables, which are highly constrained by moduli stabilization. For the simplified model of single-Kähler Calabi-Yau spaces we find that many, though not all of the consistency constraints can be satisfied. Particularly, in this simple model geometric constraints are in tension with obtaining the observed amplitude of the scalar power spectrum. However, we do find 60 efolds of inflation with a trans-Planckian field excursion which offers the hope that slightly more complicated models can lead to a fully consistent explicit construction of large field inflation of this kind.

  12. A multi-step model for facilitated unwinding of the yeast U4/U6 RNA duplex.

    PubMed

    Rodgers, Margaret L; Didychuk, Allison L; Butcher, Samuel E; Brow, David A; Hoskins, Aaron A

    2016-12-15

    The small nuclear RNA (snRNA) components of the spliceosome undergo many conformational rearrangements during its assembly, catalytic activation and disassembly. The U4 and U6 snRNAs are incorporated into the spliceosome as a base-paired complex within the U4/U6.U5 small nuclear ribonucleoprotein (tri-snRNP). U4 and U6 are then unwound in order for U6 to pair with U2 to form the spliceosome's active site. After splicing, U2/U6 is unwound and U6 annealed to U4 to reassemble the tri-snRNP. U6 rearrangements are crucial for spliceosome formation but are poorly understood. We have used single-molecule Förster resonance energy transfer and unwinding assays to identify interactions that promote U4/U6 unwinding and have studied their impact in yeast. We find that U4/U6 is efficiently unwound using DNA oligonucleotides by coupling unwinding of U4/U6 stem II with strand invasion of stem I. Unwinding is stimulated by the U6 telestem, which transiently forms in the intact U4/U6 RNA complex. Stabilization of the telestem in vivo results in accumulation of U4/U6 di-snRNP and impairs yeast growth. Our data reveal conserved mechanisms for U4/U6 unwinding and indicate telestem dynamics are critical for tri-snRNP assembly and stability.

  13. Kinetic mechanism of DNA unwinding by the BLM helicase core and molecular basis for its low processivity.

    PubMed

    Yang, Ye; Dou, Shuo-Xing; Xu, Ya-Nan; Bazeille, Nicolas; Wang, Peng-Ye; Rigolet, Pascal; Xu, Hou-Qiang; Xi, Xu Guang

    2010-02-02

    Bloom's syndrome (BS) is a rare human autosomal recessive disorder characterized by a strong predisposition to a wide range of cancers commonly affecting the general population. Understanding the functioning mechanism of the BLM protein may provide the opportunity to develop new effective therapy strategies. In this work, we studied the DNA unwinding kinetic mechanism of the helicase core of the BLM protein using various stopped-flow assays. We show that the helicase core of BLM unwinds duplex DNA as monomers even under conditions strongly favoring oligomerization. An unwinding rate of approximately 20 steps per second and a step size of 1 bp have been determined. We have observed that the helicase has a very low processivity. From dissociation and inhibition experiments, we have found that during its ATP hydrolysis cycle in DNA unwinding the helicase tends to dissociate from the DNA substrate in the ADP state. The experimental results imply that the BLM helicase core may unwind duplex DNA in an inchworm manner.

  14. A multi-step model for facilitated unwinding of the yeast U4/U6 RNA duplex

    PubMed Central

    Rodgers, Margaret L.; Didychuk, Allison L.; Butcher, Samuel E.; Brow, David A.; Hoskins, Aaron A.

    2016-01-01

    The small nuclear RNA (snRNA) components of the spliceosome undergo many conformational rearrangements during its assembly, catalytic activation and disassembly. The U4 and U6 snRNAs are incorporated into the spliceosome as a base-paired complex within the U4/U6.U5 small nuclear ribonucleoprotein (tri-snRNP). U4 and U6 are then unwound in order for U6 to pair with U2 to form the spliceosome's active site. After splicing, U2/U6 is unwound and U6 annealed to U4 to reassemble the tri-snRNP. U6 rearrangements are crucial for spliceosome formation but are poorly understood. We have used single-molecule Förster resonance energy transfer and unwinding assays to identify interactions that promote U4/U6 unwinding and have studied their impact in yeast. We find that U4/U6 is efficiently unwound using DNA oligonucleotides by coupling unwinding of U4/U6 stem II with strand invasion of stem I. Unwinding is stimulated by the U6 telestem, which transiently forms in the intact U4/U6 RNA complex. Stabilization of the telestem in vivo results in accumulation of U4/U6 di-snRNP and impairs yeast growth. Our data reveal conserved mechanisms for U4/U6 unwinding and indicate telestem dynamics are critical for tri-snRNP assembly and stability. PMID:27484481

  15. Coupling translocation with nucleic acid unwinding by NS3 helicase.

    PubMed

    Yu, Jin; Cheng, Wei; Bustamante, Carlos; Oster, George

    2010-12-03

    We present a semiquantitative model for translocation and unwinding activities of monomeric nonstructural protein 3 (NS3) helicase. The model is based on structural, biochemical, and single-molecule measurements. The model predicts that the NS3 helicase actively unwinds duplex by reducing more than 50% the free energy that stabilizes base pairing/stacking. The unwinding activity slows the movement of the helicase in a sequence-dependent manner, lowering the average unwinding efficiency to less than 1 bp per ATP cycle. When bound with ATP, the NS3 helicase can display significant translocational diffusion. This increases displacement fluctuations of the helicase, decreases the average unwinding efficiency, and enhances the sequence dependence. Also, interactions between the helicase and the duplex stabilize the helicase at the junction, facilitating the helicase's unwinding activity while preventing it from dissociating. In the presence of translocational diffusion during active unwinding, the dissociation rate of the helicase also exhibits sequence dependence. Based on unwinding velocity fluctuations measured from single-molecule experiments, we estimate the diffusion rate to be on the order of 10 s(-1). The generic features of coupling single-stranded nucleic acid translocation with duplex unwinding presented in this work may apply generally to a class of helicases.

  16. Assessment of the in vivo genotoxicity of cadmium chloride, chloroform, and D,L-menthol as coded test chemicals using the alkaline comet assay.

    PubMed

    Wada, Kunio; Fukuyama, Tomoki; Nakashima, Nobuaki; Matsumoto, Kyomu

    2015-07-01

    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM) international validation study of in vivo rat alkaline comet assays, we examined cadmium chloride, chloroform, and D,L-menthol under blind conditions as coded chemicals in the liver and stomach of Sprague-Dawley rats after 3 days of administration. Cadmium chloride showed equivocal responses in the liver and stomach, supporting previous reports of its poor mutagenic potential and non-carcinogenic effects in these organs. Treatment with chloroform, which is a non-genotoxic carcinogen, did not induce DNA damage in the liver or stomach. Some histopathological changes, such as necrosis and degeneration, were observed in the liver; however, they did not affect the comet assay results. D,L-Menthol, a non-genotoxic non-carcinogen, did not induce liver or stomach DNA damage. These results indicate that the comet assay can reflect genotoxic properties under blind conditions.

  17. Duplex unwinding and ATPase activities of the DEAD-box helicase eIF4A are coupled by eIF4G and eIF4B.

    PubMed

    Özeş, Ali R; Feoktistova, Kateryna; Avanzino, Brian C; Fraser, Christopher S

    2011-09-30

    Eukaryotic initiation factor (eIF) 4A is a DEAD-box helicase that stimulates translation initiation by unwinding mRNA secondary structure. The accessory proteins eIF4G, eIF4B, and eIF4H enhance the duplex unwinding activity of eIF4A, but the extent to which they modulate eIF4A activity is poorly understood. Here, we use real-time fluorescence assays to determine the kinetic parameters of duplex unwinding and ATP hydrolysis by these initiation factors. To ensure efficient duplex unwinding, eIF4B and eIF4G cooperatively activate the duplex unwinding activity of eIF4A. Our data reveal that eIF4H is much less efficient at stimulating eIF4A unwinding activity than eIF4B, implying that eIF4H is not able to completely substitute for eIF4B in duplex unwinding. By monitoring unwinding and ATPase assays under identical conditions, we demonstrate that eIF4B couples the ATP hydrolysis cycle of eIF4A with strand separation, thereby minimizing nonproductive unwinding events. Using duplex substrates with altered GC contents but similar predicted thermal stabilities, we further show that the rate of formation of productive unwinding complexes is strongly influenced by the local stability per base pair, in addition to the stability of the entire duplex. This finding explains how a change in the GC content of a hairpin is able to influence translation initiation while maintaining the overall predicted thermal stability.

  18. Active DNA unwinding dynamics during processive DNA replication.

    PubMed

    Morin, José A; Cao, Francisco J; Lázaro, José M; Arias-Gonzalez, J Ricardo; Valpuesta, José M; Carrascosa, José L; Salas, Margarita; Ibarra, Borja

    2012-05-22

    Duplication of double-stranded DNA (dsDNA) requires a fine-tuned coordination between the DNA replication and unwinding reactions. Using optical tweezers, we probed the coupling dynamics between these two activities when they are simultaneously carried out by individual Phi29 DNA polymerase molecules replicating a dsDNA hairpin. We used the wild-type and an unwinding deficient polymerase variant and found that mechanical tension applied on the DNA and the DNA sequence modulate in different ways the replication, unwinding rates, and pause kinetics of each polymerase. However, incorporation of pause kinetics in a model to quantify the unwinding reaction reveals that both polymerases destabilize the fork with the same active mechanism and offers insights into the topological strategies that could be used by the Phi29 DNA polymerase and other DNA replication systems to couple unwinding and replication reactions.

  19. Processivity of nucleic acid unwinding and translocation by helicases.

    PubMed

    Xie, Ping

    2016-11-01

    Helicases are a class of enzymes that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid (NA) strand separation. Besides the NA unwinding speed, another important factor for the helicase activity is the NA unwinding processivity. Here, we study the NA unwinding processivity with an analytical model that captures the phenomenology of the NA unwinding process. First, we study the processivity of the non-hexameric helicase that can unwind NA efficiently in the form of a monomer and the processivity of the hexameric helicase that can unwind DNA effectively, providing quantitative explanations of the available single-molecule experimental data. Then, we study the processivity of the non-hexameric helicases, in particular UvrD, in the form of a dimer and compare with that in the form of a monomer. The available single-molecule and some biochemical data showing that while UvrD monomer is a highly processive single-stranded DNA translocase it is inactive in DNA unwinding, whereas other biochemical data showing that UvrD is active in both single-stranded DNA translocation and DNA unwinding in the form of a monomer can be explained quantitatively and consistently. In addition, the recent single-molecule data are also explained quantitatively showing that constraining the 2B subdomain in closed conformation by intramolecular cross-linking can convert Rep monomer with a very poor DNA unwinding activity into a superhelicase that can unwind more than thousands of DNA base pairs processively, even against a large opposing force. Proteins 2016; 84:1590-1605. © 2016 Wiley Periodicals, Inc.

  20. Unwinding of circular helicoidal molecules vs. size

    NASA Astrophysics Data System (ADS)

    Zoli, Marco

    2015-04-01

    The thermodynamical stability of a set of circular double helical molecules is analyzed by path integral techniques. The minicircles differ only in i) the radius and ii) the number of base pairs (N) arranged along the molecule axis. Instead, the rise distance is kept constant. For any molecule size, the computational method simulates a broad ensemble of possible helicoidal configurations while the partition function is a sum over the path trajectories describing the base pair fluctuational states. The stablest helical repeat of every minicircle is determined by free-energy minimization. We find that, for molecules with N larger than 100, the helical repeat grows linearly with size and the twist number is constant. On the other hand, by reducing the size below 100 base pairs, the double helices sharply unwind and the twist number drops to one for N = 20. This is predicted as the minimum size for the existence of helicoidal molecules in the closed form. The helix unwinding appears as a strategy to release the bending stress associated to the circularization of the molecules.

  1. Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase.

    PubMed

    Kaku, Yoshihiro; Noguchi, Akira; Marsh, Glenn A; Barr, Jennifer A; Okutani, Akiko; Hotta, Kozue; Bazartseren, Boldbaatar; Fukushi, Shuetsu; Broder, Christopher C; Yamada, Akio; Inoue, Satoshi; Wang, Lin-Fa

    2012-01-01

    Nipah virus (NiV), Paramyxoviridae, Henipavirus, is classified as a biosafety level (BSL) 4 pathogen, along with the closely related Hendra virus (HeV). A novel serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing secreted alkaline phosphatase (SEAP) and pseudotyped with NiV F/G proteins (VSV-NiV-SEAP). A unique characteristic of this novel assay is the ability to obtain neutralization titers by measuring SEAP activity in supernatant using a common ELISA plate reader. This confers a remarkable advantage over the first generation of NiV-pseudotypes expressing green fluorescent protein or luciferase, which require expensive and specific measuring equipment. Using panels of NiV- and HeV-specific sera from various species, the VSV-NiV-SEAP assay demonstrated neutralizing antibody status (positive/negative) consistent with that obtained by conventional live NiV test, and gave higher antibody titers than the latter. Additionally, when screening sixty-six fruit bat sera at one dilution, the VSV-NiV-SEAP assay produced identical results to the live NiV test and only required a very small amount (2μl) of sera. The results suggest that this novel VSV-NiV-SEAP assay is safe, useful for high-throughput screening of sera using an ELISA plate reader, and has high sensitivity and specificity.

  2. JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for detection of genotoxic carcinogens: II. Summary of definitive validation study results.

    PubMed

    Uno, Yoshifumi; Kojima, Hajime; Omori, Takashi; Corvi, Raffaella; Honma, Masamistu; Schechtman, Leonard M; Tice, Raymond R; Beevers, Carol; De Boeck, Marlies; Burlinson, Brian; Hobbs, Cheryl A; Kitamoto, Sachiko; Kraynak, Andrew R; McNamee, James; Nakagawa, Yuzuki; Pant, Kamala; Plappert-Helbig, Ulla; Priestley, Catherine; Takasawa, Hironao; Wada, Kunio; Wirnitzer, Uta; Asano, Norihide; Escobar, Patricia A; Lovell, David; Morita, Takeshi; Nakajima, Madoka; Ohno, Yasuo; Hayashi, Makoto

    2015-07-01

    The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this exercise was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The study protocol was optimized in the pre-validation studies, and then the definitive (4th phase) validation study was conducted in two steps. In the 1st step, assay reproducibility was confirmed among laboratories using four coded reference chemicals and the positive control ethyl methanesulfonate. In the 2nd step, the predictive capability was investigated using 40 coded chemicals with known genotoxic and carcinogenic activity (i.e., genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic carcinogens, and non-genotoxic non-carcinogens). Based on the results obtained, the in vivo comet assay is concluded to be highly capable of identifying genotoxic chemicals and therefore can serve as a reliable predictor of rodent carcinogenicity.

  3. Three-dimensional mean flow experimental study of 'vortex unwinding'

    NASA Technical Reports Server (NTRS)

    Mcginley, Catherine B.

    1988-01-01

    A seven-hole flow-angularity pressure probe was used to assess the effectiveness of vortex unwinding in a turbulent boundary layer. The vortex center moved in the spanwise and vertical direction with increasing longitudinal distance. Initially, the vortex moved rapidly; it achieved 60 percent of its total 41 mm spanwise travel before it had completed half of its travel in the longitudinal direction. The flow was studied behind the single vortex combined with the most successful unwinder. The unwinder was found to be very effective in reducing the amount of secondary flow in the boundary layer.

  4. Unwinding and rewinding: double faces of helicase?

    PubMed

    Wu, Yuliang

    2012-01-01

    Helicases are enzymes that use ATP-driven motor force to unwind double-stranded DNA or RNA. Recently, increasing evidence demonstrates that some helicases also possess rewinding activity-in other words, they can anneal two complementary single-stranded nucleic acids. All five members of the human RecQ helicase family, helicase PIF1, mitochondrial helicase TWINKLE, and helicase/nuclease Dna2 have been shown to possess strand-annealing activity. Moreover, two recently identified helicases-HARP and AH2 have only ATP-dependent rewinding activity. These findings not only enhance our understanding of helicase enzymes but also establish the presence of a new type of protein: annealing helicases. This paper discusses what is known about these helicases, focusing on their biochemical activity to zip and unzip double-stranded DNA and/or RNA, their possible regulation mechanisms, and biological functions.

  5. Prokaryotic DNA ligases unwind superhelical DNA.

    PubMed

    Ivanchenko, M; van Holde, K; Zlatanova, J

    1996-09-13

    We have studied the effect on DNA topology of binding of prokaryotic DNA ligases (T4 and E. coli) to superhelical or nicked circular DNA. Performing topoisomerase I-mediated relaxation in the presence of increasing amounts of T4 ligase led to a shift in the topoisomer distribution to increasingly more negative values. This result suggested that T4 ligase unwound the DNA and was further substantiated by ligation of nicked circular molecules by E. coli DNA ligase in the presence of increasing amounts of T4 ligase. Such an experiment was possible since the two DNA ligases require different cofactors for enzymatic activity. Performing a similar experiment with reverse partners, using E. coli DNA ligase as ligand, and T4 ligase as sealing agent, we observed that the E. coli enzyme also unwound the DNA. Thus, prokaryotic DNA ligases can be added to an ever-growing list of DNA-binding proteins that unwind the DNA upon binding.

  6. Assessment of DNA damage of Lewis lung carcinoma cells irradiated by carbon ions and X-rays using alkaline comet assay

    NASA Astrophysics Data System (ADS)

    Li, Ping; Zhou, Li-Bin; Jin, Xiao-Dong; He, Jing; Dai, Zhong-Ying; Zhou, Guang-Ming; Gao, Qing-Xiang; Li, Sha; Li, Qiang

    2008-01-01

    DNA damage and cell reproductive death determined by alkaline comet and clonogenic survival assays were examined in Lewis lung carcinoma cells after exposure to 89.63 MeV/u carbon ion and 6 MV X-ray irradiations, respectively. Based on the survival data, Lewis lung carcinoma cells were verified to be more radiosensitive to the carbon ion beam than to the X-ray irradiation. The relative biological effectiveness (RBE) value, which was up to 1.77 at 10% survival level, showed that the DNA damage induced by the high-LET carbon ion beam was more remarkable than that induced by the low-LET X-ray irradiation. The dose response curves of “Tail DNA (%)” (TD) and “Olive tail moment” (OTM) for the carbon ion irradiation showed saturation beyond about 8 Gy. This behavior was not found in the X-ray curves. Additionally, the carbon ion beam produced a lower survival fraction at 2 Gy (SF2) value and a higher initial Olive tail moment 2 Gy (OTM2) than those for the X-ray irradiation. These results suggest that carbon ion beams having high-LET values produced more severe cell reproductive death and DNA damage in Lewis lung carcinoma cells in comparison with X-rays and comet assay might be an effective predictive test even combining with clonogenic assay to assess cellular radiosensitivity.

  7. Re-analysis results using medians of the data from the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay.

    PubMed

    Uno, Yoshifumi; Omori, Takashi

    2015-07-01

    The data from the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay were reported and analyzed statistically using the simple means of % tail DNA. However, OECD test guideline TG 489 recommends use of the median for data analysis due to the hierarchical nature of the data. Comparison between the simple mean approach and the median based approach for positive/negative/equivocal chemical calls was conducted using the % tail DNA data for the 40 chemicals tested in the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay, using liver and stomach as target organs. In the liver, two genotoxic chemicals, o-anisidine and 9-aminoacridine hydrochloride monohydrate, were positive using the median based approach but negative using the simple mean approach, and two genotoxic chemicals, 2-acetylaminofluorene and busulfan were equivocal using the median based approach but negative using the simple mean approach. In contrast, cadmium chloride (genotoxic carcinogen) was equivocal in both organs using the median based approach, while positive and equivocal in liver and stomach, respectively, using the simple mean approach. Two data sets of sodium arsenite showed equivocal and negative results for liver using the median based approach, although both data sets were equivocal using the simple mean approach. Overall, there are no large differences in terms of the genotoxic call between both approaches. However, the median based approach recommended in OECD TG 489 has an advantage toward higher precision within the groups treated with a test chemical, whereas the approach might show the lower values for the effect.

  8. Lack of marked cyto- and genotoxicity of cristobalite in devitrified (heated) alkaline earth silicate wools in short-term assays with cultured primary rat alveolar macrophages.

    PubMed

    Ziemann, Christina; Harrison, Paul T C; Bellmann, Bernd; Brown, Robert C; Zoitos, Bruce K; Class, Philippe

    2014-02-01

    Alkaline earth silicate (AES) wools are low-biopersistence high-temperature insulation wools. Following prolonged periods at high temperatures they may devitrify, producing crystalline silica (CS) polymorphs, including cristobalite, classified as carcinogenic to humans. Here we investigated the cytotoxic and genotoxic significance of cristobalite present in heated AES wools. Primary rat alveolar macrophages were incubated in vitro for 2 h with 200 µg/cm² unheated/heated calcium magnesium silicate wools (CMS1, CMS2, CMS3; heat-treated for 1 week at, or 4 weeks 150 °C below, their respective classification temperatures) or magnesium silicate wool (MS; heated for 24 h at 1260 °C). Types and quantities of CS formed, and fiber size distribution and shape were determined by X-ray diffraction and electron microscopy. Lactate dehydrogenase release and alkaline and hOGG1-modified comet assays were used, ± aluminum lactate (known to quench CS effects), for cytotoxicity/genotoxicity screening. Cristobalite content of wools increased with heating temperature and duration, paralleled by decreases in fiber length and changes in fiber shape. No marked cytotoxicity, and nearly no (CMS) or only slight (MS) DNA-strand break induction was observed, compared to the CS-negative control Al₂O₃, whereas DQ12 as CS-positive control was highly active. Some samples induced slight oxidative DNA damage, but no biological endpoint significantly correlated with free CS, quartz, or cristobalite. In conclusion, heating of AES wools mediates changes in CS content and fiber length/shape. While changes in fiber morphology can impact biological activity, cristobalite content appears minor or of no relevance to the intrinsic toxicity of heated AES wools in short-term assays with rat alveolar macrophages.

  9. Genotoxicity of Thermopsis turcica on Allium cepa L. roots revealed by alkaline comet and random amplified polymorphic DNA assays.

    PubMed

    Ciğerci, İbrahim Hakkı; Cenkci, Süleyman; Kargıoğlu, Mustafa; Konuk, Muhsin

    2016-08-01

    This study was undertaken to evaluate genotoxic potential of Thermopsis turcica aqueous extracts on the roots of onion bulb (Allium cepa L.) by comet assay and random amplified polymorphic DNA technique. The Allium root growth inhibition test indicated that the EC50 and 2×EC50 values were 8 and 16 mg/ml concentrations of T. turcica aqueous extracts, respectively. The negative control (distilled water), positive control (methyl methane sulfonate, 10 mg/l) and 8 and 16 mg/ml concentrations of T. turcica extracts were introduced to the roots of onion bulbs for 24 and 96 h. The root growth, DNA damage in root cells and randomly amplified polymorphic DNA (RAPD) profiles of root tissue were used as endpoints of the genotoxicity. The comet assay clearly indicated that dose-dependent single strand DNA breaks in the root nuclei of onions were determined for the treatment concentrations of T. turcica extracts. In comparison to RAPD profile of negative control group, RAPD polymorphisms became evident as disappearance and/or appearance of RAPD bands in treated roots. The diagnostic and phenetic numerical analyses of RAPD profiles obviously indicated dose-dependent genotoxicity induced by Thermopsis extracts. In conclusion, the results clearly indicated that water extract of T. turcica has genotoxic potential on the roots of onion bulbs as shown by comet assay and RAPD technique.

  10. Shifting nutrient-mediated interactions between algae and bacteria in a microcosm: evidence from alkaline phosphatase assay.

    PubMed

    Liu, Huali; Zhou, Yiyong; Xiao, Wenjuan; Ji, Lei; Cao, Xiuyun; Song, Chunlei

    2012-05-20

    The impacts of different nutrient additions (N + P, N + P + C, 4N + P, 4N + P + C, N + 2P) on the growth of algae and bacteria were studied in a microcosm experiment. Since alkaline phosphatase activity (APA) provides an indication of phosphorus deficiency, the higher value for algal APA in the treatments with excess nitrogen and for bacterial APA in the treatments with excess carbon suggested that, algal and bacterial phosphorus-limited status were induced by abundant nitrogen and carbon input, respectively. Bacterial phosphorus-limited status was weakened due to higher bacterial competition for phosphorus, compared to algae. In comparison with the bacterial and specific bacterial APA, higher values of algal and specific algal APA were found, which showed a gradual increase that coincided with the increase of chlorophyll a concentration. This fact indicated not only a stronger phosphorus demand by algae than by bacteria, but also a complementary relationship for phosphorus demand between algae and bacteria. However, this commensalism could be interfered by glucose input resulting in the decline of chlorophyll a concentration. Furthermore, the correlation between bacterial numbers and chlorophyll a concentration was positive in treatments without carbon and blurry in treatments with carbon. These observations validate a hypothesis that carbon addition can stimulate bacterial growth justifying bacterial nutrient demand, which decreases the availability of nutrients to algae and affects nutrient relationship between algae and bacteria. However, this interference would terminate after algal and bacterial adaption to carbon input.

  11. An investigation of the potential effect of sperm nuclear vacuoles in human spermatozoa on DNA fragmentation using a neutral and alkaline Comet assay.

    PubMed

    Pastuszek, E; Kiewisz, J; Skowronska, P; Liss, J; Lukaszuk, M; Bruszczynska, A; Jakiel, G; Lukaszuk, K

    2017-03-01

    Presence of vacuoles and degree of sperm DNA damage are considered to be the basic factors used for the assessment of sperm fertilization capacity. We aimed to investigate the link between these two parameters. According to our knowledge, this is the first study where the Comet assay was used to assess the degree of DNA fragmentation of sperm categorized by Motile Sperm Organelle Morphology Examination (MSOME) Grades. Semen samples from 10 patients were assessed. Spermatozoa were graded into four MSOME groups according to the Vanderzwalmen's criteria. A total of 3930 motile spermatozoa were selected one-by-one using an inverted microscope and transferred onto two different slides. The degree of DNA fragmentation was analyzed by alkaline and neutral Comet assay. Results of the neutral Comet assay showed that Grade I spermatozoa (absence of vacuoles) presented significantly lower dsDNA fragmentation level (mean: 3.13 ± 1.17%) than Grade II (maximum of two small vacuoles; mean: 10.34 ± 2.65%), Grade III (more than two small vacuoles or at least one large vacuole; mean: 23.88 ± 8.37%), and Grade IV (large vacuoles associated with abnormal head shapes or other abnormalities; mean: 36.94 ± 7.78%; p < 0.05). Results of the alkaline Comet assay showed that Grade I spermatozoa had significantly lower DNA (ssDNA + dsDNA) fragmentation level (mean: 8.33 ± 3.62%) than Grade III (mean: 25.64 ± 9.15%) and Grade IV (mean: 40.10 ± 9.10%, p < 0.05), but not significantly lower than Grade II (mean: 12.73 ± 5.06%; p > 0.05). Probably, the vacuoles may be responsible for double strand DNA breaks rather than single strand DNA breaks (only 2.39% spermatozoa in MSOME Grade II, 1.76% in III, and 3.16% in IV has single strand breaks). The results demonstrate that lower MSOME grading correlates with lower sperm DNA fragmentation. Therefore, the observation of sperm nuclear vacuoles using real-time optical microscopy without precise DNA fragmentation

  12. Unwinding initiation by the viral RNA helicase NPH-II.

    PubMed

    Fairman-Williams, Margaret E; Jankowsky, Eckhard

    2012-02-03

    Viral RNA helicases of the NS3/NPH-II group unwind RNA duplexes by processive, directional translocation on one of the duplex strands. The translocation is preceded by a poorly understood unwinding initiation phase. For NPH-II from vaccinia virus, unwinding initiation is rate limiting for the overall unwinding reaction. To develop a mechanistic understanding of the unwinding initiation, we studied kinetic and thermodynamic aspects of this reaction phase for NPH-II in vitro, using biochemical and single molecule fluorescence approaches. Our data show that NPH-II functions as a monomer and that different stages of the ATP hydrolysis cycle dictate distinct binding preferences of NPH-II for duplex versus single-stranded RNA. We further find that the NPH-II-RNA complex does not adopt a single conformation but rather at least two distinct conformations in each of the analyzed stages of ATP hydrolysis. These conformations interconvert with rate constants that depend on the stage of the ATP hydrolysis cycle. Our data establish a basic mechanistic framework for unwinding initiation by NPH-II and suggest that the various stages of the ATP hydrolysis cycle do not induce single, stage-specific conformations in the NPH-II-RNA complex but primarily control transitions between multiple states.

  13. Multiple Escherichia coli RecQ helicase monomers cooperate to unwind long DNA substrates: a fluorescence cross-correlation spectroscopy study.

    PubMed

    Li, Na; Henry, Etienne; Guiot, Elvire; Rigolet, Pascal; Brochon, Jean-Claude; Xi, Xu-Guang; Deprez, Eric

    2010-03-05

    The RecQ family helicases catalyze the DNA unwinding reaction in an ATP hydrolysis-dependent manner. We investigated the mechanism of DNA unwinding by the Escherichia coli RecQ helicase using a new sensitive helicase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. The FCCS-based assay can be used to measure the unwinding activity under both single and multiple turnover conditions with no limitation related to the size of the DNA strands constituting the DNA substrate. We found that the monomeric helicase was sufficient to perform the unwinding of short DNA substrates. However, a significant increase in the activity was observed using longer DNA substrates, under single turnover conditions, originating from the simultaneous binding of multiple helicase monomers to the same DNA molecule. This functional cooperativity was strongly dependent on several factors, including DNA substrate length, the number and size of single-stranded 3'-tails, and the temperature. Regarding the latter parameter, a strong cooperativity was observed at 37 degrees C, whereas only modest or no cooperativity was observed at 25 degrees C regardless of the nature of the DNA substrate. Consistently, the functional cooperativity was found to be tightly associated with a cooperative DNA binding mode. We also showed that the cooperative binding of helicase to the DNA substrate indirectly accounts for the sigmoidal dependence of unwinding activity on ATP concentration, which also occurs only at 37 degrees C but not at 25 degrees C. Finally, we further examined the influences of spontaneous DNA rehybridization (after helicase translocation) and the single-stranded DNA binding property of helicase on the unwinding activity as detected in the FCCS assay.

  14. Evaluation of 4,4'-diaminodiphenyl ether in the rat comet assay: Part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of in vivo rat alkaline comet assay.

    PubMed

    Priestley, Catherine C; Walker, Joanne S; O'Donovan, Michael R; Doherty, Ann T

    2015-07-01

    As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay, 4,4'-diaminodiphenyl ether (DPE), a known rodent genotoxic carcinogen, was tested in this laboratory. Sprague Dawley rats (7-9 weeks of age) were given three oral doses of DPE, 24 and 21 h apart and liver or stomach sampled 3h after the final dose. Under the conditions of the test, no increases in DNA damage in liver and stomach were observed with DPE (up to 200 mg/kg/day). A dose-dependent decrease in DNA migration, compared to vehicle controls, was noted for DPE in rat stomach. Further analysis is required to elucidate fully whether this decrease is a consequence of the mode of action or due to the toxicity of DPE. What is perhaps surprising is the inability of the comet assay to detect a known rat genotoxic carcinogen in liver. Further investigation is needed to clarify whether this apparent lack of response results from limited tissue exposure or metabolic differences between species. This finding highlights a need for careful consideration of study design when evaluating assay performance as a measure of in vivo genotoxicity.

  15. A fluorometric assay for alkaline phosphatase activity based on β-cyclodextrin-modified carbon quantum dots through host-guest recognition.

    PubMed

    Tang, Cong; Qian, Zhaosheng; Huang, Yuanyuan; Xu, Jiamin; Ao, Hang; Zhao, Meizhi; Zhou, Jin; Chen, Jianrong; Feng, Hui

    2016-09-15

    A convenient, reliable and highly sensitive assay for alkaline phosphatase (ALP) activity in the real-time manner is developed based on β-cyclodextrin-modified carbon quantum dots (β-CD-CQDs) nanoprobe through specific host-guest recognition. Carbon quantum dots were first functionalized with 3-aminophenyl boronic acid to produce boronic acid-functionalized CQDs, and then further modified with hydropropyl β-cyclodextrins (β-CD) through B-O bonds to form β-CD-CQDs nanoprobe. p-Nitrophenol phosphate disodium salt is used as the substrate of ALP, and can hydrolyze to p-nitrophenol under the catalysis of ALP. The resulting p-nitrophenol can enter the cavity of β-CD moiety in the nanoprobe due to their specific host-guest recognition, where photoinduced electron transfer process between p-nitrophenol and CQDs takes place to efficiently quench the fluorescence of the probe. The correlation between quenched fluorescence and ALP level can be used to establish quantitative evaluation of ALP activity in a broad range from 3.4 to 100.0U/L with the detection limit of 0.9U/L. This assay shows a high sensitivity to ALP even in the presence of a very high concentration of glucose. This study demonstrates a good electron donor/acceptor pair, which can be used to design general detection strategy through PET process, and also broadens the application of host-guest recognition for enzymes detection in clinical practice.

  16. Detection of in vivo DNA damage induced by ethanol in multiple organs of pregnant mice using the alkaline single cell gel electrophoresis (Comet) assay.

    PubMed

    Kido, Ryoko; Sato, Itaru; Tsuda, Shuji

    2006-01-01

    Ethanol is principal ingredient of alcohol beverage, but considered as human carcinogen, and has neurotoxicity. Alcohol consumption during pregnancy often causes fetal alcohol syndrome. The DNA damage is one of the important factors in carcinogenicity or teratogenicity. To detect the DNA damage induced by ethanol, we used an in vivo alkaline single cell gel electrophoresis (Comet) assay in pregnant mice organs and embryos. Pregnant ICR mice on Day 7 of gestation were treated with 2, 4 or 8 g/kg ethanol, and maternal organs/tissues and embryos were subjected to the Comet assay at 4, 8, 12 and 24 hr after ethanol treatment. Four and 8 g/kg ethanol induced DNA damage in brain, lung and embryos at 4 or 8 hr after the treatment. Two g/kg ethanol did not cause any DNA damage, and 8 g/kg ethanol only increased the duration of DNA damage without distinct increase in the degree of the damage. No significant DNA damage was observed in the liver. To detect the effect of acetaldehyde, disulfiram, acetaldehyde dehydrogenase inhibitor, was administered before 4 g/kg ethanol treatment. No significant increase of DNA damage was observed in the disulfiram pre-treated group. These data indicate that ethanol induces DNA damage, which might be related to ethanol toxicity. Since pre-treatment of disulfiram did not increase DNA damage, DNA damage observed in this study might not be the effect of acetaldehyde.

  17. Investigation of sodium arsenite, thioacetamide, and diethanolamine in the alkaline comet assay: Part of the JaCVAM comet validation exercise.

    PubMed

    Beevers, Carol; Henderson, Debbie; Lillford, Lucinda

    2015-07-01

    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined sodium arsenite, thioacetamide, and diethanolamine. Using the JaCVAM approved study protocol version 14.2, each chemical was tested in male rats up to maximum tolerated dose levels and DNA damage in the liver and stomach was assessed approximately 3h after the final administration by gavage. Histopathology assessments of liver and stomach sections from the same animals were also examined for evidence of cytotoxicity or necrosis. No evidence of DNA damage was observed in the stomach of animals treated with sodium arsenite at 7.5, 15, or 30 mg/kg/day. However, equivocal findings were found in the liver, where increases in DNA migration were observed in two independent experiments, but not in all treated animals and not at the same dose levels. Thioacetamide caused an increase in DNA migration in the stomach of rats treated at 19, 38, and 75 mg/kg/day, but not in the liver, despite evidence of marked hepatotoxicity following histopathology assessments. No evidence of DNA damage was observed in the stomach or liver of animals treated with diethanolamine at 175, 350, or 700 mg/kg/day.

  18. The eukaryotic initiation factor eIF4H facilitates loop-binding, repetitive RNA unwinding by the eIF4A DEAD-box helicase.

    PubMed

    Sun, Yingjie; Atas, Evrim; Lindqvist, Lisa; Sonenberg, Nahum; Pelletier, Jerry; Meller, Amit

    2012-07-01

    Eukaryotic translation initiation is a highly regulated process in protein synthesis. The principal translation initiation factor eIF4AI displays helicase activity, unwinding secondary structures in the mRNAs 5'-UTR. Single molecule fluorescence resonance energy transfer (sm-FRET) is applied here to directly observe and quantify the helicase activity of eIF4AI in the presence of the ancillary RNA-binding factor eIF4H. Results show that eIF4H can significantly enhance the helicase activity of eIF4AI by strongly binding both to loop structures within the RNA transcript as well as to eIF4AI. In the presence of ATP, the eIF4AI/eIF4H complex exhibits persistent rapid and repetitive cycles of unwinding and re-annealing. ATP titration assays suggest that this process consumes a single ATP molecule per cycle. In contrast, helicase unwinding activity does not occur in the presence of the non-hydrolysable analog ATP-γS. Based on our sm-FRET results, we propose an unwinding mechanism where eIF4AI/eIF4H can bind directly to loop structures to destabilize duplexes. Since eIF4AI is the prototypical example of a DEA(D/H)-box RNA helicase, it is highly likely that this unwinding mechanism is applicable to a myriad of DEAD-box helicases employed in RNA metabolism.

  19. Assessing the genotoxic potentials of roxarsone in V79 cells using the alkaline Comet assay and micronucleus test.

    PubMed

    Zhang, Yumei; Ying, Jun; Chen, Jun; Hu, Chenyun

    2012-01-24

    Until recently, knowledge about the genotoxicity of roxarsone in vitro or in vivo was limited. This study assessed the genotoxicity of roxarsone in an in vitro system. Roxarsone was tested for potential genotoxicity on V79 cells by a Comet assay and a micronucleus (MN) test, exposing the cells to roxarsone (1-500 μM) and to sodium arsenite (NaAsO₂, 20 μM) solutions for 3-48 h. Roxarsone was found to be cytotoxic when assessed with a commercial cell counting kit (CCK-8) used to evaluate cell viability, and moderately genotoxic in the Comet assay and micronucleus test used to assess DNA damage. The Comet metrics (percentages TDNA, TL, TM) increased significantly in a time- and concentration-dependent manner in roxarsone-treated samples compared with PBS controls (P<0.05), while the data from samples treated with 20 μM NaAsO₂ were comparable to those from 500 μM roxarsone-treated samples. The MN frequency of V79 cells treated with roxarsone was higher than that in the negative control but lower than the frequency in cells treated with 20 μM NaAsO₂. A dose- and time-dependent response in MN induction was observed at 10, 50, 100 and 500 μM doses of roxarsone after 12-48 h exposure time. The DNA damage in V79 cells treated with 500 μM roxarsone was similar to cells exposed to 20 μM NaAsO₂. The uptake of cells was correlated with the DNA damage caused by roxarsone. This investigation depicts the genotoxic potentials of roxarsone to V79 cells, which could lead to further advanced studies on the genotoxicity of roxarsone.

  20. Mcm subunits can assemble into two different active unwinding complexes.

    PubMed

    Kanter, Diane M; Bruck, Irina; Kaplan, Daniel L

    2008-11-07

    The replication fork helicase in eukaryotes is a large complex that is composed of Mcm2-7, Cdc45, and GINS. The Mcm2-7 proteins form a heterohexameric ring that hydrolyzes ATP and provide the motor function for this unwinding complex. A comprehensive study of how individual Mcm subunit biochemical activities relate to unwinding function has not been accomplished. We studied the mechanism of the Mcm4-Mcm6-Mcm7 complex, a useful model system because this complex has helicase activity in vitro. We separately purified each of three Mcm subunits until they were each nuclease-free, and we then examined the biochemical properties of different combinations of Mcm subunits. We found that Mcm4 and Mcm7 form an active unwinding assembly. The addition of Mcm6 to Mcm4/Mcm7 results in the formation of an active Mcm4/Mcm6/Mcm7 helicase assembly. The Mcm4-Mcm7 complex forms a ringed-shaped hexamer that unwinds DNA with 3' to 5' polarity by a steric exclusion mechanism, similar to Mcm4/Mcm6/Mcm7. The Mcm4-Mcm7 complex has a high level of ATPase activity that is further stimulated by DNA. The ability of different Mcm mixtures to form rings or exhibit DNA stimulation of ATPase activity correlates with the ability of these complexes to unwind DNA. The Mcm4/Mcm7 and Mcm4/Mcm6/Mcm7 assemblies can open to load onto circular DNA to initiate unwinding. We conclude that the Mcm subunits are surprisingly flexible and dynamic in their ability to interact with one another to form active unwinding complexes.

  1. Monitoring of DNA breakage in embryonic stages of the African catfish Clarias gariepinus (Burchell, 1822) after exposure to lead nitrate using alkaline comet assay.

    PubMed

    Osman, Alaa G M; Mekkawy, Imam A; Verreth, Johan; Wuertz, Sven; Kloas, Werner; Kirschbaum, Frank

    2008-12-01

    Increasing lead contamination in Egyptian ecosystems and high lead concentrations in food items have raised concern for human health and stimulated studies on monitoring ecotoxicological impact of lead-caused genotoxicity. In this work, the alkaline comet assay was modified for monitoring DNA strand breakage in sensitive early life stages of the African catfish Clarias gariepinus. Following exposure to 100, 300, and 500 microg/L lead nitrate, DNA strand breakage was quantified in embryos at 30, 48, 96, 144, and 168 h post-fertilization (PFS). For quantitative analysis, four commonly used parameters (tail % DNA, %TDNA; head % DNA, %HDNA; tail length, TL; tail moment, TM) were analyzed in 96 nuclei (in triplicates) at each sampling point. The parameter %TDNA revealed highest resolution and lowest variation. A strong correlation between lead concentration, time of exposure, and DNA strand breakage was observed. Here, genotoxicity detected by comet assay preceded the manifested malformations assessed with conventional histology. Qualitative evaluation was carried out using five categories are as follows: undamaged (%TDNA < or = 10%), low damaged (10% < %TDNA < or = 25%), median damaged (25 < %TDNA < or = 50%), highly damaged (50 < %TDNA < or = 75%), and extremely damaged (%TDNA > 75%) nuclei confirming a dose and time-dependent shift towards increased frequencies of highly and extremely damaged nuclei. A protective capacity provided by a hardened chorion is a an interesting finding in this study as DNA damage in the prehatching stages 30 h-PFS and 48 h-PFS was low in all treatments (qualitative and quantitative analyses). These results clearly show that the comet assay is a sensitive tool for the detection of genotoxicity in vulnerable early life stages of the African catfish and is a method more sensitive than histological parameters for monitoring genotoxic effects.

  2. Telomeres: structures in need of unwinding.

    PubMed

    Paeschke, Katrin; McDonald, Karin R; Zakian, Virginia A

    2010-09-10

    Telomeres protect the ends of eukaryotic chromosomes from being recognized and processed as double strand breaks. In most organisms, telomeric DNA is highly repetitive with a high GC-content. Moreover, the G residues are concentrated in the strand running 3'-5' from the end of the chromosome towards its center. This G-rich strand is extended to form a 3' single-stranded tail that can form unusual secondary structures such as T-loops and G-quadruplex DNA. Both the duplex repeats and the single-stranded G-tail are assembled into stable protein-DNA complexes. The unique architecture, high GC content, and multi-protein association create particularly stable protein-DNA complexes that are a challenge for replication, recombination, and transcription. Helicases utilize the energy of nucleotide hydrolysis to unwind base paired nucleic acids and, in some cases, to displace proteins from them. The telomeric functions of helicases from the RecQ, Pifl, FANCJ, and DNA2 families are reviewed in this article. We summarize data showing that perturbation of their telomere activities can lead to telomere dysfunction and genome instability and in some cases human disease.

  3. BENZO[A]PYRENE AND ITS K-REGION DIOL INDUCE DNA DAMAGE IN C3H10T1/2C18 CELLS AS MEASURED BY THE ALKALINE SINGLE CELL GEL (COMET) ASSAY

    EPA Science Inventory


    160. Benzo[a]pyrene and its K-region diol induce DNA damage in C3HlOTl/2Cl8 cells as measured by the alkaline single cell gel (Comet) assay

    In a continuing series of studies on the genotoxicity ofK-region dihydrodiols of polycyclic aromatic hydrocarbons, we have repo...

  4. Model of ribosome translation and mRNA unwinding.

    PubMed

    Xie, Ping

    2013-05-01

    A ribosome is an enzyme that catalyzes translation of the genetic information encoded in messenger RNA (mRNA) into proteins. Besides translation through the single-stranded mRNA, the ribosome is also able to translate through the duplex region of mRNA via unwinding the duplex. Here, based on our proposed ribosome translation model, we study analytically the dynamics of Escherichia coli ribosome translation through the duplex region of mRNA, and compare with the available single molecule experimental data. It is shown that the ribosome uses only one active mechanism (mechanical unwinding), rather than two active mechanisms (open-state stabilization and mechanical unwinding), as proposed before, to unwind the duplex. The reduced rate of translation through the duplex region is due to the occurrence of futile transitions, which are induced by the energy barrier from the duplex unwinding to the forward translocation along the single-stranded mRNA. Moreover, we also present predicted results of the average translation rate versus the external force acting on the ribosome translating through the duplex region and through the single-stranded region of mRNA, which can be easily tested by future experiments.

  5. Sensitive electrochemical assay of alkaline phosphatase activity based on TdT-mediated hemin/G-quadruplex DNAzyme nanowires for signal amplification.

    PubMed

    Liu, Yunqing; Xiong, Erhu; Li, Xiaoyu; Li, Junjing; Zhang, Xiaohua; Chen, Jinhua

    2017-01-15

    Taking TdT-mediated hemin/G-quadruplex DNAzyme nanowires as NADH oxidase and HRP-mimicking DNAzyme, a novel DNA-based electrochemical method has been developed for sensitive and selective assay of alkaline phosphatase (AP) activity. The double-stranded DNA (dsDNA) probe consisted of thiol-functionalized DNA1 and 3'-phosphorylated DNA2, was immobilized on a gold nanoparticles (AuNPs) modified glassy carbon (GC) electrode. In the presence of AP, 3'-phosphoryl end of DNA2 was dephosphorylated. Terminal deoxynucletidyl transferase (TdT) catalyzed the sequential addition of deoxynucleotides (dTTPs) at 3'-OH end of DNA2 to extend DNA2 with a poly-T sequence. Then, G-rich DNA3 strand hybridized with the poly-T sequence of DNA2. Upon addition of hemin, the hemin/G-quadruplex DNAzyme was formed. In the presence of NADH, the hemin/G-quadruplex DNAzyme oxidased NADH to NAD(+), accompanied by the formation of H2O2 which was further catalyzed by hemin/G-quadruplex DNAzyme (served as a HRP-mimicking DNAzyme) with the thionine (Thi) as electron transfer mediator, leading to the amplified electrochemical signal. Under optimized conditions, the response peak current was linear with the concentration of AP in the range from 0.1UL(-1) to 5UL(-1) with the detection limit of 0.03UL(-1). Also, the developed biosensor possessed good selectivity, reproducibility and stability, and simple sensing structure, showing promising practical applications in AP activity assay.

  6. Elastic coupling between RNA degradation and unwinding by an exoribonuclease.

    PubMed

    Lee, Gwangrog; Bratkowski, Matthew A; Ding, Fang; Ke, Ailong; Ha, Taekjip

    2012-06-29

    Rrp44 (Dis3) is a key catalytic subunit of the yeast exosome complex and can processively digest structured RNA one nucleotide at a time in the 3' to 5' direction. Its motor function is powered by the energy released from the hydrolytic nuclease reaction instead of adenosine triphosphate hydrolysis as in conventional helicases. Single-molecule fluorescence analysis revealed that instead of unwinding RNA in single base pair steps, Rrp44 accumulates the energy released by multiple single nucleotide step hydrolysis reactions until about four base pairs are unwound in a burst. Kinetic analyses showed that RNA unwinding, not cleavage or strand release, determines the overall RNA degradation rate and that the unwinding step size is determined by the nonlinear elasticity of the Rrp44/RNA complex, but not by duplex stability.

  7. 3'- to 5' DNA unwinding by TIP49b proteins.

    PubMed

    Papin, Christophe; Humbert, Odile; Kalashnikova, Anna; Eckert, Kelvin; Morera, Solange; Käs, Emmanuel; Grigoriev, Mikhail

    2010-06-01

    TIP49b (reptin) is an essential eukaryotic AAA+ ATPase involved in a variety of cellular processes, such as chromatin remodeling during double-strand break repair, transcriptional regulation, control of cell proliferation and small nucleolar RNA biogenesis. How it acts at the molecular level remains largely unknown. In the present study, we show that both human TIP49b and its yeast orthologue, Rvb2p, cooperatively bind single-stranded DNA as monomers. Binding stimulates a slow ATPase activity and supports a 3'- to 5' DNA unwinding activity that requires a 3'-protruding tail >or= 30 nucleotides. The data obtained indicate that DNA unwinding of 3'- to 5' junctions is also constrained by the length of flanking duplex DNA. By contrast, TIP49b hexamers were found to be inactive for ATP hydrolysis and DNA unwinding, suggesting that, in cells, protein factors that remain unknown might be required to recycle these into an active form.

  8. Analysis of possible genotoxicity of the herbicide flurochloridone and its commercial formulations: Endo III and Fpg alkaline comet assays in Chinese hamster ovary (CHO-K1) cells.

    PubMed

    Soloneski, Sonia; Nikoloff, Noelia; Larramendy, Marcelo L

    2016-02-01

    Cytotoxic and genotoxic effects of flurochloridone (FLC) and its formulations Twin Pack Gold(®) and Rainbow(®) were evaluated in CHO-K1 cells. Using the alkaline single-cell gel electrophoresis (SCGE) assay, we observed that FLC (15 μg/ml), Twin Pack Gold(®) or Rainbow(®) induced primary DNA damage, increasing the frequency of damaged nucleoids. Vitamin E pretreatment did not modify the effect. Decreased cell viability was observed only in Twin Pack Gold(®)-treated cultures and was significantly ameliorated by vitamin E. Post-treatment of herbicide-damaged CHO-K1 cells with the enzymes Endo III or Fpg did not increase FLC-, Twin Pack Gold(®)-, or Rainbow(®)-induced DNA damage. These results demonstrate that neither FLC nor FLC-based formulations induce DNA damage through hydroxyl radical or lipid alkoxyl radical production, and that the induced DNA lesions were not related to oxidative damage at the purine/pyrimidine level. Our observations strongly suggest that the cytotoxic effects observed after Twin Pack Gold(®) exposure are due to the excipients contained within the technical formulation rather than FLC itself.

  9. Genotoxicity of the herbicide imazethapyr in mammalian cells by oxidative DNA damage evaluation using the Endo III and FPG alkaline comet assays.

    PubMed

    Soloneski, Sonia; Ruiz de Arcaute, Celeste; Nikoloff, Noelia; Larramendy, Marcelo L

    2017-03-07

    We evaluated the role of oxidative stress in the genotoxic damage induced by imazethapyr (IMZT) and its formulation Pivot® in mammalian CHO-K1 cell line. Using the alkaline comet assay, we observed that a concentration of 0.1 μg/mL of IMZT or Pivot® was able to induce DNA damage by increasing the frequency of damaged nucleoids. To test whether the DNA lesions were caused by oxidative stress, the DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), which convert base damage to strand breaks, were used. Our results demonstrate that after treatment of CHO-K1 cells with the pure active ingredient as well as the commercial formulation Pivot®, an increase in DNA strand breaks was observed after incubation of both Endo III and Fpg enzymes, indicating that both compounds induce DNA damage involving both pyrimidine and purine-based oxidations, at least in CHO-K1 cells. Our findings confirm the genotoxic potential of IMZT and suggest that this herbicide formulation must be employed with great caution, especially not only for exposed occupational workers but also for other living species.

  10. In vivo genotoxicity of ortho-phenylphenol, biphenyl, and thiabendazole detected in multiple mouse organs by the alkaline single cell gel electrophoresis assay.

    PubMed

    Sasaki, Y F; Saga, A; Akasaka, M; Yoshida, K; Nishidate, E; Su, Y Q; Matsusaka, N; Tsuda, S

    1997-12-12

    In Japan, ortho-phenylphenol (OPP), biphenyl (BP), and thiabendazole (2-(4'-thiazolyl)benzimidazole, TBZ) are commonly used as a postharvest treatment to preserve imported citrus fruits during transport and storage. We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of those agents in mouse stomach, liver, kidney, bladder, lung, brain, and bone marrow. CD-1 male mice were sacrificed 3, 8, and 24 h after oral administration of the test compounds. OPP (2000 mg/kg) induced DNA damage in the stomach, liver, kidney, bladder, and lung, BP (2000 mg/kg) and TBZ (200 mg/kg) induced DNA damage in all the organs studied. For OPP, increased DNA damage peaked at 3-8 h and tended to decrease at 24 h. For BP, on the contrary, increased DNA migration peaked at 24 h. That delay may have been due to the fact that OPP is metabolized by cytochrome 450 and prostaglandin H synthase to phenylbenzoquinone (PBQ), a DNA binding metabolite, and BP is metabolized to PBQ via OPP and m-phenylphenol. The positive response to TBZ, an aneugen, supports the in vivo DNA-damaging action of TBZ.

  11. Evaluation of DNA Single and Double Strand Breaks in Women with Cervical Neoplasia Based on Alkaline and Neutral Comet Assay Techniques

    PubMed Central

    Cortés-Gutiérrez, Elva I.; Hernández-Garza, Fernando; García-Pérez, Jorge O.; Dávila-Rodríguez, Martha I.; Aguado-Barrera, Miguel E.; Cerda-Flores, Ricardo M.

    2012-01-01

    A hospital-based unmatched case-control study was performed in order to determine the relation of DNA single (ssb) and double (dsb) strand breaks in women with and without cervical neoplasia. Cervical epithelial cells of 30 women: 10 with low grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 without cervical lesions were evaluated using alkaline and neutral comet assays. A significant increase in global DNA damage (ssb + dsb) and dsb was observed in patients with HG-SIL (48.90 ± 12.87 and 23.50 ± 13.91), patients with LG-SIL (33.60 ± 14.96 and 11.20 ± 5.71), and controls (21.70 ± 11.87 and 5.30 ± 5.38; resp.). Pearson correlation coefficient reveled a strong relation between the levels ssb and dsb (r2 = 0.99, P = 0.03, and r2 = 0.94, P = 0.16, resp.) and progression of neoplasia. The increase of dsb damage in patients with HG-SIL was confirmed by DNA breakage detection-FISH (DBD-FISH) on neutral comets. Our results argue in favor of a real genomic instability in women with cervical neoplasia, which was strengthened by our finding of a higher proportion of DNA dsb. PMID:23093842

  12. Inhibition of dsDNA-templated copper nanoparticles by pyrophosphate as a label-free fluorescent strategy for alkaline phosphatase assay.

    PubMed

    Zhang, Liangliang; Zhao, Jingjin; Duan, Min; Zhang, Hua; Jiang, Jianhui; Yu, Ruqin

    2013-04-16

    On the basis of the inhibition of double strand DNA (dsDNA)-templated fluorescent copper nanoparticles (CuNPs) by pyrophosphate (PPi), a novel label-free turn-on fluorescent strategy to detect alkaline phosphatase (ALP) under physiological conditions has been developed. This method relies on the strong interaction between PPi and Cu(2+), which would hamper the effective formation of fluorescent CuNPs, leading to low fluorescence intensity. The ALP-catalyzed PPi hydrolysis would disable the complexation between Cu(2+) and PPi, facilitating the formation of fluorescent CuNPs through the reduction by ascorbate in the presence of dsDNA templates. Thus, the fluorescence intensity was recovered, and the fluorescence enhancement was related to the concentration of ALP. This method is cost-effective and convenient without any labels or complicated operations. The present strategy exhibits a high sensitivity and the turn-on mode provides a high selectivity for the ALP assay. Additionally, the inhibition effect of phosphate on the ALP activity was also studied. The proposed method using a PPi substrate may hold a potential application in diagnosis of ALP-related diseases or evaluation of ALP functions in biological systems.

  13. Mutual inhibition of RecQ molecules in DNA unwinding.

    PubMed

    Pan, Bing-Yi; Dou, Shuo-Xing; Yang, Ye; Xu, Ya-Nan; Bugnard, Elisabeth; Ding, Xiu-Yan; Zhang, Lingyun; Wang, Peng-Ye; Li, Ming; Xi, Xu Guang

    2010-05-21

    Helicases make conformational changes and mechanical movements through hydrolysis of NTP to unwind duplex DNA (or RNA). Most helicases require a single-stranded overhang for loading onto the duplex DNA substrates. Some helicases have been observed to exhibit an enhanced unwinding efficiency with increasing length of the single-stranded DNA tail both by preventing reannealing of the unwound DNA and by compensating for premature dissociation of the leading monomers. Here we report a previously unknown mutual inhibition of neighboring monomers in DNA unwinding by the monomeric Escherichia coli RecQ helicase. With single molecule fluorescence resonance energy transfer microscopy, we observed that the unwinding initiation of RecQ at saturating concentrations was more delayed for a long rather than a short tailed DNA. In stopped-flow kinetic studies under both single and multiple turnover conditions, the unwinding efficiency decreased with increasing enzyme concentration for long tailed substrates. In addition, preincubation of RecQ and DNA in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate was observed to alleviate the inhibition. We propose that the mutual inhibition effect results from a forced closure of cleft between the two RecA-like domains of a leading monomer by a trailing one, hence the forward movements of both monomers are stalled by prohibition of ATP binding to the leading one. This effect represents direct evidence for the relative movements of the two RecA-like domains of RecQ in DNA unwinding. It may occur for all superfamily I and II helicases possessing two RecA-like domains.

  14. Unwinding of a carbon nanoscroll due to high speed rotation

    NASA Astrophysics Data System (ADS)

    Yin, Hang; Cai, Kun

    2015-10-01

    A carbon nanoscroll (CNS) can be formed easily by rolling a graphene sheet around a carbon nanotube (CNT) [Zhang and Li, 2010, APL, 97, 081909]. When the CNS is driven by the rotary CNT to rotate at a high speed, the attractive interaction within the CNS or between the CNS and CNT is crippled by the centrifugal force on the CNS. The unwinding of CNS is triggered when the kinetic energy increment approaches to the variation of interaction energy of the system during CNS formation. Numerical experiments also indicate that the unwinding of CNS happens earlier when the CNT has a higher rotational speed or the system is at a higher temperature.

  15. Clinical utility of a wheat-germ precipitation assay for determination of bone alkaline phosphatase concentrations in patients with different metabolic bone diseases.

    PubMed

    Braga, V; Dorizzi, R; Brocco, G; Rossini, M; Zamberlan, N; Gatti, D; Adami, S

    1995-07-01

    Bone alkaline phosphatase was evaluated by wheat-germ lectin precipitation in several clinical conditions. The study included 33 premenopausal healthy women, 46 postmenopausal apparently healthy women, 19 growing children, 24 patients with Paget's disease, 31 patients with primary hyperparathyroidism and 66 patients with hepatobiliary diseases. In postmenopausal women the mean T score (i.e.: the number of SD below or above the mean for premenopausal women) was 2.6 +/- 1.3 (SD) for bone alkaline phosphatase and 1.61 +/- 1.21 for total alkaline phosphatase (p < 0.001). The T score for bone alkaline phosphatase provided a better discrimination from normals for both Paget's disease (22.1 +/- 27.8 versus 12.8 +/- 16 p < 0.001) and primary hyperparathyroidism (8.2 +/- 4.3 versus 4.6 +/- 3.7 p < 0.005 for bone alkaline phosphatase and total alkaline phosphatase respectively). After treatment with intravenous bisphosphonate the percent decrease of bone alkaline phosphatase was larger than that of total alkaline phosphatase both in patients with Paget's disease (-46% versus -72% p < 0.01) and in patients with primary hyperparathyroidism (-21% versus -47% p < 0.02) and an estimate of the precision (delta mean/SD of the delta mean) for bone alkaline phosphatase was 1.9-3.7 times higher than that of total alkaline phosphatase. In twelve osteoporotic patients treated for six months with oral alendronate the decrease in bone turnover was detected with significantly higher precision with bone alkaline phosphatase than with total alkaline phosphatase (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

  16. CdS/TiO2-fluorescein isothiocyanate nanoparticles as fluorescence resonance energy transfer probe for the determination of trace alkaline phosphatase based on affinity adsorption assay.

    PubMed

    Liu, Jia-Ming; Lin, Li-ping; Jiao, Li; Cui, Ma-Lin; Wang, Xin-Xing; Zhang, Li-Hong; Zheng, Zhi-Yong

    2012-08-30

    The CdS/TiO(2)-fluorescein isothiocyanate (FITC) luminescent nanoparticles (CdS/TiO(2)-FITC) with the particle size of 20 nm have been synthesized by sol-gel method. CdS/TiO(2)-FITC could emit the fluorescence of both FITC and CdS/TiO(2). The fluorescence resonance energy transfer (FRET) occurred between the donor CdS/TiO(2) and the acceptor FITC in the CdS/TiO(2)-FITC. Taking advantages of the excellent characteristics of FRET, a new CdS/TiO(2)-FITC FRET labeling reagent and a CdS/TiO(2)-FITC-wheat germ agglutinin (CdS/TiO(2)-FITC-WGA) fluorescent probe have been developed. The FRET occurring between the donor CdS/TiO(2) and the acceptor FITC in the labelled product CdS/TiO(2)-FITC-WGA-AP, formed in the affinity adsorption reaction between the WGA in this CdS/TiO(2)-FITC-WGA fluorescent probe and alkaline phosphatase (AP), sharply enhanced the fluorescence signal of FITC and quench the fluorescence signal of CdS/TiO(2). Moreover, the ΔF (the change of the fluorescence signal) of FITC and CdS/TiO(2) were proportional to the content of AP, respectively. Thus, a new method that CdS/TiO(2)-fluorescein isothiocyanate nanoparticles for the determination of trace AP based on FRET-affinity adsorption assay has been established. The limit of quantification (LOQ) of the method was 1.3×10(-17) g AP mL(-1) for CdS/TiO(2) and 1.1×10(-17) g AP mL(-1) for FITC, respectively. This sensitive, rapid, high selective and precise method has been applied to the determination of AP in human serum and the prediction of human disease with the results agreed well with enzyme-linked immunosorbent assay (ELISA) in Zhangzhou Municipal Hospital of Fujian Province. Simultaneously, the reaction mechanism for the determination of AP was also discussed.

  17. RNA unwinding by NS3 helicase: a statistical approach.

    PubMed

    Arunajadai, Srikesh G

    2009-09-22

    The study of double-stranded RNA unwinding by helicases is a problem of basic scientific interest. One such example is provided by studies on the hepatitis C virus (HCV) NS3 helicase using single molecule mechanical experiments. HCV currently infects nearly 3% of the world population and NS3 is a protein essential for viral genome replication. The objective of this study is to model the RNA unwinding mechanism based on previously published data and study its characteristics and their dependence on force, ATP and NS3 protein concentration. In this work, RNA unwinding by NS3 helicase is hypothesized to occur in a series of discrete steps and the steps themselves occurring in accordance with an underlying point process. A point process driven change point model is employed to model the RNA unwinding mechanism. The results are in large agreement with findings in previous studies. A gamma distribution based renewal process was found to model well the point process that drives the unwinding mechanism. The analysis suggests that the periods of constant extension observed during NS3 activity can indeed be classified into pauses and subpauses and that each depend on the ATP concentration. The step size is independent of external factors and seems to have a median value of 11.37 base pairs. The steps themselves are composed of a number of substeps with an average of about 4 substeps per step and an average substep size of about 3.7 base pairs. An interesting finding pertains to the stepping velocity. Our analysis indicates that stepping velocity may be of two kinds- a low and a high velocity.

  18. Kinetics and thermodynamics of phenotype: unwinding and rewinding the nucleosome.

    PubMed

    Mack, Andrew H; Schlingman, Daniel J; Ilagan, Robielyn P; Regan, Lynne; Mochrie, Simon G J

    2012-11-09

    Chromatin "remodeling" is widely accepted as the mechanism that permits access to DNA by the transcription machinery. To date, however, there has been no experimental measurement of the changes in the kinetics and thermodynamics of the DNA-histone octamer association that are required to remodel chromatin so that transcription may occur. Here, we present the results of optical tweezer measurements that compare the kinetic and thermodynamic properties of nucleosomes composed of unmodified histones with those of nucleosomes that contain a mutant histone H4 (H4-R45H), which has been shown to allow SWI/SNF remodeling factor-independent transcription from the yeast HO promoter in vivo. Our measurements, carried out in a force-clamp mode, determine the force-dependent unwinding and rewinding rates of the nucleosome inner turn. At each force studied, nucleosomes containing H4-R45H unwind more rapidly and rewind more slowly than nucleosomes containing unmodified H4, indicating that the latter are the more stable. Extrapolation to forces at which the winding and unwinding rates are equal determines the absolute free energy of the nucleosome inner turn to be -32k(B)T for nucleosomes containing unmodified H4 and -27k(B)T for nucleosomes containing H4-R45H. Thus, the "loosening" or "remodeling" caused by this point mutation, which is demonstrated to be sufficient to allow transcriptional machinery access to the HO promoter (in the absence of other remodeling factors), is 5k(B)T. The correlation between the free energy of the nucleosome inner turn and the sin (SWI/SNF-independent) transcription suggests that, beyond partial unwinding, complete histone unwinding may play a role in transcriptional activation.

  19. A fluorescence enhancement assay for cellular DNA damage. [X Radiation

    SciTech Connect

    Kanter, P.M.; Schwartz, H.S.

    1982-07-01

    A fluorescence procedure is described for quantitative measurement of DNA damage in mammalian cells. The technique is based upon the time-dependent partial alkaline unwinding of cellular DNA followed by determination of duplex:total DNA ratios with bisbenzamide, which has a differential molar fluorescence with single-stranded and duplex DNA. The method is rapid, does not require radioactive labeling of DNA, and is sufficiently sensitive to detect damage induced with 100 rads of X-irradiation. This method is standardized with respect to the alkaline unwinding unit, Mn0, and the unwinding constant, beta. Results obtained with this new technique and with hydroxylapatite chromatography for physical separation of single- and double-stranded DNA were confirmatory. The utility of the technique was demonstrated by detection of dose-related damage with X-irradiation and a variety of antineoplastic agents in unlabeled murine leukemia cells.

  20. Effects of co-exposure to extremely low frequency (50 Hz) magnetic fields and xenobiotics determined in vitro by the alkaline comet assay.

    PubMed

    Villarini, Milena; Moretti, Massimo; Scassellati-Sforzolini, Giuseppina; Boccioli, Bruno; Pasquini, Rossana

    2006-05-15

    In the present study, we used human peripheral blood leukocytes from 4 different donors, to investigate in vitro the possible genotoxic and/or co-genotoxic activity of extremely low frequency magnetic fields (ELF-MF) at 3 mT intensity. Two model mutagens were used to study the possible interaction between ELF-MF and xenobiotics: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4-nitroquinoline N-oxide (4NQO). Primary DNA damage was evaluated by the alkaline single-cell microgel-electrophoresis ("comet") assay. Control cells (leukocytes not exposed to ELF-MF, nor treated with genotoxins) from the different blood donors showed a comparable level of basal DNA damage, whereas the contribution of individual susceptibility toward ELF-MF and the tested genotoxic compounds led to differences in the extent of DNA damage observed following exposure to the genotoxins, both in the presence and in the absence of an applied ELF-MF. A 3 mT ELF-MF alone was unable to cause direct primary DNA damage. In leukocytes exposed to ELF-MF and genotoxins, the extent of MNNG-induced DNA damage increased with exposure duration compared to sham-exposed cells. The opposite was observed in cells treated with 4NQO. In this case the extent of 4NQO-induced DNA damage was somewhat reduced in leukocytes exposed to ELF-MF compared to sham-exposed cells. Moreover, in cells exposed to ELF-MF an increased concentration of GSH was always observed, compared to sham-exposed cells. Since following GSH conjugation the genotoxic pattern of MNNG and 4NQO is quite different, an influence of ELF-MF on the activity of the enzyme involved in the synthesis of GSH leading to different activation/deactivation of the model mutagens used was hypothesized to explain the different trends observed in MNNG and 4NQO genotoxic activity in the presence of an applied ELF-MF. The possibility that ELF-MF might interfere with the genotoxic activity of xenobiotics has important implications, since human populations are likely to be

  1. Unwinding of a single quantized vortex from a wire

    SciTech Connect

    Schwarz, K.W. )

    1993-05-01

    The dynamical behavior of a quantized vortex partially attached to a wire is studied theoretically, with the aim of interpreting recent experiments on quantized circulation in superfluid [sup 3]He-B. The geometry considered consists of a thin wire running parallel to the axis of a circular cylinder enclosing the wire. The circulation is assumed to run part way up the wire, and then to enter the fluid as a free vortex which eventually terminates on the outer wall. It is found that such a vortex achieves a state of steady precession around the wire, accompanied by a steady unwinding motion down the wire due to frictional effects. For an off-center wire, both the precession rate and the unwinding rate develop oscillatory components. Various particulars, such as the effects of friction, of moving the wire off center, and of pinning, are investigated. Excellent agreement is obtained between experiment, analytical theory, and numerical calculations.

  2. Structural Mechanisms of Hexameric Helicase Loading, Assembly, and Unwinding.

    PubMed

    Trakselis, Michael A

    2016-01-01

    Hexameric helicases control both the initiation and the elongation phase of DNA replication. The toroidal structure of these enzymes provides an inherent challenge in the opening and loading onto DNA at origins, as well as the conformational changes required to exclude one strand from the central channel and activate DNA unwinding. Recently, high-resolution structures have not only revealed the architecture of various hexameric helicases but also detailed the interactions of DNA within the central channel, as well as conformational changes that occur during loading. This structural information coupled with advanced biochemical reconstitutions and biophysical methods have transformed our understanding of the dynamics of both the helicase structure and the DNA interactions required for efficient unwinding at the replisome.

  3. Structural Mechanisms of Hexameric Helicase Loading, Assembly, and Unwinding

    PubMed Central

    Trakselis, Michael A.

    2016-01-01

    Hexameric helicases control both the initiation and the elongation phase of DNA replication. The toroidal structure of these enzymes provides an inherent challenge in the opening and loading onto DNA at origins, as well as the conformational changes required to exclude one strand from the central channel and activate DNA unwinding. Recently, high-resolution structures have not only revealed the architecture of various hexameric helicases but also detailed the interactions of DNA within the central channel, as well as conformational changes that occur during loading. This structural information coupled with advanced biochemical reconstitutions and biophysical methods have transformed our understanding of the dynamics of both the helicase structure and the DNA interactions required for efficient unwinding at the replisome. PMID:26918187

  4. The N domain of Argonaute drives duplex unwinding during RISC assembly.

    PubMed

    Kwak, Pieter Bas; Tomari, Yukihide

    2012-01-10

    Small RNAs, such as microRNAs and small interfering RNAs, act through Argonaute (Ago) proteins as a part of RNA-induced silencing complexes (RISCs). To make RISCs, Ago proteins bind and subsequently unwind small RNA duplexes, finally leaving one strand stably incorporated. Here we identified the N domain of human AGO2 as the initiator of duplex unwinding during RISC assembly. We discovered that a functional N domain is strictly required for small RNA duplex unwinding but not for precedent duplex loading or subsequent target cleavage. We postulate that RISC assembly is tripartite, comprising (i) RISC loading, whereby Ago undergoes conformational opening and loads a small RNA duplex, forming pre-RISC; (ii) wedging, whereby the end of the duplex is pried open through active wedging by the N domain, in preparation for unwinding; and (iii) unwinding, whereby the passenger strand is removed through slicer-dependent or slicer-independent unwinding, forming mature RISC.

  5. Topological Winding and Unwinding in Metastable Bose-Einstein Condensates

    SciTech Connect

    Kanamoto, Rina; Carr, Lincoln D.; Ueda, Masahito

    2008-02-15

    Topological winding and unwinding in a quasi-one-dimensional metastable Bose-Einstein condensate are shown to be manipulated by changing the strength of interaction or the frequency of rotation. Exact diagonalization analysis reveals that quasidegenerate states emerge spontaneously near the transition point, allowing a smooth crossover between topologically distinct states. On a mean-field level, the transition is accompanied by formation of gray solitons, or density notches, which serve as an experimental signature of this phenomenon.

  6. Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase: SUBSTRATE SPECIFICITY, DNA BRANCH MIGRATION, AND ABILITY TO OVERCOME BLOCKADES TO DNA UNWINDING.

    PubMed

    Khan, Irfan; Crouch, Jack D; Bharti, Sanjay Kumar; Sommers, Joshua A; Carney, Sean M; Yakubovskaya, Elena; Garcia-Diaz, Miguel; Trakselis, Michael A; Brosh, Robert M

    2016-07-01

    Mutations in the c10orf2 gene encoding the human mitochondrial DNA replicative helicase Twinkle are linked to several rare genetic diseases characterized by mitochondrial defects. In this study, we have examined the catalytic activity of Twinkle helicase on model replication fork and DNA repair structures. Although Twinkle behaves as a traditional 5' to 3' helicase on conventional forked duplex substrates, the enzyme efficiently dissociates D-loop DNA substrates irrespective of whether it possesses a 5' or 3' single-stranded tailed invading strand. In contrast, we report for the first time that Twinkle branch-migrates an open-ended mobile three-stranded DNA structure with a strong 5' to 3' directionality preference. To determine how well Twinkle handles potential roadblocks to mtDNA replication, we tested the ability of the helicase to unwind substrates with site-specific oxidative DNA lesions or bound by the mitochondrial transcription factor A. Twinkle helicase is inhibited by DNA damage in a unique manner that is dependent on the type of oxidative lesion and the strand in which it resides. Novel single molecule FRET binding and unwinding assays show an interaction of the excluded strand with Twinkle as well as events corresponding to stepwise unwinding and annealing. TFAM inhibits Twinkle unwinding, suggesting other replisome proteins may be required for efficient removal. These studies shed new insight on the catalytic functions of Twinkle on the key DNA structures it would encounter during replication or possibly repair of the mitochondrial genome and how well it tolerates potential roadblocks to DNA unwinding.

  7. Routes to DNA accessibility: alternative pathways for nucleosome unwinding.

    PubMed

    Schlingman, Daniel J; Mack, Andrew H; Kamenetska, Masha; Mochrie, Simon G J; Regan, Lynne

    2014-07-15

    The dynamic packaging of DNA into chromatin is a key determinant of eukaryotic gene regulation and epigenetic inheritance. Nucleosomes are the basic unit of chromatin, and therefore the accessible states of the nucleosome must be the starting point for mechanistic models regarding these essential processes. Although the existence of different unwound nucleosome states has been hypothesized, there have been few studies of these states. The consequences of multiple states are far reaching. These states will behave differently in all aspects, including their interactions with chromatin remodelers, histone variant exchange, and kinetic properties. Here, we demonstrate the existence of two distinct states of the unwound nucleosome, which are accessible at physiological forces and ionic strengths. Using optical tweezers, we measure the rates of unwinding and rewinding for these two states and show that the rewinding rates from each state are different. In addition, we show that the probability of unwinding into each state is dependent on the applied force and ionic strength. Our results demonstrate not only that multiple unwound states exist but that their accessibility can be differentially perturbed, suggesting possible roles for these states in gene regulation. For example, different histone variants or modifications may facilitate or suppress access to DNA by promoting unwinding into one state or the other. We anticipate that the two unwound states reported here will be the basis for future models of eukaryotic transcriptional control.

  8. A distinct triplex DNA unwinding activity of ChlR1 helicase.

    PubMed

    Guo, Manhong; Hundseth, Kristian; Ding, Hao; Vidhyasagar, Venkatasubramanian; Inoue, Akira; Nguyen, Chi-Hung; Zain, Rula; Lee, Jeremy S; Wu, Yuliang

    2015-02-20

    Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in genome maintenance. The DNA triplex helix structures that form by Hoogsteen or reverse Hoogsteen hydrogen bonding are examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human ChlR1 helicase to destabilize DNA triplexes. Biochemical studies demonstrated that ChlR1 efficiently melted both intermolecular and intramolecular DNA triplex substrates in an ATP-dependent manner. Compared with other substrates such as replication fork and G-quadruplex DNA, triplex DNA was a preferred substrate for ChlR1. Also, compared with FANCJ, a helicase of the same family, the triplex resolving activity of ChlR1 is unique. On the other hand, the mutant protein from a Warsaw breakage syndrome patient failed to unwind these triplexes. A previously characterized triplex DNA-specific antibody (Jel 466) bound triplex DNA structures and inhibited ChlR1 unwinding activity. Moreover, cellular assays demonstrated that there were increased triplex DNA content and double-stranded breaks in ChlR1-depleted cells, but not in FANCJ(-/-) cells, when cells were treated with a triplex stabilizing compound benzoquinoquinoxaline, suggesting that ChlR1 melting of triple-helix structures is distinctive and physiologically important to defend genome integrity. On the basis of our results, we conclude that the abundance of ChlR1 known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form in the human genome.

  9. JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: I. Summary of pre-validation study results.

    PubMed

    Uno, Yoshifumi; Kojima, Hajime; Omori, Takashi; Corvi, Raffaella; Honma, Masamistu; Schechtman, Leonard M; Tice, Raymond R; Burlinson, Brian; Escobar, Patricia A; Kraynak, Andrew R; Nakagawa, Yuzuki; Nakajima, Madoka; Pant, Kamala; Asano, Norihide; Lovell, David; Morita, Takeshi; Ohno, Yasuo; Hayashi, Makoto

    2015-07-01

    The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this validation effort was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The purpose of the pre-validation studies (i.e., Phase 1 through 3), conducted in four or five laboratories with extensive comet assay experience, was to optimize the protocol to be used during the definitive validation study.

  10. Recombination hotspots and single-stranded DNA binding proteins couple DNA translocation to DNA unwinding by the AddAB helicase-nuclease.

    PubMed

    Yeeles, Joseph T P; van Aelst, Kara; Dillingham, Mark S; Moreno-Herrero, Fernando

    2011-06-24

    AddAB is a helicase-nuclease that processes double-stranded DNA breaks for repair by homologous recombination. This process is modulated by Chi recombination hotspots: specific DNA sequences that attenuate the nuclease activity of the translocating AddAB complex to promote downstream recombination. Using a combination of kinetic and imaging techniques, we show that AddAB translocation is not coupled to DNA unwinding in the absence of single-stranded DNA binding proteins because nascent single-stranded DNA immediately re-anneals behind the moving enzyme. However, recognition of recombination hotspot sequences during translocation activates unwinding by coupling these activities, thereby ensuring the downstream formation of single-stranded DNA that is required for RecA-mediated recombinational repair. In addition to their implications for the mechanism of double-stranded DNA break repair, these observations may affect our implementation and interpretation of helicase assays and our understanding of helicase mechanisms in general.

  11. Evaluation of methyl methanesulfonate, 2,6-diaminotoluene and 5-fluorouracil: Part of the Japanese center for the validation of alternative methods (JaCVAM) international validation study of the in vivo rat alkaline comet assay.

    PubMed

    Plappert-Helbig, Ulla; Junker-Walker, Ursula; Martus, Hans-Joerg

    2015-07-01

    As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined methyl methanesulfonate, 2,6-diaminotoluene, and 5-fluorouracil under coded test conditions. Rats were treated orally with the maximum tolerated dose (MTD) and two additional descending doses of the respective compounds. In the MMS treated groups liver and stomach showed significantly elevated DNA damage at each dose level and a significant dose-response relationship. 2,6-diaminotoluene induced significantly elevated DNA damage in the liver at each dose and a statistically significant dose-response relationship whereas no DNA damage was obtained in the stomach. 5-fluorouracil did not induce DNA damage in either liver or stomach.

  12. Helicase processivity and not the unwinding velocity exhibits universal increase with force.

    PubMed

    Pincus, David L; Chakrabarti, Shaon; Thirumalai, D

    2015-07-21

    Helicases, involved in a number of cellular functions, are motors that translocate along single-stranded nucleic acid and couple the motion to unwinding double-strands of a duplex nucleic acid. The junction between double- and single-strands creates a barrier to the movement of the helicase, which can be manipulated in vitro by applying mechanical forces directly on the nucleic acid strands. Single-molecule experiments have demonstrated that the unwinding velocities of some helicases increase dramatically with increase in the external force, while others show little response. In contrast, the unwinding processivity always increases when the force increases. The differing responses of the unwinding velocity and processivity to force have lacked explanation. By generalizing a previous model of processive unwinding by helicases, we provide a unified framework for understanding the dependence of velocity and processivity on force and the nucleic acid sequence. We predict that the sensitivity of unwinding processivity to external force is a universal feature that should be observed in all helicases. Our prediction is illustrated using T7 and NS3 helicases as case studies. Interestingly, the increase in unwinding processivity with force depends on whether the helicase forces basepair opening by direct interaction or if such a disruption occurs spontaneously due to thermal fluctuations. Based on the theoretical results, we propose that proteins like single-strand binding proteins associated with helicases in the replisome may have coevolved with helicases to increase the unwinding processivity even if the velocity remains unaffected.

  13. Helicase Processivity and Not the Unwinding Velocity Exhibits Universal Increase with Force

    PubMed Central

    Pincus, David L.; Chakrabarti, Shaon; Thirumalai, D.

    2015-01-01

    Helicases, involved in a number of cellular functions, are motors that translocate along single-stranded nucleic acid and couple the motion to unwinding double-strands of a duplex nucleic acid. The junction between double- and single-strands creates a barrier to the movement of the helicase, which can be manipulated in vitro by applying mechanical forces directly on the nucleic acid strands. Single-molecule experiments have demonstrated that the unwinding velocities of some helicases increase dramatically with increase in the external force, while others show little response. In contrast, the unwinding processivity always increases when the force increases. The differing responses of the unwinding velocity and processivity to force have lacked explanation. By generalizing a previous model of processive unwinding by helicases, we provide a unified framework for understanding the dependence of velocity and processivity on force and the nucleic acid sequence. We predict that the sensitivity of unwinding processivity to external force is a universal feature that should be observed in all helicases. Our prediction is illustrated using T7 and NS3 helicases as case studies. Interestingly, the increase in unwinding processivity with force depends on whether the helicase forces basepair opening by direct interaction or if such a disruption occurs spontaneously due to thermal fluctuations. Based on the theoretical results, we propose that proteins like single-strand binding proteins associated with helicases in the replisome may have coevolved with helicases to increase the unwinding processivity even if the velocity remains unaffected. PMID:26200858

  14. From unwinding to clamping - the DEAD box RNA helicase family.

    PubMed

    Linder, Patrick; Jankowsky, Eckhard

    2011-07-22

    RNA helicases of the DEAD box family are present in all eukaryotic cells and in many bacteria and Archaea. These highly conserved enzymes are required for RNA metabolism from transcription to degradation and are therefore important players in gene expression. DEAD box proteins use ATP to unwind short duplex RNA in an unusual fashion and remodel RNA-protein complexes, but they can also function as ATP-dependent RNA clamps to provide nucleation centres that establish larger RNA-protein complexes. Structural, mechanistic and molecular biological studies have started to reveal how these conserved proteins can perform such diverse functions and how accessory proteins have a central role in their regulation.

  15. Evaluation of the mutagenicity and genotoxic potential of carvacrol and thymol using the Ames Salmonella test and alkaline, Endo III- and FPG-modified comet assays with the human cell line Caco-2.

    PubMed

    LLana-Ruiz-Cabello, Maria; Maisanaba, Sara; Puerto, Maria; Prieto, Ana I; Pichardo, Silvia; Jos, Ángeles; Cameán, Ana M

    2014-10-01

    Currently, direct antimicrobial and antioxidant additives derived from essential oils are used in food packaging and are perceived by consumers as low-health-risk compounds. In this study, we investigated the potential mutagenicity and genotoxicity of carvacrol and thymol, major compounds in several essential oils, using the Ames Salmonella test and the alkaline, Endo III- and formamidopyrimidine glycosylase (FPG)-modified comet assays, respectively. Thymol did not show any mutagenic activity at any concentration assayed (0-250 μM), whereas carvacrol exhibited mutagenic potential, displaying greater activity in presence of the metabolic fraction (29-460 μM). The genotoxic effects were evaluated in the human colon carcinoma cell line Caco-2, and the standard comet assay revealed that neither carvacrol (0-460 μM) nor thymol (0-250 μM) had any affects at 24 and 48 h. The FPG-modified comet assay showed that the highest concentration of carvacrol (460 μM) caused DNA damage, indicating damage to the purine bases. These results should be used to identify the appropriate concentrations of carvacrol and thymol as additives in food packaging. Moreover, further studies are necessary to explore the safety and/or the toxicity mechanisms of these compounds.

  16. Unwinding by local strand separation is critical for the function of DEAD-box proteins as RNA chaperones

    PubMed Central

    Campo, Mark Del; Mohr, Sabine; Jiang, Yue; Jia, Huijue; Jankowsky, Eckhard

    2009-01-01

    The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are broadly acting RNA chaperones that function in mitochondria to stimulate group I and group II intron splicing and activate mRNA translation. Previous studies showed that the S. cerevisiae cytosolic/nuclear DEAD-box protein Ded1p could stimulate group II intron splicing in vitro. Here, we show that Ded1p complements the mitochondrial translation and group I and II intron splicing defects in mss116Δ strains, stimulates the in vitro splicing of group I as well as group II introns, and functions indistinguishably from CYT-19 to resolve different non-native secondary and/or tertiary structures in the Tetrahymena thermophila LSU-ΔP5abc group I intron. The Escherichia coli DEAD-box protein SrmB also stimulates group I and II intron splicing in vitro, while the E. coli DEAD-box protein DbpA and vaccinia virus DExH-box protein NPH-II gave little if any group I or II intron splicing stimulation in vitro or in vivo. The four DEAD-box proteins that stimulate group I and II intron splicing unwind RNA duplexes by local strand separation and have little or no specificity, as judged by RNA-binding assays and stimulation of their ATPase activity by diverse RNAs. By contrast, DbpA binds group I and II intron RNAs non-specifically, but its ATPase activity is activated specifically by a helical segment of E. coli 23S rRNA, and NPH-II unwinds RNAs by directional translocation. The ability of DEAD-box proteins to stimulate group I and II intron splicing correlates primarily with their RNA-unwinding activity, which for the protein preparations used here was greatest for Mss116p, followed by Ded1p, CYT-19, and SrmB. Further, this correlation holds for all group I and II intron RNAs tested, implying a fundamentally similar mechanism for both types of introns. Our results support the hypothesis that DEAD-box proteins have an inherent ability to function as RNA chaperones by virtue of their

  17. Recruitment, Duplex Unwinding and Protein-Mediated Inhibition of the Dead-Box RNA Helicase Dbp2 at Actively Transcribed Chromatin.

    PubMed

    Ma, Wai Kit; Paudel, Bishnu P; Xing, Zheng; Sabath, Ivan G; Rueda, David; Tran, Elizabeth J

    2016-03-27

    RNA helicases play fundamental roles in modulating RNA structures and facilitating RNA-protein (RNP) complex assembly in vivo. Previously, our laboratory demonstrated that the DEAD-box RNA helicase Dbp2 in Saccharomyces cerevisiae is required to promote efficient assembly of the co-transcriptionally associated mRNA-binding proteins Yra1, Nab2, and Mex67 onto poly(A)(+)RNA. We also found that Yra1 associates directly with Dbp2 and functions as an inhibitor of Dbp2-dependent duplex unwinding, suggestive of a cycle of unwinding and inhibition by Dbp2. To test this, we undertook a series of experiments to shed light on the order of events for Dbp2 in co-transcriptional mRNP assembly. We now show that Dbp2 is recruited to chromatin via RNA and forms a large, RNA-dependent complex with Yra1 and Mex67. Moreover, single-molecule fluorescence resonance energy transfer and bulk biochemical assays show that Yra1 inhibits unwinding in a concentration-dependent manner by preventing the association of Dbp2 with single-stranded RNA. This inhibition prevents over-accumulation of Dbp2 on mRNA and stabilization of a subset of RNA polymerase II transcripts. We propose a model whereby Yra1 terminates a cycle of mRNP assembly by Dbp2.

  18. G-quadruplex hinders translocation of BLM helicase on DNA: a real-time fluorescence spectroscopic unwinding study and comparison with duplex substrates.

    PubMed

    Liu, Jia-quan; Chen, Chang-yue; Xue, Yong; Hao, Yu-hua; Tan, Zheng

    2010-08-04

    Sequences with the potential to form G-quadruplex structures are spread throughout genomic DNA. G-quadruplexes in promoter regions can play regulatory roles in gene expression. Expression of protein-encoding genes involves processing of DNA and RNA molecules at the level of transcription and translation, respectively. In order to examine how the G-quadruplex affects processing of nucleic acids, we established a real-time fluorescent assay and studied the unwinding of intramolecular G-quadruplex formed by the human telomere, ILPR and PSMA4 sequences by the BLM helicase. Through comparison with their corresponding duplex substrates, we found that the unwinding of intramolecular G-quadruplex structures was much less efficient than that of the duplexes. This result is in contrast to previous reports that multistranded intermolecular G-quadruplexes are far better substrates for the BLM and other RecQ family helicases. In addition, the unwinding efficiency varied significantly among the G-quadruplex structures, which correlated with the stability of the structures. These facts suggest that G-quadruplex has the capability to modulate the processing of DNA and RNA molecules in a stability-dependent manner and, as a consequence, may provide a mechanism to play regulatory roles in events such as gene expression.

  19. H/sub 2/O/sub 2/ as a DNA fragmenting agent in the alkaline elution interstrand crosslinking and DNA-protein crosslinking assays

    SciTech Connect

    Szmigiero, L.; Studzian, K.

    1988-01-01

    A method for DNA fragmentation by H/sub 2/O/sub 2/ in the DNA alkaline elution procedure is described. Treatment of cell suspensions for 1 h with 100 microM H/sub 2/O/sub 2/ or 5 mM H/sub 2/O/sub 2/ at 0-1 degree C resulted in DNA breakage equivalent to doses of 300 and 3000 rad of gamma-rays, respectively. The elution profiles were reproducible and H/sub 2/O/sub 2/ was used for measurements of interstrand crosslinks and DNA-protein crosslinks induced in HeLa cells by mitomycin C, cis-diamminedichloroplatinum(II), and trans-diamminedichloroplatinum(II). The comparison of data obtained with the use of H/sub 2/O/sub 2/ and gamma-rays has shown that both methods have similar sensitivity and reproducibility.

  20. RNA unwinding by the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex.

    PubMed

    Jia, Huijue; Wang, Xuying; Anderson, James T; Jankowsky, Eckhard

    2012-05-08

    Many RNA-processing events in the cell nucleus involve the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex, which contains the poly(A) polymerase Trf4p, the Zn-knuckle protein Air2p, and the RNA helicase Mtr4p. TRAMP polyadenylates RNAs designated for processing by the nuclear exosome. In addition, TRAMP functions as an exosome cofactor during RNA degradation, and it has been speculated that this role involves disruption of RNA secondary structure. However, it is unknown whether TRAMP displays RNA unwinding activity. It is also not clear how unwinding would be coordinated with polyadenylation and the function of the RNA helicase Mtr4p in modulating poly(A) addition. Here, we show that TRAMP robustly unwinds RNA duplexes. The unwinding activity of Mtr4p is significantly stimulated by Trf4p/Air2p, but the stimulation of Mtr4p does not depend on ongoing polyadenylation. Nonetheless, polyadenylation enables TRAMP to unwind RNA substrates that it otherwise cannot separate. Moreover, TRAMP displays optimal unwinding activity on substrates with a minimal Mtr4p binding site comprised of adenylates. Our results suggest a model for coordination between unwinding and polyadenylation activities by TRAMP that reveals remarkable synergy between helicase and poly(A) polymerase.

  1. The excluded DNA strand is SEW important for hexameric helicase unwinding.

    PubMed

    Carney, Sean M; Trakselis, Michael A

    2016-10-01

    Helicases are proposed to unwind dsDNA primarily by translocating on one strand to sterically exclude and separate the two strands. Hexameric helicases in particular have been shown to encircle one strand while physically excluding the other strand. In this article, we will detail experimental methods used to validate specific interactions with the excluded strand on the exterior surface of hexameric helicases. Both qualitative and quantitative methods are described to identify an excluded strand interaction, determine the exterior interacting residues, and measure the dynamics of binding. The implications of exterior interactions with the nontranslocating strand are discussed and include forward unwinding stabilization, regulation of the unwinding rate, and DNA damage sensing.

  2. Biological Significance of Unwinding Capability of Nuclear Matrix-Associating DNAs

    NASA Astrophysics Data System (ADS)

    Bode, J.; Kohwi, Y.; Dickinson, L.; Joh, T.; Klehr, D.; Mielke, C.; Kohwi-Shigematsu, T.

    1992-01-01

    Matrix attachment regions (MARs) are thought to separate chromatin into topologically constrained loop domains. A MAR located 5' of the human β-interferon gene becomes stably base-unpaired under superhelical strain, as do the MARs flanking the immunoglobulin heavy chain gene enhancer; in both cases a nucleation site exists for DNA unwinding. Concatemerized oligonucleotides containing the unwinding nucleation site exhibited a strong affinity for the nuclear scaffold and augmented SV40 promoter activity in stable transformants. Mutated concatemerized oligonucleotides resisted unwinding, showed weak affinity for the nuclear scaffold, and did not enhance promoter activity. These results suggest that the DNA feature capable of relieving superhelical strain is important for MAR functions.

  3. HUMAN DHX9 HELICASE UNWINDS TRIPLE HELICAL DNA STRUCTURES☟

    PubMed Central

    Jain, Aklank; Bacolla, Albino; Chakraborty, Prasun; Grosse, Frank; Vasquez, Karen M.

    2010-01-01

    Naturally occurring poly(purine·pyrimidine) rich regions in the human genome are prone to adopt non-canonical DNA structures such as intramolecular triplexes (i.e. H-DNA). Such structure-forming sequences are abundant and can regulate the expression of several diseases-linked genes. In addition, the use of triplex-forming oligonucleotides (TFOs) to modulate gene structure and function has potential as an approach to targeted gene therapy. Previously, we found that endogenous H-DNA structures can induce DNA double-strand breaks and promote genomic rearrangements. Herein, we find that the DHX9 helicase co-immunoprecipitates with triplex DNA structures in mammalian cells, suggesting a role in the maintenance of genome stability. We tested this postulate by assessing the helicase activity of purified human DHX9 on various duplex and triplex DNA substrates in vitro. DHX9 displaced the third strand from a specific triplex DNA structure and catalyzed the unwinding with a 3′→5′ polarity with respect to the displaced third strand. Helicase activity required a 3′-single-stranded overhang on the third strand and was dependent on ATP hydrolysis. The reaction kinetics consisted of a pre-steady-state burst phase followed by a linear, steady-state pseudo-zero-order-reaction. In contrast, very little, if any helicase activity was detected on blunt triplexes, triplexes with 5′-overhangs, blunt duplexes, duplexes with overhangs, or forked duplex substrates. Thus, triplex structures containing a 3′-overhang represent preferred substrates for DHX9, where it removes the strand with Hoogsteen hydrogen-bonded bases. Our results suggest the involvement of DHX9 in maintaining genome integrity by unwinding mutagenic triplex DNA structures. PMID:20669935

  4. OBSERVATIONAL EVIDENCE OF UNWINDING AND CHIRALITY CHANGING IN PENUMBRAL FILAMENTS BY HINODE

    SciTech Connect

    Su Jiangtao; Zhang Hongqi; Zhang Yin; He Han; Liu, Yu; Mao Xinjie

    2010-02-10

    In this paper, we present a very important and quite controversial observational property of penumbral filaments that may exhibit twisting motions and change their chirality. Using high-resolution observations from the Solar Optical Telescope on board Hinode, we study the unwinding and twisting motions in the penumbral filaments of active region NOAA 10930. Penumbral filaments, including those to the northwest of the magnetic polarity inversion line (PIL) and those inside the PIL region, are found to make unwinding motions associated with the decrease of their right-handed twists. After unwinding, the filaments inside the PIL region are found to twist continuously to develop left-handed twists in them. Moreover, we find the filament elongating, expanding, splitting, and the screw pitch decreasing in the unwinding process and, getting shorter, shrinking, merging, and the screw pitch increasing in the twisting process. These observational results indicate that penumbral filaments are more inclined to be twisted magnetic flux tubes in nature.

  5. Characterization of the Caenorhabditis elegans HIM-6/BLM helicase: unwinding recombination intermediates.

    PubMed

    Jung, Hana; Lee, Jin A; Choi, Seoyoon; Lee, Hyunwoo; Ahn, Byungchan

    2014-01-01

    Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3'-5' direction and needs at least five 3' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates.

  6. A DNA unwinding element and an ARS consensus comprise a replication origin within a yeast chromosome.

    PubMed Central

    Huang, R Y; Kowalski, D

    1993-01-01

    We have defined a replication origin, ORI305, within chromosome III of Saccharomyces cerevisiae by means of mutational analysis. cis-acting elements required for origin activity in the chromosome, as assayed by two-dimensional gel electrophoresis of replication intermediates, are the same as those required for the function of an autonomously replicating sequence, ARS305, in a plasmid. Essential elements include (i) an 11 bp sequence that is a near match to the ARS consensus and (ii) a broad sequence directly 3' to the consensus near match. Origin function is inactivated by point mutations in the essential near match sequence, suggesting that the sequence contributes to specifying the origin in the chromosome. Other consensus near matches with different sequences are present but are not required. The essential 3'-flanking sequence exhibits DNA helical instability and is sensitive to deletion mutations that stabilize the DNA helix. The wild-type 3'-flanking sequence can be functionally substituted by dissimilar sequences that also exhibit helical instability. The requirement for DNA helical instability indicates that the essential 3'-flanking sequence serves as a DNA unwinding element in the chromosome. Images PMID:8223462

  7. Evaluation of p-phenylenediamine, o-phenylphenol sodium salt, and 2,4-diaminotoluene in the rat comet assay as part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay.

    PubMed

    De Boeck, Marlies; van der Leede, Bas-jan; De Vlieger, Kathleen; Geys, Helena; Vynckier, An; Van Gompel, Jacky

    2015-07-01

    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay (comet assay), p-phenylenediamine dihydrochloride (PPD), o-phenylphenol sodium salt (OPP), and 2,4-diaminotoluene (2,4-DAT), were analyzed in this laboratory as coded test chemicals. Male Sprague-Dawley rats (7-9 weeks of age) were given three oral doses of the test compounds, 24 and 21 h apart and liver and stomach were sampled 3h after the final dose administration. Under the conditions of the test, no increases in DNA damage were observed in liver and stomach with PPD and OPP up to 100 and 1000 mg/kg/day, respectively. 2,4-DAT, a known genotoxic carcinogen, induced a weak but reproducible, dose-related and statistically significant increase in DNA damage in liver cells while no increases were observed in stomach cells.

  8. Copper(II) facilitates bleomycin-mediated unwinding of plasmid DNA

    SciTech Connect

    Levy, M.J.; Hecht, S.M.

    1988-04-19

    The unwinding of plasmid DNA by bleomycin A/sub 2/ (BLM A/sub 2/) was investigated by use of two-dimensional gel electrophoresis. It was found that Cu/sup 2 +/ ions greatly facilitated the unwinding of topoisomers of plasmid DNA by BLM A/sub 2/ at concentrations where cupric ions along had no effect on DNA supercoiling. The concentration of BLM A/sub 2/ required for observable unwinding was reduced at least 100-fold in the presence of equimolar Cu/sup 2 +/. A plot of (Cu/sup 2 +/) vs extent of DNA unwinding in the presence of 10/sup -4/ M BLM A/sub 2/ gave a curve consistent with the action of cupric ions on BLM in an allosteric fashion, possibly rearranging the drug into a conformation that facilitates DNA unwinding. The participation of the metal center in enhancing DNA unwinding via direct ionic interaction with one or more negatively charged groups on the DNA duplex also seems possible. Further analysis of the structural factors required for BLM-mediated DNA unwinding was carried out with Cu/sup 2 +/ + BLM demethyl A/sub 2/, the latter of which differs from BLM A/sub 2/ only in that it lacks a methyl group, and associated positive charge, at the C-terminus. Cu(II) x BLM demethyl A/sub 2/ was found to be much less effective than Cu(II) x BLM A/sub 2/ as a DNA unwinding agent, emphasizing the strong dependence of this process on the presence of positively charged groups within the BLM molecule. These findings constitute the first direct evidence that the metal center of BLM can participate in DNA interaction, as well as in the previously recognized role of oxygen binding and activation.

  9. AP1 enhances polyomavirus DNA replication by promoting T-antigen-mediated unwinding of DNA.

    PubMed Central

    Guo, W; Tang, W J; Bu, X; Bermudez, V; Martin, M; Folk, W R

    1996-01-01

    An early step in the initiation of polyomavirus DNA replication is viral large-T-antigen-mediated unwinding of the origin. We report that components of the AP1 transcription factor, Fos and Jun, interact with T antigen in vitro to enhance unwinding of the viral origin. This provides a biochemical basis for the capacity of AP1 to activate viral DNA replication in vivo. PMID:8763994

  10. Severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase.

    PubMed

    Adedeji, Adeyemi O; Singh, Kamalendra; Calcaterra, Nicholas E; DeDiego, Marta L; Enjuanes, Luis; Weiss, Susan; Sarafianos, Stefan G

    2012-09-01

    Severe acute respiratory syndrome (SARS) is a highly contagious disease, caused by SARS coronavirus (SARS-CoV), for which there are no approved treatments. We report the discovery of a potent inhibitor of SARS-CoV that blocks replication by inhibiting the unwinding activity of the SARS-CoV helicase (nsp13). We used a Förster resonance energy transfer (FRET)-based helicase assay to screen the Maybridge Hitfinder chemical library. We identified and validated a compound (SSYA10-001) that specifically blocks the double-stranded RNA (dsRNA) and dsDNA unwinding activities of nsp13, with 50% inhibitory concentrations (IC(50)s) of 5.70 and 5.30 μM, respectively. This compound also has inhibitory activity (50% effective concentration [EC(50)] = 8.95 μM) in a SARS-CoV replicon assay, with low cytotoxicity (50% cytotoxic concentration [CC(50)] = >250 μM), suggesting that the helicase plays a still unidentified critical role in the SARS-CoV life cycle. Enzyme kinetic studies on the mechanism of nsp13 inhibition revealed that SSYA10-001 acts as a noncompetitive inhibitor of nsp13 with respect to nucleic acid and ATP substrates. Moreover, SSYA10-001 does not affect ATP hydrolysis or nsp13 binding to the nucleic acid substrate. SSYA10-001 did not inhibit hepatitis C virus (HCV) helicase, other bacterial and viral RNA-dependent RNA polymerases, or reverse transcriptase. These results suggest that SSYA10-001 specifically blocks nsp13 through a novel mechanism and is less likely to interfere with the functions of cellular enzymes that process nucleic acids or ATP. Hence, it is possible that SSYA10-001 inhibits unwinding by nsp13 by affecting conformational changes during the course of the reaction or translocation on the nucleic acid. SSYA10-001 will be a valuable tool for studying the specific role of nsp13 in the SARS-CoV life cycle, which could be a model for other nidoviruses and also a candidate for further development as a SARS antiviral target.

  11. Cooperative translocation enhances the unwinding of duplex DNA by SARS coronavirus helicase nsP13.

    PubMed

    Lee, Na-Ra; Kwon, Hyun-Mi; Park, Kkothanahreum; Oh, Sangtaek; Jeong, Yong-Joo; Kim, Dong-Eun

    2010-11-01

    SARS coronavirus encodes non-structural protein 13 (nsP13), a nucleic acid helicase/NTPase belonging to superfamily 1 helicase, which efficiently unwinds both partial-duplex RNA and DNA. In this study, unwinding of DNA substrates that had different duplex lengths and 5'-overhangs was examined under single-turnover reaction conditions in the presence of excess enzyme. The amount of DNA unwound decreased significantly as the length of the duplex increased, indicating a poor in vitro processivity. However, the quantity of duplex DNA unwound increased as the length of the single-stranded 5'-tail increased for the 50-bp duplex. This enhanced processivity was also observed for duplex DNA that had a longer single-stranded gap in between. These results demonstrate that nsP13 requires the presence of a long 5'-overhang to unwind longer DNA duplexes. In addition, enhanced DNA unwinding was observed for gapped DNA substrates that had a 5'-overhang, indicating that the translocated nsP13 molecules pile up and the preceding helicase facilitate DNA unwinding. Together with the propensity of oligomer formation of nsP13 molecules, we propose that the cooperative translocation by the functionally interacting oligomers of the helicase molecules loaded onto the 5'-overhang account for the observed enhanced processivity of DNA unwinding.

  12. Yeast Pif1 helicase exhibits a one-base-pair stepping mechanism for unwinding duplex DNA.

    PubMed

    Ramanagoudr-Bhojappa, Ramanagouda; Chib, Shubeena; Byrd, Alicia K; Aarattuthodiyil, Suja; Pandey, Manjula; Patel, Smita S; Raney, Kevin D

    2013-05-31

    Kinetic analysis of the DNA unwinding and translocation activities of helicases is necessary for characterization of the biochemical mechanism(s) for this class of enzymes. Saccharomyces cerevisiae Pif1 helicase was characterized using presteady state kinetics to determine rates of DNA unwinding, displacement of streptavidin from biotinylated DNA, translocation on single-stranded DNA (ssDNA), and ATP hydrolysis activities. Unwinding of substrates containing varying duplex lengths was fit globally to a model for stepwise unwinding and resulted in an unwinding rate of ∼75 bp/s and a kinetic step size of 1 base pair. Pif1 is capable of displacing streptavidin from biotinylated oligonucleotides with a linear increase in the rates as the length of the oligonucleotides increased. The rate of translocation on ssDNA was determined by measuring dissociation from varying lengths of ssDNA and is essentially the same as the rate of unwinding of dsDNA, making Pif1 an active helicase. The ATPase activity of Pif1 on ssDNA was determined using fluorescently labeled phosphate-binding protein to measure the rate of phosphate release. The quantity of phosphate released corresponds to a chemical efficiency of 0.84 ATP/nucleotides translocated. Hence, when all of the kinetic data are considered, Pif1 appears to move along DNA in single nucleotide or base pair steps, powered by hydrolysis of 1 molecule of ATP.

  13. Results of the International Validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: Individual data for 1,2-dibromoethane, p-anisidine, and o-anthranilic acid in the 2nd step of the 4th phase Validation Study under the JaCVAM initiative.

    PubMed

    Takasawa, Hironao; Takashima, Rie; Narumi, Kazunori; Kawasako, Kazufumi; Hattori, Akiko; Kawabata, Masayoshi; Hamada, Shuichi

    2015-07-01

    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative International Validation Study of an in vivo rat alkaline comet assay, we examined 1,2-dibromoethane (DBE), p-anisidine (ASD), and o-anthranilic acid (ANT) to investigate the effectiveness of the comet assay in detecting genotoxic carcinogens. Each of the three test chemicals was administered to 5 male Sprague-Dawley rats per group by oral gavage at 48, 24, and 3h before specimen preparation. Single cells were collected from the liver and glandular stomach at 3h after the final dosing, and the specimens prepared from these two organs were subjected to electrophoresis under alkaline conditions (pH>13). The percentage of DNA intensity in the comet tail was then assessed using an image analysis system. A micronucleus (MN) assay was also conducted using these three test chemicals with the bone marrow (BM) cells collected from the same animals simultaneously used in the comet assay, i.e., combination study of the comet assay and BM MN assay. A genotoxic (Ames positive) rodent carcinogen, DBE gave a positive result in the comet assay in the present study, while a genotoxic (Ames positive) non-carcinogen, ASD and a non-genotoxic (Ames negative) non-carcinogen, ANT showed negative results in the comet assay. All three chemicals produced negative results in the BM MN assay. While the comet assay findings in the present study were consistent with those obtained from the rodent carcinogenicity studies for the three test chemicals, we consider the positive result in the comet assay for DBE to be particularly meaningful, given that this chemical produced a negative result in the BM MN assay. Therefore, the combination study of the comet assay and BM MN assay is a useful method to detect genotoxic carcinogens that are undetectable with the BM MN assay alone.

  14. Structural basis for replication origin unwinding by an initiator primase of plasmid ColE2-P9: duplex DNA unwinding by a single protein.

    PubMed

    Itou, Hiroshi; Yagura, Masaru; Shirakihara, Yasuo; Itoh, Tateo

    2015-02-06

    Duplex DNA is generally unwound by protein oligomers prior to replication. The Rep protein of plasmid ColE2-P9 (34 kDa) is an essential initiator for plasmid DNA replication. This protein binds the replication origin (Ori) in a sequence-specific manner as a monomer and unwinds DNA. Here we present the crystal structure of the DNA-binding domain of Rep (E2Rep-DBD) in complex with Ori DNA. The structure unveils the basis for Ori-specific recognition by the E2Rep-DBD and also reveals that it unwinds DNA by the concerted actions of its three contiguous structural modules. The structure also shows that the functionally unknown PriCT domain, which forms a compact module, plays a central role in DNA unwinding. The conservation of the PriCT domain in the C termini of some archaeo-eukaryotic primases indicates that it probably plays a similar role in these proteins. Thus, this is the first report providing the structural basis for the functional importance of the conserved PriCT domain and also reveals a novel mechanism for DNA unwinding by a single protein.

  15. Evidence for a functional dimeric form of the PcrA helicase in DNA unwinding.

    PubMed

    Yang, Ye; Dou, Shuo-Xing; Ren, Hua; Wang, Peng-Ye; Zhang, Xing-Dong; Qian, Min; Pan, Bing-Yi; Xi, Xu Guang

    2008-04-01

    PcrA helicase, a member of the superfamily 1, is an essential enzyme in many bacteria. The first crystal structures of helicases were obtained with PcrA. Based on structural and biochemical studies, it was proposed and then generally believed that PcrA is a monomeric helicase that unwinds DNA by an inchworm mechanism. But a functional state of PcrA from unwinding kinetics studies has been lacking. In this work, we studied the kinetic mechanism of PcrA-catalysed DNA unwinding with fluorometric stopped-flow method under both single- and multiple-turnover conditions. It was found that the PcrA-catalysed DNA unwinding depended strongly on the PcrA concentration as well as on the 3'-ssDNA tail length of the substrate, indicating that an oligomerization was indispensable for efficient unwinding. Study of the effect of ATP concentration on the unwinding rate gave a Hill coefficient of approximately 2, suggesting strongly that PcrA functions as a dimer. It was further determined that PcrA unwound DNA with a step size of 4 bp and a rate of approximately 9 steps per second. Surprisingly, it was observed that PcrA unwound 12-bp duplex substrates much less efficiently than 16-bp ones, highlighting the importance of protein-DNA duplex interaction in the helicase activity. From the present studies, it is concluded that PcrA is a dimeric helicase with a low processivity in vitro. Implications of the experimental results for the DNA unwinding mechanism of PcrA are discussed.

  16. Unwinding the State subsidisation of private health insurance in Ireland.

    PubMed

    Turner, Brian

    2015-10-01

    Ireland's private health insurance market provides primarily supplementary health insurance for hospital services, operating alongside a public hospital system to which residents have universal access entitlements, subject to some copayments for those without a medical card. The State subsidises the purchase of private health insurance through measures including tax relief on premiums and not charging the full economic cost for private beds in public hospitals. Furthermore, privately insured patients occupying public beds in public hospitals did not, until 2014, incur charges for such accommodation, apart from modest statutory charges. In the Budget in October 2013, a number of measures were announced that began to unwind these subsidies. Although it was initially feared that these measures would add to premium inflation, leading in turn to further discontinuation of health insurance, the evidence suggests that premium inflation has eased and take-up has stabilised, although some of this may have been due to the introduction of lifetime community rating in May 2015. Nevertheless, it would appear that the restriction on the subsidisation of private health insurance has not had a significant adverse effect on the market, while it has reduced an inequitable cross-subsidy.

  17. Unwinding motion of a twisted active region filament

    SciTech Connect

    Yan, X. L.; Xue, Z. K.; Kong, D. F.; Liu, J. H.; Xu, C. L.

    2014-12-10

    To better understand the structures of active region filaments and the eruption process, we study an active region filament eruption in active region NOAA 11082 in detail on 2010 June 22. Before the filament eruption, the opposite unidirectional material flows appeared in succession along the spine of the filament. The rising of the filament triggered two B-class flares at the upper part of the filament. As the bright material was injected into the filament from the sites of the flares, the filament exhibited a rapid uplift accompanying the counterclockwise rotation of the filament body. From the expansion of the filament, we can see that the filament consisted of twisted magnetic field lines. The total twist of the filament is at least 5π obtained by using a time slice method. According to the morphology change during the filament eruption, it is found that the active region filament was a twisted flux rope and its unwinding motion was like a solar tornado. We also find that there was a continuous magnetic helicity injection before and during the filament eruption. It is confirmed that magnetic helicity can be transferred from the photosphere to the filament. Using the extrapolated potential fields, the average decay index of the background magnetic fields over the filament is 0.91. Consequently, these findings imply that the mechanism of solar filament eruption could be due to the kink instability and magnetic helicity accumulation.

  18. Genotoxicity of three food processing contaminants in transgenic mice expressing human sulfotransferases 1A1 and 1A2 as assessed by the in vivo alkaline single cell gel electrophoresis assay

    PubMed Central

    Høie, Anja Hortemo; Svendsen, Camilla; Brunborg, Gunnar; Glatt, Hansruedi; Alexander, Jan; Meinl, Walter

    2015-01-01

    The food processing contaminants 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), 5‐hydroxymethylfurfural (HMF) and 2,5 dimethylfuran (DMF) are potentially both mutagenic and carcinogenic in vitro and/or in vivo, although data on DMF is lacking. The PHIP metabolite N‐hydroxy‐PhIP and HMF are bioactivated by sulfotransferases (SULTs). The substrate specificity and tissue distribution of SULTs differs between species. A single oral dose of PhIP, HMF or DMF was administered to wild‐type (wt) mice and mice expressing human SULT1A1/1A2 (hSULT mice). DNA damage was studied using the in vivo alkaline single cell gel electrophoresis (SCGE) assay. No effects were detected in wt mice. In the hSULT mice, PhIP and HMF exposure increased the levels of DNA damage in the liver and kidney, respectively. DMF was not found to be genotoxic. The observation of increased DNA damage in hSULT mice compared with wt mice supports the role of human SULTs in the bioactivation of N‐hydroxy‐PhIP and HMF in vivo. Environ. Mol. Mutagen. 56:709–714, 2015. © 2015 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc. PMID:26270892

  19. Genotoxicity of three food processing contaminants in transgenic mice expressing human sulfotransferases 1A1 and 1A2 as assessed by the in vivo alkaline single cell gel electrophoresis assay.

    PubMed

    Høie, Anja Hortemo; Svendsen, Camilla; Brunborg, Gunnar; Glatt, Hansruedi; Alexander, Jan; Meinl, Walter; Husøy, Trine

    2015-10-01

    The food processing contaminants 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 5-hydroxymethylfurfural (HMF) and 2,5 dimethylfuran (DMF) are potentially both mutagenic and carcinogenic in vitro and/or in vivo, although data on DMF is lacking. The PHIP metabolite N-hydroxy-PhIP and HMF are bioactivated by sulfotransferases (SULTs). The substrate specificity and tissue distribution of SULTs differs between species. A single oral dose of PhIP, HMF or DMF was administered to wild-type (wt) mice and mice expressing human SULT1A1/1A2 (hSULT mice). DNA damage was studied using the in vivo alkaline single cell gel electrophoresis (SCGE) assay. No effects were detected in wt mice. In the hSULT mice, PhIP and HMF exposure increased the levels of DNA damage in the liver and kidney, respectively. DMF was not found to be genotoxic. The observation of increased DNA damage in hSULT mice compared with wt mice supports the role of human SULTs in the bioactivation of N-hydroxy-PhIP and HMF in vivo.

  20. BLM helicase measures DNA unwound before switching strands and hRPA promotes unwinding reinitiation.

    PubMed

    Yodh, Jaya G; Stevens, Benjamin C; Kanagaraj, Radhakrishnan; Janscak, Pavel; Ha, Taekjip

    2009-02-18

    Bloom syndrome (BS) is a rare genetic disorder characterized by genomic instability and a high predisposition to cancer. The gene defective in BS, BLM, encodes a member of the RecQ family of 3'-5' DNA helicases, and is proposed to function in recombinational repair during DNA replication. Here, we have utilized single-molecule fluorescence resonance energy transfer microscopy to examine the behaviour of BLM on forked DNA substrates. Strikingly, BLM unwound individual DNA molecules in a repetitive manner, unwinding a short length of duplex DNA followed by rapid reannealing and reinitiation of unwinding in several successions. Our results show that a monomeric BLM can 'measure' how many base pairs it has unwound, and once it has unwound a critical length, it reverses the unwinding reaction through strand switching and translocating on the opposing strand. Repetitive unwinding persisted even in the presence of hRPA, and interaction between wild-type BLM and hRPA was necessary for unwinding reinitiation on hRPA-coated DNA. The reported activities may facilitate BLM processing of stalled replication forks and illegitimately formed recombination intermediates.

  1. Rapid unwinding of triplet repeat hairpins by Srs2 helicase of Saccharomyces cerevisiae.

    PubMed

    Dhar, Alok; Lahue, Robert S

    2008-06-01

    Expansions of trinucleotide repeats cause at least 15 heritable human diseases. Single-stranded triplet repeat DNA in vitro forms stable hairpins in a sequence-dependent manner that correlates with expansion risk in vivo. Hairpins are therefore considered likely intermediates during the expansion process. Unwinding of a hairpin by a DNA helicase would help protect against expansions. Yeast Srs2, but not the RecQ homolog Sgs1, blocks expansions in vivo in a manner largely dependent on its helicase function. The current study tested the idea that Srs2 would be faster at unwinding DNA substrates with an extrahelical triplet repeat hairpin embedded in a duplex context. These substrates should mimic the relevant intermediate structure thought to occur in vivo. Srs2 was faster than Sgs1 at unwinding several substrates containing triplet repeat hairpins or another structured loop. In contrast, control substrates with an unstructured loop or a Watson-Crick duplex were unwound equally well by both enzymes. Results with a fluorescently labeled, three-way junction showed that Srs2 unwinding proceeds unabated through extrahelical triplet repeats. In summary, Srs2 maintains its facile unwinding of triplet repeat hairpins embedded within duplex DNA, supporting the genetic evidence that Srs2 is a key helicase in Saccharomyces cerevisiae for preventing expansions.

  2. Torsional regulation of hRPA-induced unwinding of double-stranded DNA.

    PubMed

    De Vlaminck, Iwijn; Vidic, Iztok; van Loenhout, Marijn T J; Kanaar, Roland; Lebbink, Joyce H G; Dekker, Cees

    2010-07-01

    All cellular single-stranded (ss) DNA is rapidly bound and stabilized by single stranded DNA-binding proteins (SSBs). Replication protein A, the main eukaryotic SSB, is able to unwind double-stranded (ds) DNA by binding and stabilizing transiently forming bubbles of ssDNA. Here, we study the dynamics of human RPA (hRPA) activity on topologically constrained dsDNA with single-molecule magnetic tweezers. We find that the hRPA unwinding rate is exponentially dependent on torsion present in the DNA. The unwinding reaction is self-limiting, ultimately removing the driving torsional stress. The process can easily be reverted: release of tension or the application of a rewinding torque leads to protein dissociation and helix rewinding. Based on the force and salt dependence of the in vitro kinetics we anticipate that the unwinding reaction occurs frequently in vivo. We propose that the hRPA unwinding reaction serves to protect and stabilize the dsDNA when it is structurally destabilized by mechanical stresses.

  3. Inhibition of pea chloroplast DNA helicase unwinding and ATPase activities by DNA-interacting ligands.

    PubMed

    Tuteja, N; Phan, T N

    1998-03-27

    DNA helicases unwind the duplex DNA in an ATP dependent manner and thus play an essential role in DNA replication, repair, recombination and transcription. Any DNA-interacting ligand which will modulate DNA helicase activity may interrupt practically all kinds of DNA transactions. There are no studies on the effect of various cytotoxic DNA-interacting ligands on organelle helicases. We have determined the effect of camptothecin, VP-16 (etoposide), ellipticine, genistein, novobiocin, m-AMSA, actinomycin C1, ethidium bromide, daunorubicin and nogalamycin on unwinding and ATPase activities of purified chloroplast DNA helicase from pea (Pisum sativum). Our study has shown that DNA-intercalating ligands actinomycin C1, ethidium bromide, daunorubicin and nogalamycin were inhibiting the DNA unwinding activity with an apparent Ki of 2.9 microM, 3.0 microM, 1.4 microM and 1.0 microM, respectively. These four inhibitors also inhibited the ATPase activity of pea chloroplast DNA helicase. These results indicate that the intercalation of the inhibitors into DNA generates a complex that impedes the translocation of chloroplast DNA helicase, resulting in both inhibition of unwinding activity and ATP hydrolysis. This study would be useful for understanding the mechanism of organelle DNA helicase unwinding and the mechanism by which these DNA-interacting ligands inhibit cellular function.

  4. Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases.

    PubMed

    Manthei, Kelly A; Hill, Morgan C; Burke, Jordan E; Butcher, Samuel E; Keck, James L

    2015-04-07

    RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. Here, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3' single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ∼90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. This bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.

  5. Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

    PubMed Central

    Manthei, Kelly A.; Hill, Morgan C.; Burke, Jordan E.; Butcher, Samuel E.; Keck, James L.

    2015-01-01

    RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. Here, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3′ single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ∼90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. This bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures. PMID:25831501

  6. Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

    SciTech Connect

    Manthei, Kelly A.; Hill, Morgan C.; Burke, Jordan E.; Butcher, Samuel E.; Keck, James L.

    2015-03-23

    RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. In this paper, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3' single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ~90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. Finally, this bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.

  7. Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase.

    PubMed

    Langston, Lance D; O'Donnell, Mike E

    2017-03-27

    Replicative helicases are ring-shaped hexamers that encircle DNA for duplex unwinding. The currently accepted view of hexameric helicase function is by steric exclusion, where the helicase encircles one DNA strand and excludes the other, acting as a wedge with an external DNA unwinding point during translocation. Accordingly, strand specific blocks only affect these helicases when placed on the tracking strand, not the excluded strand. We examined the effect of blocks on the eukaryotic CMG and, contrary to expectations, blocks on either strand inhibit CMG unwinding. A recent cryoEM structure of yeast CMG shows that duplex DNA enters the helicase and unwinding occurs in the central channel. The results of this report inform important aspects of the structure and we propose that CMG functions by a modified steric exclusion process in which both strands enter the helicase and the duplex unwinding point is internal, followed by exclusion of the non-tracking strand.

  8. Distinctions in early stage unwinding mechanisms of zwitterionic, capped, and neutral forms of different α-helical homopolymeric peptides.

    PubMed

    Pandey, Prithvi Raj; Roy, Sudip

    2012-04-26

    Molecular dynamics simulations of α-helical polyalanine, polyleucine, polylysine, and poly(glutamic acid) with different forms of terminal groups in water at 300 K showed sharp distinctions in their unwinding mechanisms. Zwitterionic, capped, and neutral forms of polyalanine, polyleucine, and polylysine have been explored to elucidate their unwinding mechanism at very early stage, e.g., initial time window. Role of water in the unwinding mechanisms of the various helices has been envisaged. Also, it is evident from our calculations that the short- and long-range nonbonded interactions among the side chains is an important factor determining the unwinding mechanisms of the various homopolymeric α-helices. These findings can be helpful in constructing predictive models for understanding of the unwinding of α-helical proteins and peptides.

  9. Single-molecule studies reveal reciprocating of WRN helicase core along ssDNA during DNA unwinding.

    PubMed

    Wu, Wen-Qiang; Hou, Xi-Miao; Zhang, Bo; Fossé, Philippe; René, Brigitte; Mauffret, Olivier; Li, Ming; Dou, Shuo-Xing; Xi, Xu-Guang

    2017-03-07

    Werner syndrome is caused by mutations in the WRN gene encoding WRN helicase. A knowledge of WRN helicase's DNA unwinding mechanism in vitro is helpful for predicting its behaviors in vivo, and then understanding their biological functions. In the present study, for deeply understanding the DNA unwinding mechanism of WRN, we comprehensively characterized the DNA unwinding properties of chicken WRN helicase core in details, by taking advantages of single-molecule fluorescence resonance energy transfer (smFRET) method. We showed that WRN exhibits repetitive DNA unwinding and translocation behaviors on different DNA structures, including forked, overhanging and G-quadruplex-containing DNAs with an apparently limited unwinding processivity. It was further revealed that the repetitive behaviors were caused by reciprocating of WRN along the same ssDNA, rather than by complete dissociation from and rebinding to substrates or by strand switching. The present study sheds new light on the mechanism for WRN functioning.

  10. Single-molecule studies reveal reciprocating of WRN helicase core along ssDNA during DNA unwinding

    PubMed Central

    Wu, Wen-Qiang; Hou, Xi-Miao; Zhang, Bo; Fossé, Philippe; René, Brigitte; Mauffret, Olivier; Li, Ming; Dou, Shuo-Xing; Xi, Xu-Guang

    2017-01-01

    Werner syndrome is caused by mutations in the WRN gene encoding WRN helicase. A knowledge of WRN helicase’s DNA unwinding mechanism in vitro is helpful for predicting its behaviors in vivo, and then understanding their biological functions. In the present study, for deeply understanding the DNA unwinding mechanism of WRN, we comprehensively characterized the DNA unwinding properties of chicken WRN helicase core in details, by taking advantages of single-molecule fluorescence resonance energy transfer (smFRET) method. We showed that WRN exhibits repetitive DNA unwinding and translocation behaviors on different DNA structures, including forked, overhanging and G-quadruplex-containing DNAs with an apparently limited unwinding processivity. It was further revealed that the repetitive behaviors were caused by reciprocating of WRN along the same ssDNA, rather than by complete dissociation from and rebinding to substrates or by strand switching. The present study sheds new light on the mechanism for WRN functioning. PMID:28266653

  11. Kinetic mechanism for DNA unwinding by multiple molecules of Dda helicase aligned on DNA.

    PubMed

    Eoff, Robert L; Raney, Kevin D

    2010-06-01

    Helicases catalyze the separation of double-stranded nucleic acids to form single-stranded intermediates. Using transient state kinetic methods, we have determined the kinetic properties of DNA unwinding under conditions that favor a monomeric form of the Dda helicase as well as conditions that allow multiple molecules to function on the same substrate. Multiple helicase molecules can align like a train on the DNA track. The number of base pairs unwound in a single binding event for Dda is increased from approximately 19 bp for the monomeric form to approximately 64 bp when as many as four Dda molecules are aligned on the same substrate, while the kinetic step size (3.2 +/- 0.7 bp) and unwinding rate (242 +/- 25 bp/s) appear to be independent of the number of Dda molecules present on a given substrate. The data support a model in which the helicase molecules bound to the same substrate move along the DNA track independently during DNA unwinding. The observed increase in processivity arises from the increased probability that at least one of the helicases will completely unwind the DNA prior to dissociation. These results are in contrast to previous reports in which multiple Dda molecules on the same track greatly enhanced the rate and amplitude for displacement of protein blocks on the track. Therefore, only when the progress of the lead molecule in the train is impeded by some type of block, such as a protein bound to DNA, do the trailing molecules interact with the lead molecule to overcome the block. The fact that trailing helicase molecules have little impact on the lead molecule in the train during routine DNA unwinding suggests that the trailing molecules are moving at rates similar to that of the lead molecule. This result implicates a step in the translocation mechanism as contributing greatly to the overall rate-limiting step for unwinding of duplex DNA.

  12. Variola type IB DNA topoisomerase: DNA binding and supercoil unwinding using engineered DNA minicircles.

    PubMed

    Anderson, Breeana G; Stivers, James T

    2014-07-08

    Type IB topoisomerases unwind positive and negative DNA supercoils and play a key role in removing supercoils that would otherwise accumulate at replication and transcription forks. An interesting question is whether topoisomerase activity is regulated by the topological state of the DNA, thereby providing a mechanism for targeting the enzyme to highly supercoiled DNA domains in genomes. The type IB enzyme from variola virus (vTopo) has proven to be useful in addressing mechanistic questions about topoisomerase function because it forms a reversible 3'-phosphotyrosyl adduct with the DNA backbone at a specific target sequence (5'-CCCTT-3') from which DNA unwinding can proceed. We have synthesized supercoiled DNA minicircles (MCs) containing a single vTopo target site that provides highly defined substrates for exploring the effects of supercoil density on DNA binding, strand cleavage and ligation, and unwinding. We observed no topological dependence for binding of vTopo to these supercoiled MC DNAs, indicating that affinity-based targeting to supercoiled DNA regions by vTopo is unlikely. Similarly, the cleavage and religation rates of the MCs were not topologically dependent, but topoisomers with low superhelical densities were found to unwind more slowly than highly supercoiled topoisomers, suggesting that reduced torque at low superhelical densities leads to an increased number of cycles of cleavage and ligation before a successful unwinding event. The K271E charge reversal mutant has an impaired interaction with the rotating DNA segment that leads to an increase in the number of supercoils that were unwound per cleavage event. This result provides evidence that interactions of the enzyme with the rotating DNA segment can restrict the number of supercoils that are unwound. We infer that both superhelical density and transient contacts between vTopo and the rotating DNA determine the efficiency of supercoil unwinding. Such determinants are likely to be important in

  13. Transient DNA damage induced by high-frequency electromagnetic fields (GSM 1.8 GHz) in the human trophoblast HTR-8/SVneo cell line evaluated with the alkaline comet assay.

    PubMed

    Franzellitti, Silvia; Valbonesi, Paola; Ciancaglini, Nicola; Biondi, Carla; Contin, Andrea; Bersani, Ferdinando; Fabbri, Elena

    2010-01-05

    One of the most controversial issue regarding high-frequency electromagnetic fields (HF-EMF) is their putative capacity to affect DNA integrity. This is of particular concern due to the increasing use of HF-EMF in communication technologies, including mobile phones. Although epidemiological studies report no detrimental effects on human health, the possible disturbance generated by HF-EMF on cell physiology remains controversial. In addition, the question remains as to whether cells are able to compensate their potential effects. We have previously reported that a 1-h exposure to amplitude-modulated 1.8 GHz sinusoidal waves (GSM-217 Hz, SAR=2 W/kg) largely used in mobile telephony did not cause increased levels of primary DNA damage in human trophoblast HTR-8/SVneo cells. Nevertheless, further investigations on trophoblast cell responses after exposure to GSM signals of different types and durations were considered of interest. In the present work, HTR-8/SVneo cells were exposed for 4, 16 or 24h to 1.8 GHz continuous wave (CW) and different GSM signals, namely GSM-217 Hz and GSM-Talk (intermittent exposure: 5 min field on, 10 min field off). The alkaline comet assay was used to evaluate primary DNA damages and/or strand breaks due to uncompleted repair processes in HF-EMF exposed samples. The amplitude-modulated signals GSM-217 Hz and GSM-Talk induced a significant increase in comet parameters in trophoblast cells after 16 and 24h of exposure, while the un-modulated CW was ineffective. However, alterations were rapidly recovered and the DNA integrity of HF-EMF exposed cells was similar to that of sham-exposed cells within 2h of recovery in the absence irradiation. Our data suggest that HF-EMF with a carrier frequency and modulation scheme typical of the GSM signal may affect the DNA integrity.

  14. Rickettsia Phylogenomics: Unwinding the Intricacies of Obligate Intracellular Life

    PubMed Central

    Gillespie, Joseph J.; Williams, Kelly; Shukla, Maulik; Snyder, Eric E.; Nordberg, Eric K.; Ceraul, Shane M.; Dharmanolla, Chitti; Rainey, Daphne; Soneja, Jeetendra; Shallom, Joshua M.; Vishnubhat, Nataraj Dongre; Wattam, Rebecca; Purkayastha, Anjan; Czar, Michael; Crasta, Oswald; Setubal, Joao C.; Azad, Abdu F.; Sobral, Bruno S.

    2008-01-01

    ORFs from 10 rickettsial genomes across robust phylogeny estimation. The data, available at PATRIC (PathoSystems Resource Integration Center), provide novel information for unwinding the intricacies associated with Rickettsia pathogenesis, expanding the range of potential diagnostic, vaccine and therapeutic targets. PMID:19194535

  15. Fluorescent primuline derivatives inhibit hepatitis C virus NS3-catalyzed RNA unwinding, peptide hydrolysis and viral replicase formation.

    PubMed

    Ndjomou, Jean; Kolli, Rajesh; Mukherjee, Sourav; Shadrick, William R; Hanson, Alicia M; Sweeney, Noreena L; Bartczak, Diana; Li, Kelin; Frankowski, Kevin J; Schoenen, Frank J; Frick, David N

    2012-11-01

    The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (2012) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye primuline. This study further characterizes a subset of these primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset contained potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with K(d)s that reflect their potency in assays. The fluorescent properties of the primuline derivatives both in vitro and in cells are also described. The primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC(50)=5±2μM). In cells, the most effective primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.

  16. Grip it and rip it: structural mechanisms of DNA helicase substrate binding and unwinding.

    PubMed

    Bhattacharyya, Basudeb; Keck, James L

    2014-11-01

    Maintenance and faithful transmission of genomic information depends on the efficient execution of numerous DNA replication, recombination, and repair pathways. Many of the enzymes that catalyze steps within these pathways require access to sequence information that is buried in the interior of the DNA double helix, which makes DNA unwinding an essential cellular reaction. The unwinding process is mediated by specialized molecular motors called DNA helicases that couple the chemical energy derived from nucleoside triphosphate hydrolysis to the otherwise non-spontaneous unwinding reaction. An impressive number of high-resolution helicase structures are now available that, together with equally important mechanistic studies, have begun to define the features that allow this class of enzymes to function as molecular motors. In this review, we explore the structural features within DNA helicases that are used to bind and unwind DNA. We focus in particular on "aromatic-rich loops" that allow some helicases to couple single-stranded DNA binding to ATP hydrolysis and "wedge/pin" elements that provide mechanical tools for DNA strand separation when connected to translocating motor domains.

  17. Single-molecule fluorescence reveals the unwinding stepping mechanism of replicative helicase.

    PubMed

    Syed, Salman; Pandey, Manjula; Patel, Smita S; Ha, Taekjip

    2014-03-27

    Bacteriophage T7 gp4 serves as a model protein for replicative helicases that couples deoxythymidine triphosphate (dTTP) hydrolysis to directional movement and DNA strand separation. We employed single-molecule fluorescence resonance energy transfer methods to resolve steps during DNA unwinding by T7 helicase. We confirm that the unwinding rate of T7 helicase decreases with increasing base pair stability. For duplexes containing >35% guanine-cytosine (GC) base pairs, we observed stochastic pauses every 2-3 bp during unwinding. The dwells on each pause were distributed nonexponentially, consistent with two or three rounds of dTTP hydrolysis before each unwinding step. Moreover, we observed backward movements of the enzyme on GC-rich DNAs at low dTTP concentrations. Our data suggest a coupling ratio of 1:1 between base pairs unwound and dTTP hydrolysis, and they further support the concept that nucleic acid motors can have a hierarchy of different-sized steps or can accumulate elastic energy before transitioning to a subsequent phase.

  18. Insights into the mechanism of a G-quadruplex-unwinding DEAH-box helicase.

    PubMed

    Chen, Michael C; Murat, Pierre; Abecassis, Keren; Ferré-D'Amaré, Adrian R; Balasubramanian, Shankar

    2015-02-27

    The unwinding of nucleic acid secondary structures within cells is crucial to maintain genomic integrity and prevent abortive transcription and translation initiation. DHX36, also known as RHAU or G4R1, is a DEAH-box ATP-dependent helicase highly specific for DNA and RNA G-quadruplexes (G4s). A fundamental mechanistic understanding of the interaction between helicases and their G4 substrates is important to elucidate G4 biology and pave the way toward G4-targeted therapies. Here we analyze how the thermodynamic stability of G4 substrates affects binding and unwinding by DHX36. We modulated the stability of the G4 substrates by varying the sequence and the number of G-tetrads and by using small, G4-stabilizing molecules. We found an inverse correlation between the thermodynamic stability of the G4 substrates and rates of unwinding by DHX36. In stark contrast, the ATPase activity of the helicase was largely independent of substrate stability pointing toward a decoupling mechanism akin to what has been observed for many double-stranded DEAD-box RNA helicases. Our study provides the first evidence that DHX36 uses a local, non-processive mechanism to unwind G4 substrates, reminiscent of that of eukaryotic initiation factor 4A (eIF4A) on double-stranded substrates.

  19. Fluorescence methods to study DNA translocation and unwinding kinetics by nucleic acid motors.

    PubMed

    Fischer, Christopher J; Tomko, Eric J; Wu, Colin G; Lohman, Timothy M

    2012-01-01

    Translocation of nucleic acid motor proteins (translocases) along linear nucleic acids can be studied by monitoring either the time course of the arrival of the motor protein at one end of the nucleic acid or the kinetics of ATP hydrolysis by the motor protein during translocation using pre-steady state ensemble kinetic methods in a stopped-flow instrument. Similarly, the unwinding of double-stranded DNA or RNA by helicases can be studied in ensemble experiments by monitoring either the kinetics of the conversion of the double-stranded nucleic acid into its complementary single strands by the helicase or the kinetics of ATP hydrolysis by the helicase during unwinding. Such experiments monitor translocation of the enzyme along or unwinding of a series of nucleic acids labeled at one position (usually the end) with a fluorophore or a pair of fluorophores that undergo changes in fluorescence intensity or efficiency of fluorescence resonance energy transfer (FRET). We discuss how the pre-steady state kinetic data collected in these ensemble experiments can be analyzed by simultaneous global nonlinear least squares (NLLS) analysis using simple sequential "n-step" mechanisms to obtain estimates of the macroscopic rates and processivities of translocation and/or unwinding, the rate-limiting step(s) in these mechanisms, the average "kinetic step-size," and the stoichiometry of coupling ATP binding and hydrolysis to movement along the nucleic acid.

  20. Brr2 plays a role in spliceosomal activation in addition to U4/U6 unwinding.

    PubMed

    Zhang, Lingdi; Li, Xueni; Hill, Ryan C; Qiu, Yan; Zhang, Wenzheng; Hansen, Kirk C; Zhao, Rui

    2015-03-31

    Brr2 is a DExD/H-box RNA helicase that is responsible for U4/U6 unwinding, a critical step in spliceosomal activation. Brr2 is a large protein (∼250 kD) that consists of an N-terminal domain (∼500 residues) with unknown function and two Hel308-like modules that are responsible for RNA unwinding. Here we demonstrate that removal of the entire N-terminal domain is lethal to Saccharomyces cerevisiae and deletion of the N-terminal 120 residues leads to splicing defects and severely impaired growth. This N-terminal truncation does not significantly affect Brr2's helicase activity. Brr2-Δ120 can be successfully assembled into the tri-snRNP (albeit at a lower level than the WT Brr2) and the spliceosomal B complex. However, the truncation significantly impairs spliceosomal activation, leading to a dramatic reduction of U5, U6 snRNAs and accumulation of U1 snRNA in the B(act) complex. The N-terminal domain of Brr2 does not seem to be directly involved in regulating U1/5'ss unwinding. Instead, the N-terminal domain seems to be critical for retaining U5 and U6 snRNPs during/after spliceosomal activation through its interaction with snRNAs and possibly other spliceosomal proteins, revealing a new role of Brr2 in spliceosomal activation in addition to U4/U6 unwinding.

  1. A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication.

    PubMed

    Pacek, Marcin; Walter, Johannes C

    2004-09-15

    In vertebrates, MCM2-7 and Cdc45 are required for DNA replication initiation, but it is unknown whether they are also required for elongation, as in yeast. Moreover, although MCM2-7 is a prime candidate for the eukaryotic replicative DNA helicase, a demonstration that MCM2-7 unwinds DNA during replication is lacking. Here, we use Xenopus egg extracts to investigate the roles of MCM7 and Cdc45 in DNA replication. A fragment of the retinoblastoma protein, Rb(1-400), was used to neutralize MCM7, and antibodies were used to neutralize Cdc45. When added immediately after origin unwinding, or after significant DNA synthesis, both inhibitors blocked further DNA replication, indicating that MCM7 and Cdc45 are required throughout replication elongation in vertebrates. We next exploited the fact that inhibition of DNA polymerase by aphidicolin causes extensive chromosome unwinding, likely due to uncoupling of the replicative DNA helicase. Strikingly, Rb(1-400) and Cdc45 antibodies both abolished unwinding by the uncoupled helicase. These results provide new support for the model that MCM2-7 is the replicative DNA helicase, and they indicate that Cdc45 functions as a helicase co-factor.

  2. Single-molecule kinetics of the eukaryotic initiation factor 4AI upon RNA unwinding.

    PubMed

    Sun, Yingjie; Atas, Evrim; Lindqvist, Lisa M; Sonenberg, Nahum; Pelletier, Jerry; Meller, Amit

    2014-07-08

    The eukaryotic translation initiation factor 4AI (eIF4AI) is the prototypical DEAD-box RNA helicase. It has a "dumbbell" structure consisting of two domains connected by a flexible linker. Previous studies demonstrated that eIF4AI, in conjunction with eIF4H, bind to loop structures and repetitively unwind RNA hairpins. Here, we probe the conformational dynamics of eIF4AI in real time using single-molecule FRET. We demonstrate that eIF4AI/eIF4H complex can repetitively unwind RNA hairpins by transitioning between an eIF4AI "open" and a "closed" conformation using the energy derived from ATP hydrolysis. Our experiments directly track the conformational changes in the catalytic cycle of eIF4AI and eIF4H, and this correlates precisely with the kinetics of RNA unwinding. Furthermore, we show that the small-molecule eIF4A inhibitor hippuristanol locks eIF4AI in the closed conformation, thus efficiently inhibiting RNA unwinding. These results indicate that the large conformational changes undertaken by eIF4A during the helicase catalytic cycle are rate limiting.

  3. A unified model of nucleic acid unwinding by the ribosome and the hexameric and monomeric DNA helicases.

    PubMed

    Xie, Ping

    2015-09-07

    DNA helicases are enzymes that use the chemical energy to separate DNA duplex into their single-stranded forms. The ribosome, which catalyzes the translation of messenger RNAs (mRNAs) into proteins, can also unwind mRNA duplex. According to their structures, the DNA helicases can fall broadly into hexameric and monomeric forms. A puzzling issue for the monomeric helicases is that although they have similar structures, in vitro biochemical data showed convincingly that in the monomeric forms some have very weak DNA unwinding activities, some have relatively high unwinding activities while others have high unwinding activities. However, in the dimeric or oligomeric forms all of them have high unwinding activities. In addition, in the monomeric forms all of them can translocate efficiently along the single-stranded DNA (ssDNA). Here, we propose a model of the translocation along the ssDNA and DNA unwinding by the monomeric helicases, providing a consistent explanation of these in vitro experimental data. Moreover, by comparing the present model for the monomeric helicases with the model for the hexameric helicases and that for the ribosome which were proposed before, a unified model of nucleic acid unwinding by the three enzymes is proposed.

  4. Mutations altering the interplay between GkDnaC helicase and DNA reveal an insight into helicase unwinding.

    PubMed

    Lo, Yu-Hua; Liu, Shih-Wei; Sun, Yuh-Ju; Li, Hung-Wen; Lizz, Hung-Wen; Hsiao, Chwan-Deng

    2011-01-01

    Replicative helicases are essential molecular machines that utilize energy derived from NTP hydrolysis to move along nucleic acids and to unwind double-stranded DNA (dsDNA). Our earlier crystal structure of the hexameric helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in complex with single-stranded DNA (ssDNA) suggested several key residues responsible for DNA binding that likely play a role in DNA translocation during the unwinding process. Here, we demonstrated that the unwinding activities of mutants with substitutions at these key residues in GkDnaC are 2-4-fold higher than that of wild-type protein. We also observed the faster unwinding velocities in these mutants using single-molecule experiments. A partial loss in the interaction of helicase with ssDNA leads to an enhancement in helicase efficiency, while their ATPase activities remain unchanged. In strong contrast, adding accessory proteins (DnaG or DnaI) to GkDnaC helicase alters the ATPase, unwinding efficiency and the unwinding velocity of the helicase. It suggests that the unwinding velocity of helicase could be modulated by two different pathways, the efficiency of ATP hydrolysis or protein-DNA interaction.

  5. Kinetics of DNA unwinding by the RecD2 helicase from Deinococcus radiodurans.

    PubMed

    Shadrick, William R; Julin, Douglas A

    2010-06-04

    RecD2 from Deinococcus radiodurans is a superfamily 1 DNA helicase that is homologous to the Escherichia coli RecD protein but functions outside the context of RecBCD enzyme. We report here on the kinetics of DNA unwinding by RecD2 under single and multiple turnover conditions. There is little unwinding of 20-bp substrates by preformed RecD2-dsDNA complexes when excess ssDNA is present to trap enzyme molecules not bound to the substrate. A shorter 12-bp substrate is unwound rapidly under single turnover conditions. The 12-bp unwinding reaction could be simulated with a mechanism in which the DNA is unwound in two kinetic steps with rate constant of k(unw) = 5.5 s(-1) and a dissociation step from partially unwound DNA of k(off) = 1.9 s(-1). These results indicate a kinetic step size of about 3-4 bp, unwinding rate of about 15-20 bp/s, and low processivity (p = 0.74). The reaction time courses with 20-bp substrates, determined under multiple turnover conditions, could be simulated with a four-step mechanism and rate constant values very similar to those for the 12-bp substrate. The results indicate that the faster unwinding of a DNA substrate with a forked end versus only a 5'-terminal single-stranded extension can be accounted for by a difference in the rate of enzyme binding to the DNA substrates. Analysis of reactions done with different RecD2 concentrations indicates that the enzyme forms an inactive dimer or other oligomer at high enzyme concentrations. RecD2 oligomers can be detected by glutaraldehyde cross-linking but not by size exclusion chromatography.

  6. Unwinding and rewinding the nucleosome inner turn: force dependence of the kinetic rate constants.

    PubMed

    Mochrie, S G J; Mack, A H; Schlingman, D J; Collins, R; Kamenetska, M; Regan, L

    2013-01-01

    A simple model for the force-dependent unwinding and rewinding rates of the nucleosome inner turn is constructed and quantitatively compared to the results of recent measurements [A. H. Mack et al., J. Mol. Biol. 423, 687 (2012)]. First, a coarse-grained model for the histone-DNA free-energy landscape that incorporates both an elastic free-energy barrier and specific histone-DNA bonds is developed. Next, a theoretical expression for the rate of transitions across a piecewise linear free-energy landscape with multiple minima and maxima is presented. Then, the model free-energy landscape, approximated as a piecewise linear function, and the theoretical expression for the transition rates are combined to construct a model for the force-dependent unwinding and rewinding rates of the nucleosome inner turn. Least-mean-squares fitting of the model rates to the rates observed in recent experiments rates demonstrates that this model is able to well describe the force-dependent unwinding and rewinding rates of the nucleosome inner turn, observed in the recent experiments, except at the highest forces studied, where an additional ad hoc term is required to describe the data, which may be interpreted as an indication of an alternate high-force nucleosome disassembly pathway, that bypasses simple unwinding. The good agreement between the measurements and the model at lower forces demonstrates that both specific histone-DNA contacts and an elastic free-energy barrier play essential roles for nucleosome winding and unwinding, and quantifies their relative contributions.

  7. Mechanism of nucleic acid unwinding by SARS-CoV helicase.

    PubMed

    Adedeji, Adeyemi O; Marchand, Bruno; Te Velthuis, Aartjan J W; Snijder, Eric J; Weiss, Susan; Eoff, Robert L; Singh, Kamalendra; Sarafianos, Stefan G

    2012-01-01

    The non-structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a helicase that separates double-stranded RNA (dsRNA) or DNA (dsDNA) with a 5' → 3' polarity, using the energy of nucleotide hydrolysis. We determined the minimal mechanism of helicase function by nsp13. We showed a clear unwinding lag with increasing length of the double-stranded region of the nucleic acid, suggesting the presence of intermediates in the unwinding process. To elucidate the nature of the intermediates we carried out transient kinetic analysis of the nsp13 helicase activity. We demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 base-pairs (bp) each, with a catalytic rate of 30 steps per second. Therefore the net unwinding rate is ~280 base-pairs per second. We also showed that nsp12, the SARS-CoV RNA-dependent RNA polymerase (RdRp), enhances (2-fold) the catalytic efficiency of nsp13 by increasing the step size of nucleic acid (RNA/RNA or DNA/DNA) unwinding. This effect is specific for SARS-CoV nsp12, as no change in nsp13 activity was observed when foot-and-mouth-disease virus RdRp was used in place of nsp12. Our data provide experimental evidence that nsp13 and nsp12 can function in a concerted manner to improve the efficiency of viral replication and enhance our understanding of nsp13 function during SARS-CoV RNA synthesis.

  8. ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors

    SciTech Connect

    Ray, B.K.; Lawson, T.G.; Kramer, J.C.; Cladaras, M.H.; Grifo, J.A.; Abramson, R.D.; Merrick, W.C.; Thach, R.E.

    1985-06-25

    Interaction of protein synthesis initiation factors with mRNA has been studied in order to characterize early events in the eukaryotic translation pathway. Individual reovirus mRNAs labeled with /sup 32/P in the alpha position relative to the m7G cap and eukaryotic initiation factor (eIF)-4A, -4B, and -4F purified from rabbit reticulocytes were employed. It was found that eIF-4A causes a structural change in mRNA, as evidenced by a nuclease sensitivity test: addition of high concentrations of eIF-4A greatly increase the nuclease sensitivity of the mRNA, suggesting that this factor can melt or ''unwind'' mRNA structure. ATP is required for this reaction. At low concentrations of eIF-4A, addition of eIF-4B is required for maximal unwinding activity. Thus eIF-4B enhances eIF-4A activity. Addition of eIF-4F also makes the mRNA sensitive to nuclease indicating a similar unwinding role to that of eIF-4A. Stoichiometric comparisons indicate that eIF-4F is more than 20-fold more efficient than eIF-4A in catalyzing this reaction. The unwinding activity of eIF-4F is inhibited by m7GDP, while that of eIF-4A is not. This suggests that eIF-4A functions independent of the 5' cap structure. These results also suggest that the unwinding activity of eIF-4F is located in the 46,000-dalton polypeptide of this complex, which has shown by others to be similar or identical to eIF-4A.

  9. The SV40 large T-antigen origin binding domain directly participates in DNA unwinding.

    PubMed

    Foster, Erin C; Simmons, Daniel T

    2010-03-16

    The origin binding domain (OBD) of SV40 large T-ag serves a critical role during initiation of DNA replication to position T-ag on the origin. After origin recognition, T-ag forms a double hexamer over the origin. Within each hexamer, the OBD adopts a lock washer structure where the origin recognizing A1 and B2 loops face toward the helicase domain and likely become unavailable for binding DNA. In this study, we investigated the role of the central channel of the OBD hexamer in DNA replication by analyzing the effects of mutations of residues lining the channel. All mutants showed binding defects with origin DNA and ssDNA especially at low protein concentrations, but only half were defective at supporting DNA replication in vitro. All mutants were normal in unwinding linear origin DNA fragments. However, replication defective mutants failed to unwind a small origin containing circular DNA whereas replication competent mutants did so normally. The presence of RPA and/or pol/prim restored circular DNA unwinding activity of compromised mutants probably by interacting with the separated DNA strands on the T-ag surface. We interpret these results to indicate a role for the OBD central channel in binding and threading ssDNA during unwinding of circular SV40 DNA. Mixing experiments suggested that only one monomer in an OBD hexamer was necessary for DNA unwinding. We present a model of DNA threading through the T-ag complex illustrating how single-stranded DNA could pass close to a trough generated by key residues in one monomer of the OBD hexamer.

  10. Unwinding and rewinding the nucleosome inner turn: Force dependence of the kinetic rate constants

    NASA Astrophysics Data System (ADS)

    Mochrie, S. G. J.; Mack, A. H.; Schlingman, D. J.; Collins, R.; Kamenetska, M.; Regan, L.

    2013-01-01

    A simple model for the force-dependent unwinding and rewinding rates of the nucleosome inner turn is constructed and quantitatively compared to the results of recent measurements [A. H. Mack , J. Mol. Biol.JMOBAK0022-283610.1016/j.jmb.2012.08.021 423, 687 (2012)]. First, a coarse-grained model for the histone-DNA free-energy landscape that incorporates both an elastic free-energy barrier and specific histone-DNA bonds is developed. Next, a theoretical expression for the rate of transitions across a piecewise linear free-energy landscape with multiple minima and maxima is presented. Then, the model free-energy landscape, approximated as a piecewise linear function, and the theoretical expression for the transition rates are combined to construct a model for the force-dependent unwinding and rewinding rates of the nucleosome inner turn. Least-mean-squares fitting of the model rates to the rates observed in recent experiments rates demonstrates that this model is able to well describe the force-dependent unwinding and rewinding rates of the nucleosome inner turn, observed in the recent experiments, except at the highest forces studied, where an additional ad hoc term is required to describe the data, which may be interpreted as an indication of an alternate high-force nucleosome disassembly pathway, that bypasses simple unwinding. The good agreement between the measurements and the model at lower forces demonstrates that both specific histone-DNA contacts and an elastic free-energy barrier play essential roles for nucleosome winding and unwinding, and quantifies their relative contributions.

  11. Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: the effect of the 3' arm and the stability of the dsDNA on the unwinding activity of the Escherichia coli DnaB helicase.

    PubMed

    Galletto, Roberto; Jezewska, Maria J; Bujalowski, Wlodzimierz

    2004-10-08

    The effect of two structural elements of a replication DNA fork substrate, the length of the 3' arm of the fork and the stability of the double-stranded DNA (dsDNA) part, on the kinetics of the dsDNA unwinding by the Escherichia coli hexameric helicase DnaB protein has been examined under single turnover conditions using the rapid quench-flow technique. The length of the 3' arm of the replication fork, i.e. the number of nucleotides in the arm, is a major structural factor that controls the unwinding rate and processivity of the helicase. The data show the existence of an optimal length of the 3' arm where there is the highest unwinding rate and processivity, indicating that during the unwinding process, the helicase transiently interacts with the 3' arm at a specific distance on the arm with respect to the duplex part of the DNA. Moreover, the area on the enzyme that engages in interactions has also a discrete size. For DNA substrates with the 3' arm containing 14, or less, nucleotide residues, the DnaB helicase becomes a completely distributive enzyme. However, the 3' arm is not a "specific activating cofactor" in the unwinding reaction. Rather, the 3' arm plays a role as a mechanical fulcrum for the enzyme, necessary to provide support for the advancing large helicase molecule on the opposite strand of the DNA. Binding of ATP is necessary to engage the 3' arm with the DnaB helicase, but it does not change the initial distribution of complexes of the enzyme with the DNA fork substrate. Stability of the dsDNA has a significant effect on the unwinding rate and processivity. The unwinding rate constant is a decreasing linear function of the fractional content of GC base-pairs in the dsDNA, indicating that the activation of the unwinding step is proportional to the stability of the nucleic acid.

  12. Direct single-molecule observations of DNA unwinding by SV40 large tumor antigen under a negative DNA supercoil state.

    PubMed

    Takahashi, Shunsuke; Motooka, Shinya; Kawasaki, Shohei; Kurita, Hirofumi; Mizuno, Takeshi; Matsuura, Shun-Ichi; Hanaoka, Fumio; Mizuno, Akira; Oshige, Masahiko; Katsura, Shinji

    2017-01-05

    Superhelices, which are induced by the twisting and coiling of double-helical DNA in chromosomes, are thought to affect transcription, replication, and other DNA metabolic processes. In this study, we report the effects of negative supercoiling on the unwinding activity of simian virus 40 large tumor antigen (SV40 TAg) at a single-molecular level. The supercoiling density of linear DNA templates was controlled using magnetic tweezers and monitored using a fluorescent microscope in a flow cell. SV40 TAg-mediated DNA unwinding under relaxed and negative supercoil states was analyzed by the direct observation of both single- and double-stranded regions of single DNA molecules. Increased negative superhelicity stimulated SV40 TAg-mediated DNA unwinding more strongly than a relaxed state; furthermore, negative superhelicity was associated with an increased probability of SV40 TAg-mediated DNA unwinding. These results suggest that negative superhelicity helps to regulate the initiation of DNA replication.

  13. AvrXa27 binding influences unwinding of the double-stranded DNA in the UPT box.

    PubMed

    Zhao, Jing; Zhang, Bo; Jiang, Junpeng; Liu, Nanv; Wei, Qi; Xi, Xuguang; Fu, Jing

    2017-03-04

    Transcription-Activator Like (TAL) effectors, delivered by Xanthomonas pathogens bind specifically to UP-regulated by TAL effectors (UPT) box of the host gene promoter to arouse disease or trigger defense response. This type of protein-DNA interaction model has been applied in site-directed genome editing. However, the off-target effects of TAL have severely hindered the development of this promising technology. To better exploit the specific interaction and to deeper understand the TAL-induced host transcription rewiring, the binding between the central repeat region (CRR) of the TAL effector AvrXa27 and its UPT box variants was studied by kinetics analysis and TAL-blocked helicase unwinding assay. The results revealed that while AvrXa27 exhibited the highest affinity to the wild type UPT box, it could also bind to mutated UPT box variants, implying the possibility of non-specific interactions. Furthermore, some of these non-specific combinations restricted the helicase-elicited double-stranded DNA (dsDNA) separation to a greater extent. Our findings provide insight into the mechanism of TAL transcriptional activation and are beneficial to TAL-mediated genome modification.

  14. Pathway of ATP utilization and duplex rRNA unwinding by the DEAD-box helicase, DbpA.

    PubMed

    Henn, Arnon; Cao, Wenxiang; Licciardello, Nicholas; Heitkamp, Sara E; Hackney, David D; De La Cruz, Enrique M

    2010-03-02

    DEAD-box RNA helicase proteins use the energy of ATP hydrolysis to drive the unwinding of duplex RNA. However, the mechanism that couples ATP utilization to duplex RNA unwinding is unknown. We measured ATP utilization and duplex RNA unwinding by DbpA, a non-processive bacterial DEAD-box RNA helicase specifically activated by the peptidyl transferase center (PTC) of 23S rRNA. Consumption of a single ATP molecule is sufficient to unwind and displace an 8 base pair rRNA strand annealed to a 32 base pair PTC-RNA "mother strand" fragment. Strand displacement occurs after ATP binding and hydrolysis but before P(i) product release. P(i) release weakens binding to rRNA, thereby facilitating the release of the unwound rRNA mother strand and the recycling of DbpA for additional rounds of unwinding. This work explains how ATPase activity of DEAD-box helicases is linked to RNA unwinding.

  15. Nucleic acid unwinding by hepatitis C virus and bacteriophage t7 helicases is sensitive to base pair stability.

    PubMed

    Donmez, Ilker; Rajagopal, Vaishnavi; Jeong, Yong-Joo; Patel, Smita S

    2007-07-20

    Helicases are motor enzymes that convert the chemical energy of NTP hydrolysis into mechanical force for motion and nucleic acid strand separation. Within the cell, helicases process a range of nucleic acid sequences. It is not known whether this composite rate of moving and opening the strands of nucleic acids depends on the base sequence. Our presteady state kinetic studies of helicases from two classes, the ring-shaped T7 helicase and two forms of non-ring-shaped hepatitis C virus (HCV) helicase, show that both the unwinding rate and processivity depend on the sequence and decrease as the nucleic acid stability increases. The DNA unwinding activity of T7 helicase and the RNA unwinding activity of HCV helicases decrease steeply with increasing base pair stability. On the other hand, the DNA unwinding activity of HCV helicases is less sensitive to base pair stability. These results predict that helicases will fall into a spectrum of modest to high sensitivity to base pair stability depending on their biological role in the cell. Modeling of the dependence provided the degree of the active involvement of helicase in base pair destabilization during the unwinding process and distinguished between passive and active mechanisms of unwinding.

  16. DNA unwinding proteins of an eukaryotic organism. Final report, July 1977-April 1981

    SciTech Connect

    Patel, G.L.

    1982-06-01

    A rat liver protein, which preferentially binds in vitro to single-stranded DNA, and destabilizes the DNA double helix to promote its unwinding or melting was investigated. Proteins of this class isolated from viral, prokaryotic, and eukaryotic sources have been referred to as DNA melting, unwinding, helix-destabilizing, or single-strand binding proteins. The rat liver protein that we have purified and studied here is referred to as a helix-destabilizing protein (HDP). The specific objectives proposed were to: (1) purify and characterize physico-chemically one or more rat liver HDP; (2) prepare specific anti-HDP sera for immunological approaches; (3) characterize the HDP interaction with DNA; and (4) study the HDP metabolism in normal and regenerating liver. A novel conceptual contribution was made with regards to the biological roles of this class of proteins. Activities and accomplishments during this entire contract period are summarized. (ERB)

  17. Processive translocation and DNA unwinding by individual RecBCD enzyme molecules

    NASA Astrophysics Data System (ADS)

    Bianco, Piero R.; Brewer, Laurence R.; Corzett, Michele; Balhorn, Rod; Yeh, Yin; Kowalczykowski, Stephen C.; Baskin, Ronald J.

    2001-01-01

    RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 +/- 172 base pairs per second (0.30µms-1), with as many as 42,300base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.

  18. Enhancement of topoisomerase I-mediated unwinding of supercoiled DNA by the radioprotector WR-33278

    SciTech Connect

    Holwitt, E.A.; Koda, E.; Swenberg, C.E. )

    1990-10-01

    The radioprotector WR-33278, the disulfide of WR-1065 (N-(2-mercaptoethyl)-1,3-diaminopropane), is shown to stimulate eukaryotic topoisomerase I unwinding of negatively supercoiled DNA. This observation suggests the possibility that some protection may be conferred to DNA either by a decrease in its supercoiled state or by altering directly other enzymatic processes. This is the first report of a radioprotective compound stimulating an enzyme involved in DNA structure and synthesis.

  19. Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A single-chain variable fragment (scFv) and alkaline phosphatase (AP) fusion protein for detection of O, O-diethyl organophosphorus pesticides (OPs) was produced and characterized. The scFv gene was prepared by cloning VL and VH genes from a hybridoma cell secreting monoclonal antibody with broad-s...

  20. G-quadruplexes significantly stimulate Pif1 helicase-catalyzed duplex DNA unwinding.

    PubMed

    Duan, Xiao-Lei; Liu, Na-Nv; Yang, Yan-Tao; Li, Hai-Hong; Li, Ming; Dou, Shuo-Xing; Xi, Xu-Guang

    2015-03-20

    The evolutionarily conserved G-quadruplexes (G4s) are faithfully inherited and serve a variety of cellular functions such as telomere maintenance, gene regulation, DNA replication initiation, and epigenetic regulation. Different from the Watson-Crick base-pairing found in duplex DNA, G4s are formed via Hoogsteen base pairing and are very stable and compact DNA structures. Failure of untangling them in the cell impedes DNA-based transactions and leads to genome instability. Cells have evolved highly specific helicases to resolve G4 structures. We used a recombinant nuclear form of Saccharomyces cerevisiae Pif1 to characterize Pif1-mediated DNA unwinding with a substrate mimicking an ongoing lagging strand synthesis stalled by G4s, which resembles a replication origin and a G4-structured flap in Okazaki fragment maturation. We find that the presence of G4 may greatly stimulate the Pif1 helicase to unwind duplex DNA. Further studies reveal that this stimulation results from G4-enhanced Pif1 dimerization, which is required for duplex DNA unwinding. This finding provides new insights into the properties and functions of G4s. We discuss the observed activation phenomenon in relation to the possible regulatory role of G4s in the rapid rescue of the stalled lagging strand synthesis by helping the replicator recognize and activate the replication origin as well as by quickly removing the G4-structured flap during Okazaki fragment maturation.

  1. Identification and characterization of RNA duplex unwinding and ATPase activities of an alphatetravirus superfamily 1 helicase.

    PubMed

    Wang, Qinrong; Han, Yajuan; Qiu, Yang; Zhang, Shaoqiong; Tang, Fenfen; Wang, Yan; Zhang, Jiamin; Hu, Yuanyang; Zhou, Xi

    2012-11-25

    Dendrolimus punctatus tetravirus (DpTV) belongs to the genus omegatetravirus of the Alphatetraviridae family. Sequence analysis predicts that DpTV replicase contains a putative helicase domain (Hel). However, the helicase activity in alphatetraviruses has never been formally determined. In this study, we determined that DpTV Hel is a functional RNA helicase belonging to superfamily-1 helicase with 5'-3' dsRNA unwinding directionality. Further characterization determined the length requirement of the 5' single-stranded tail on the RNA template and the optimal reaction conditions for the unwinding activity of DpTV Hel. Moreover, DpTV Hel also contains NTPase activity. The ATPase activity of DpTV Hel could be significantly stimulated by dsRNA, and dsRNA could partially rescue the ATPase activity abolishment caused by mutations. Our study is the first to identify an alphatetravirus RNA helicase and further characterize its dsRNA unwinding and NTPase activities in detail and should foster our understanding of DpTV and other alphatetraviruses.

  2. The Borrelia burgdorferi telomere resolvase, ResT, possesses ATP-dependent DNA unwinding activity.

    PubMed

    Huang, Shu Hui; Cozart, McKayla R; Hart, Madison A; Kobryn, Kerri

    2016-12-09

    Spirochetes of the genus Borrelia possess unusual genomes harboring multiple linear and circular replicons. The linear replicons are terminated by covalently closed hairpin (hp) telomeres. Hairpin telomeres are formed from replicated intermediates by the telomere resolvase, ResT, in a phosphoryl transfer reaction with mechanistic similarities to those promoted by type 1B topoisomerases and tyrosine recombinases. There is growing evidence that ResT is multifunctional. Upon ResT depletion DNA replication unexpectedly ceases. Additionally, ResT possesses RecO-like biochemical activities being able to promote single-strand annealing on both free ssDNA and ssDNA complexed with cognate single-stranded DNA binding protein. We report here that ResT possesses DNA-dependent ATPase activity that promotes DNA unwinding with a 3'-5' polarity. ResT can unwind a variety of substrates including synthetic replication forks and D-loops. We demonstrate that ResT's twin activities of DNA unwinding and annealing can drive regression of a model replication fork. These properties are similar to those of the RecQ helicase of the RecF pathway involved in DNA gap repair. We propose that ResT's combination of activities implicates it in replication and recombination processes operating on the linear chromosome and plasmids of Borrelia burgdorferi.

  3. The mechanism of ATP-dependent RNA unwinding by DEAD box proteins.

    PubMed

    Hilbert, Manuel; Karow, Anne R; Klostermeier, Dagmar

    2009-12-01

    DEAD box proteins catalyze the ATP-dependent unwinding of double-stranded RNA (dsRNA). In addition, they facilitate protein displacement and remodeling of RNA or RNA/protein complexes. Their hallmark feature is local destabilization of RNA duplexes. Here, we summarize current data on the DEAD box protein mechanism and present a model for RNA unwinding that integrates recent data on the effect of ATP analogs and mutations on DEAD box protein activity. DEAD box proteins share a conserved helicase core with two flexibly linked RecA-like domains that contain all helicase signature motifs. Variable flanking regions contribute to substrate binding and modulate activity. In the presence of ATP and RNA, the helicase core adopts a compact, closed conformation with extensive interdomain contacts and high affinity for RNA. In the closed conformation, the RecA-like domains form a catalytic site for ATP hydrolysis and a continuous RNA binding site. A kink in the backbone of the bound RNA locally destabilizes the duplex. Rearrangement of this initial complex generates a hydrolysis- and unwinding-competent state. From this complex, the first RNA strand can dissociate. After ATP hydrolysis and phosphate release, the DEAD box protein returns to a low-affinity state for RNA. Dissociation of the second RNA strand and reopening of the cleft in the helicase core allow for further catalytic cycles.

  4. Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation.

    PubMed

    Simon, Michael J; Sokoloski, Joshua E; Hao, Linxuan; Weiland, Elizabeth; Lohman, Timothy M

    2016-07-31

    Escherichia coli RecBCD is a DNA helicase/nuclease that functions in double-stranded DNA break repair. RecBCD possesses two motors (RecB, a 3' to 5' translocase, and RecD, a 5' to 3' translocase). Current DNA unwinding models propose that motor translocation is tightly coupled to base pair melting. However, some biochemical evidence suggests that DNA melting of multiple base pairs may occur separately from single-stranded DNA translocation. To test this hypothesis, we designed DNA substrates containing reverse backbone polarity linkages that prevent ssDNA translocation of the canonical RecB and RecD motors. Surprisingly, we find that RecBCD can processively unwind DNA for at least 80bp beyond the reverse polarity linkages. This ability requires an ATPase active RecB motor, the RecB "arm" domain, and also the RecB nuclease domain, but not its nuclease activity. These results indicate that RecBCD can unwind duplex DNA processively in the absence of ssDNA translocation by the canonical motors and that the nuclease domain regulates the helicase activity of RecBCD.

  5. Inhibition of unwinding and ATPase activities of pea MCM6 DNA helicase by actinomycin and nogalamycin.

    PubMed

    Tran, Ngoc Quang; Pham, Xuan Hoi; Tuteja, Renu; Tuteja, Narendra

    2011-03-01

    Pea mini-chromosome maintenance 6 (MCM6) single subunit (93 kDa) forms homohexamer (560 kDa) and contains an ATP-dependent and replication fork stimulated 3' to 5' DNA unwinding activity along with intrinsic DNA-dependent ATPase and ATP-binding activities [Plant Mol. Biol. 2010; DOI: 10.1007/s11103-010-9675-7]. Here, we have determined the effect of various DNA-binding agents, such as actinomycin, nogalamycin, daunorubicin, doxorubicin, distamycin, camptothecin, cyclophosphamide, ellipticine, VP-16, novobiocin, netropsin, cisplatin, mitoxantrone and genistein on the DNA unwinding and ATPase activities of the pea MCM6 DNA helicase. The results show that actinomycin and nogalamycin inhibited the DNA helicase (apparent Ki values of 10 and 1 μM, respectively) and ATPase (apparent Ki values of 100 and 17 μM, respectively) activities. Although, daunorubicin and doxorubicin also inhibited the DNA helicase activity of pea MCM6, but with less efficiency; however, these could not inhibit the ATPase activity. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of MCM6, resulting in the inhibitions of the activities. This study could be useful in our better understanding of the mechanism of plant nuclear DNA helicase unwinding.

  6. Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

    DOE PAGES

    Manthei, Kelly A.; Hill, Morgan C.; Burke, Jordan E.; ...

    2015-03-23

    RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. In this paper, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3' single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ~90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATPmore » hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. Finally, this bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.« less

  7. Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding.

    PubMed

    Rad, Behzad; Forget, Anthony L; Baskin, Ronald J; Kowalczykowski, Stephen C

    2015-12-15

    DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40-60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.

  8. DNA unwinding produced by site-specific intrastrand cross-links of the antitumor drug cis-diamminedichloroplatinum(II)

    SciTech Connect

    Bellon, S.F.; Coleman, J.H.; Lippard, S.J. )

    1991-08-13

    The DNA unwinding produced by specific adducts of the antitumor drug cis-diamminedi-chloroplatinum(II) has been quantitatively determined. Synthetic DNA duplex oligonucleotides of varying lengths with two base pair cohesive ends were synthesized and characterized that contained site-specific intrastrand N7-purine/N7-purine cross-links. Included are cis-(Pt(NH{sub 3}){sub 2}(d(GpG))), cis-(Pt(NH){sub 3}{sub 2}(d(ApG))), and cis-(Pt(NH{sub 3}){sub 2}(d(GpTpG))) adducts, respectively referred to as cis-GG, cis-AG, and cis-GTG. Local DNA distortions at the site of platination were amplified by polymerization of these monomers and quantitatively evaluated by using polyacrylamide gel electrophoresis. The extent of DNA unwinding was determined by systematically varying the interplatinum distance, or phasing, in polymers containing the adducts. The multimer that migrates most slowly gives the optimal phasing for cooperative bending, from which the degree of unwinding can be obtained. The authors find that the cis-GG and cis-AG adducts both unwind DNA by 13{degrees}, while the cis-GTG adduct unwinds DNA by 23{degrees}. In addition, experiments are presented that support previous studies revealing that a hinge joint forms at the sites of platination in DNA molecules containing trans-GTG adducts. On the basis of an analysis of the present and other published studies of site-specifically modified DNA. The authors propose that local duplex unwinding is a major determinant in the recognition of DNA damage by the Escherichia coli (A)BC excinuclease. In addition, local duplex unwinding of 13{degrees} and bending by 35{degrees} are shown to correlate well with the recognition of platinated DNA by a previously identified damage recognition protein (DRP) in human cells.

  9. Residues in the central beta-hairpin of the DNA helicase of bacteriophage T7 are important in DNA unwinding.

    PubMed

    Satapathy, Ajit K; Kochaniak, Anna B; Mukherjee, Sourav; Crampton, Donald J; van Oijen, Antoine; Richardson, Charles C

    2010-04-13

    The ring-shaped helicase of bacteriophage T7 (gp4), the product of gene 4, has basic beta-hairpin loops lining its central core where they are postulated to be the major sites of DNA interaction. We have altered multiple residues within the beta-hairpin loop to determine their role during dTTPase-driven DNA unwinding. Residues His-465, Leu-466, and Asn-468 are essential for both DNA unwinding and DNA synthesis mediated by T7 DNA polymerase during leading-strand DNA synthesis. Gp4-K467A, gp4-K471A, and gp4-K473A form fewer hexamers than heptamers compared to wild-type helicase and alone are deficient in DNA unwinding. However, they complement for the growth of T7 bacteriophage lacking gene 4. Single-molecule studies show that these three altered helicases support rates of leading-strand DNA synthesis comparable to that observed with wild-type gp4. Gp4-K467A, devoid of unwinding activity alone, supports leading-strand synthesis in the presence of T7 DNA polymerase. We propose that DNA polymerase limits the backward movement of the helicase during unwinding as well as assisting the forward movement necessary for strand separation.

  10. Simultaneous binding to the tracking strand, displaced strand and the duplex of a DNA fork enhances unwinding by Dda helicase.

    PubMed

    Aarattuthodiyil, Suja; Byrd, Alicia K; Raney, Kevin D

    2014-10-01

    Interactions between helicases and the tracking strand of a DNA substrate are well-characterized; however, the role of the displaced strand is a less understood characteristic of DNA unwinding. Dda helicase exhibited greater processivity when unwinding a DNA fork compared to a ss/ds DNA junction substrate. The lag phase in the unwinding progress curve was reduced for the forked DNA compared to the ss/ds junction. Fewer kinetic steps were required to unwind the fork compared to the ss/ds junction, suggesting that binding to the fork leads to disruption of the duplex. DNA footprinting confirmed that interaction of Dda with a fork leads to two base pairs being disrupted whereas no disruption of base pairing was observed with the ss/ds junction. Neutralization of the phosphodiester backbone resulted in a DNA-footprinting pattern similar to that observed with the ss/ds junction, consistent with disruption of the interaction between Dda and the displaced strand. Several basic residues in the 1A domain which were previously proposed to bind to the incoming duplex DNA were replaced with alanines, resulting in apparent loss of interaction with the duplex. Taken together, these results suggest that Dda interaction with the tracking strand, displaced strand and duplex coordinates DNA unwinding.

  11. Steric exclusion and wrapping of the excluded DNA strand occurs along discrete external binding paths during MCM helicase unwinding.

    PubMed

    Graham, Brian W; Schauer, Grant D; Leuba, Sanford H; Trakselis, Michael A

    2011-08-01

    The minichromosome maintenance (MCM) helicase complex is essential for the initiation and elongation of DNA replication in both the eukaryotic and archaeal domains. The archaeal homohexameric MCM helicase from Sulfolobus solfataricus serves as a model for understanding mechanisms of DNA unwinding. In this report, the displaced 5'-tail is shown to provide stability to the MCM complex on DNA and contribute to unwinding. Mutations in a positively charged patch on the exterior surface of the MCM hexamer destabilize this interaction, alter the path of the displaced 5'-tail DNA and reduce unwinding. DNA footprinting and single-molecule fluorescence experiments support a previously unrecognized wrapping of the 5'-tail. This mode of hexameric helicase DNA unwinding is termed the steric exclusion and wrapping (SEW) model, where the 3'-tail is encircled by the helicase while the displaced 5'-tail wraps around defined paths on the exterior of the helicase. The novel wrapping mechanism stabilizes the MCM complex in a positive unwinding mode, protects the displaced single-stranded DNA tail and prevents reannealing.

  12. Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase

    PubMed Central

    Langston, Lance; O’Donnell, Mike

    2017-01-01

    Replicative helicases are ring-shaped hexamers that encircle DNA for duplex unwinding. The currently accepted view of hexameric helicase function is by steric exclusion, where the helicase encircles one DNA strand and excludes the other, acting as a wedge with an external DNA unwinding point during translocation. Accordingly, strand-specific blocks only affect these helicases when placed on the tracking strand, not the excluded strand. We examined the effect of blocks on the eukaryotic CMG and, contrary to expectations, blocks on either strand inhibit CMG unwinding. A recent cryoEM structure of yeast CMG shows that duplex DNA enters the helicase and unwinding occurs in the central channel. The results of this report inform important aspects of the structure, and we propose that CMG functions by a modified steric exclusion process in which both strands enter the helicase and the duplex unwinding point is internal, followed by exclusion of the non-tracking strand. DOI: http://dx.doi.org/10.7554/eLife.23449.001 PMID:28346143

  13. Potent inhibition of DNA unwinding and ATPase activities of pea DNA helicase 45 by DNA-binding agents.

    PubMed

    Pham, Xuan Hoi; Tuteja, Narendra

    2002-06-07

    Pea DNA helicase 45 (PDH45) is an ATP-dependent DNA unwinding enzyme, with intrinsic DNA-dependent ATPase activity [Plant J. 24 (2000) 219]. We have determined the effect of various DNA-binding agents, such as daunorubicin, ethidium bromide, ellipticine, cisplatin, nogalamycin, actinomycin C1, and camptothecin on the DNA unwinding and ATPase activities of the plant nuclear DNA helicase PDH45. The results show that all the agents except actinomycin C1, and camptothecin inhibited the helicase (apparent K(i) values ranging from 1.5 to 7.0 microM) and ATPase (apparent K(i) values ranging from 2.5 to 11.9 microM) activities. This is the first study to show the effect of various DNA-binding agents on the plant nuclear helicase and also first to demonstrate inhibition of any helicase by cisplatin. Another striking finding that the actinomycin C1 and ellipticine act differentially on PDH45 as compared to pea chloroplast helicase suggests that the mechanism of DNA unwinding could be different in nucleus and chloroplast. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of PDH45, resulting in both the inhibitions of unwinding activity and ATP hydrolysis. This study would be useful to obtain a better understanding of the mechanism of plant nuclear DNA helicase unwinding and the mechanism by which these agents can disturb genome integrity.

  14. Anodes for alkaline electrolysis

    DOEpatents

    Soloveichik, Grigorii Lev

    2011-02-01

    A method of making an anode for alkaline electrolysis cells includes adsorption of precursor material on a carbonaceous material, conversion of the precursor material to hydroxide form and conversion of precursor material from hydroxide form to oxy-hydroxide form within the alkaline electrolysis cell.

  15. Switching from single-stranded to double-stranded DNA limits the unwinding processivity of ring-shaped T7 DNA helicase.

    PubMed

    Jeong, Yong-Joo; Rajagopal, Vaishnavi; Patel, Smita S

    2013-04-01

    Phage T7 helicase unwinds double-stranded DNA (dsDNA) by encircling one strand while excluding the complementary strand from its central channel. When T7 helicase translocates on single-stranded DNA (ssDNA), it has kilobase processivity; yet, it is unable to processively unwind linear dsDNA, even 60 base-pairs long. Particularly, the GC-rich dsDNAs are unwound with lower amplitudes under single-turnover conditions. Here, we provide evidence that T7 helicase switches from ssDNA to dsDNA during DNA unwinding. The switching propensity is higher when dsDNA is GC-rich or when the 3'-overhang of forked DNA is <15 bases. Once helicase encircles dsDNA, it travels along dsDNA and dissociates from the end of linear DNA without strand separation, which explains the low unwinding amplitude of these substrates. Trapping the displaced strand with ssDNA binding protein or changing its composition to morpholino oligomer that does not interact with helicase increases the unwinding amplitude. We conclude that the displaced strand must be continuously excluded and kept away from the central channel for processive DNA unwinding. The finding that T7 helicase can switch from ssDNA to dsDNA binding mode during unwinding provides new insights into ways of limiting DNA unwinding and triggering fork regression when stalled forks need to be restarted.

  16. Purified human SUV3p exhibits multiple-substrate unwinding activity upon conformational change.

    PubMed

    Shu, Zhanyong; Vijayakumar, Sangeetha; Chen, Chi-Fen; Chen, Phang-Lang; Lee, Wen-Hwa

    2004-04-27

    Suv3 of Saccharomyces cerevisiae has been classified as a mitochondrial RNA helicase. However, the helicase domain in both yeast and human SUV3 varies considerably from the typical RNA helicase motifs. To investigate its enzymatic activities, a homogeneously purified preparation of SUV3 is required. Expression of a processed form of human SUV3 carrying an N-terminal deletion of 46 amino acids (SUV3DeltaN46) in a yeast suv3 null mutant, which otherwise fails to grow in a nonfermentable carbon source and forms petite colonies in glucose medium, rescues the null phenotype. Through a five-step chromatographic procedure, an 83 kDa SUV3DeltaN46 protein (SUV3-83) and a partially degraded 70 kDa product (SUV3-70) containing amino acids 68-685 were purified to homogeneity. Single- or double-stranded DNA and RNA stimulated ATPase activity of both proteins. SUV3-70, which retains core catalytic domains, can bind and unwind multiple duplex substrates of RNA and DNA with a 5'-3' directionality over a wide range of pH, while SUV3-83 has helicase activity at only acidic pH. ATP, but not nonhydrolyzable ATP, is essential for the unwinding activity, suggesting the requirement of the energy derived from ATP hydrolysis. Consistent with this notion, suv3 mutants containing alanine (A) or arginine (R) substitutions at the conserved lysine residue in the ATP binding site (K213) lost ATPase activity and also failed to unwind the substrates. Importantly, circular dichroism (CD) spectral analysis showed that SUV3-83, at pH 5.0, adopts a conformation similar to that of SUV3-70, suggesting a conformational change in SUV3-83 is required for its helicase activity. The physiological relevance of the multiple-substrate helicase activity of human SUV3 is discussed.

  17. Human mitochondrial DNA helicase TWINKLE is both an unwinding and annealing helicase.

    PubMed

    Sen, Doyel; Nandakumar, Divya; Tang, Guo-Qing; Patel, Smita S

    2012-04-27

    TWINKLE is a nucleus-encoded human mitochondrial (mt)DNA helicase. Point mutations in TWINKLE are associated with heritable neuromuscular diseases characterized by deletions in the mtDNA. To understand the biochemical basis of these diseases, it is important to define the roles of TWINKLE in mtDNA metabolism by studying its enzymatic activities. To this end, we purified native TWINKLE from Escherichia coli. The recombinant TWINKLE assembles into hexamers and higher oligomers, and addition of MgUTP stabilizes hexamers over higher oligomers. Probing into the DNA unwinding activity, we discovered that the efficiency of unwinding is greatly enhanced in the presence of a heterologous single strand-binding protein or a single-stranded (ss) DNA that is complementary to the unwound strand. We show that TWINKLE, although a helicase, has an antagonistic activity of annealing two complementary ssDNAs that interferes with unwinding in the absence of gp2.5 or ssDNA trap. Furthermore, only ssDNA and not double-stranded (ds)DNA competitively inhibits the annealing activity, although both DNAs bind with high affinities. This implies that dsDNA binds to a site that is distinct from the ssDNA-binding site that promotes annealing. Fluorescence anisotropy competition binding experiments suggest that TWINKLE has more than one ssDNA-binding sites, and we speculate that a surface-exposed ssDNA-specific site is involved in catalyzing DNA annealing. We propose that the strand annealing activity of TWINKLE may play a role in recombination-mediated replication initiation found in the mitochondria of mammalian brain and heart or in replication fork regression during repair of damaged DNA replication forks.

  18. Alkaline battery operational methodology

    DOEpatents

    Sholklapper, Tal; Gallaway, Joshua; Steingart, Daniel; Ingale, Nilesh; Nyce, Michael

    2016-08-16

    Methods of using specific operational charge and discharge parameters to extend the life of alkaline batteries are disclosed. The methods can be used with any commercial primary or secondary alkaline battery, as well as with newer alkaline battery designs, including batteries with flowing electrolyte. The methods include cycling batteries within a narrow operating voltage window, with minimum and maximum cut-off voltages that are set based on battery characteristics and environmental conditions. The narrow voltage window decreases available capacity but allows the batteries to be cycled for hundreds or thousands of times.

  19. Direct Fluorescent Imaging of Translocation and Unwinding by Individual DNA Helicases.

    PubMed

    Pavankumar, T L; Exell, J C; Kowalczykowski, S C

    2016-01-01

    The unique translocation and DNA unwinding properties of DNA helicases can be concealed by the stochastic behavior of enzyme molecules within the necessarily large populations used in ensemble experiments. With recent technological advances, the direct visualization of helicases acting on individual DNA molecules has contributed significantly to the current understanding of their mechanisms of action and biological functions. The combination of single-molecule techniques that enable both manipulation of individual protein or DNA molecules and visualization of their actions has made it possible to literally see novel and unique biochemical characteristics that were previously masked. Here, we describe the execution and use of single-molecule fluorescence imaging techniques, focusing on methods that include optical trapping in conjunction with epifluorescent imaging, and also surface immobilization in conjunction with total internal reflection fluorescence visualization. Combined with microchannel flow cells and microfluidic control, these methods allow individual fluorescently labeled protein and DNA molecules to be imaged and tracked, affording measurement of DNA unwinding and translocation at single-molecule resolution.

  20. TRF2 recruits RTEL1 to telomeres in S phase to promote t-loop unwinding.

    PubMed

    Sarek, Grzegorz; Vannier, Jean-Baptiste; Panier, Stephanie; Petrini, John H J; Boulton, Simon J

    2015-02-19

    The helicase RTEL1 promotes t-loop unwinding and suppresses telomere fragility to maintain the integrity of vertebrate telomeres. An interaction between RTEL1 and PCNA is important to prevent telomere fragility, but how RTEL1 engages with the telomere to promote t-loop unwinding is unclear. Here, we establish that the shelterin protein TRF2 recruits RTEL1 to telomeres in S phase, which is required to prevent catastrophic t-loop processing by structure-specific nucleases. We show that the TRF2-RTEL1 interaction is mediated by a metal-coordinating C4C4 motif in RTEL1, which is compromised by the Hoyeraal-Hreidarsson syndrome (HHS) mutation, RTEL1(R1264H). Conversely, we define a TRF2(I124D) substitution mutation within the TRFH domain of TRF2, which eliminates RTEL1 binding and phenocopies the RTEL1(R1264H) mutation, giving rise to aberrant t-loop excision, telomere length heterogeneity, and loss of the telomere as a circle. These results implicate TRF2 in the recruitment of RTEL1 to facilitate t-loop disassembly at telomeres in S phase.

  1. The Mtr4 ratchet helix and arch domain both function to promote RNA unwinding.

    PubMed

    Taylor, Lacy L; Jackson, Ryan N; Rexhepaj, Megi; King, Alejandra Klauer; Lott, Lindsey K; van Hoof, Ambro; Johnson, Sean J

    2014-12-16

    Mtr4 is a conserved Ski2-like RNA helicase and a subunit of the TRAMP complex that activates exosome-mediated 3'-5' turnover in nuclear RNA surveillance and processing pathways. Prominent features of the Mtr4 structure include a four-domain ring-like helicase core and a large arch domain that spans the core. The 'ratchet helix' is positioned to interact with RNA substrates as they move through the helicase. However, the contribution of the ratchet helix in Mtr4 activity is poorly understood. Here we show that strict conservation along the ratchet helix is particularly extensive for Ski2-like RNA helicases compared to related helicases. Mutation of residues along the ratchet helix alters in vitro activity in Mtr4 and TRAMP and causes slow growth phenotypes in vivo. We also identify a residue on the ratchet helix that influences Mtr4 affinity for polyadenylated substrates. Previous work indicated that deletion of the arch domain has minimal effect on Mtr4 unwinding activity. We now show that combining the arch deletion with ratchet helix mutations abolishes helicase activity and produces a lethal in vivo phenotype. These studies demonstrate that the ratchet helix modulates helicase activity and suggest that the arch domain plays a previously unrecognized role in unwinding substrates.

  2. Structural insights into 5' flap DNA unwinding and incision by the human FAN1 dimer.

    PubMed

    Zhao, Qi; Xue, Xiaoyu; Longerich, Simonne; Sung, Patrick; Xiong, Yong

    2014-12-11

    Human FANCD2-associated nuclease 1 (FAN1) is a DNA structure-specific nuclease involved in the processing of DNA interstrand crosslinks (ICLs). FAN1 maintains genomic stability and prevents tissue decline in multiple organs, yet it confers ICL-induced anti-cancer drug resistance in several cancer subtypes. Here we report three crystal structures of human FAN1 in complex with a 5' flap DNA substrate, showing that two FAN1 molecules form a head-to-tail dimer to locate the lesion, orient the DNA and unwind a 5' flap for subsequent incision. Biochemical experiments further validate our model for FAN1 action, as structure-informed mutations that disrupt protein dimerization, substrate orientation or flap unwinding impair the structure-specific nuclease activity. Our work elucidates essential aspects of FAN1-DNA lesion recognition and a unique mechanism of incision. These structural insights shed light on the cellular mechanisms underlying organ degeneration protection and cancer drug resistance mediated by FAN1.

  3. Biochemical analysis of the DNA unwinding and strand annealing activities catalyzed by human RECQ1.

    PubMed

    Sharma, Sudha; Sommers, Joshua A; Choudhary, Saba; Faulkner, Jinnifer Korin; Cui, Sheng; Andreoli, Lucia; Muzzolini, Laura; Vindigni, Alessandro; Brosh, Robert M

    2005-07-29

    RecQ helicases play an important role in preserving genomic integrity, and their cellular roles in DNA repair, recombination, and replication have been of considerable interest. Of the five human RecQ helicases identified, three are associated with genetic disorders characterized by an elevated incidence of cancer or premature aging: Werner syndrome, Bloom syndrome, and Rothmund-Thomson syndrome. Although the biochemical properties and protein interactions of the WRN and BLM helicases defective in Werner syndrome and Bloom syndrome, respectively, have been extensively investigated, less information is available concerning the functions of the other human RecQ helicases. We have focused our attention on human RECQ1, a DNA helicase whose cellular functions remain largely uncharacterized. In this work, we have characterized the DNA substrate specificity and optimal cofactor requirements for efficient RECQ1-catalyzed DNA unwinding and determined that RECQ1 has certain properties that are distinct from those of other RecQ helicases. RECQ1 stably bound to a variety of DNA structures, enabling it to unwind a diverse set of DNA substrates. In addition to its DNA binding and helicase activities, RECQ1 catalyzed efficient strand annealing between complementary single-stranded DNA molecules. The ability of RECQ1 to promote strand annealing was modulated by ATP binding, which induced a conformational change in the protein. The enzymatic properties of the RECQ1 helicase and strand annealing activities are discussed in the context of proposed cellular DNA metabolic pathways that are important in the maintenance of genomic stability.

  4. Structural insights into 5‧ flap DNA unwinding and incision by the human FAN1 dimer

    NASA Astrophysics Data System (ADS)

    Zhao, Qi; Xue, Xiaoyu; Longerich, Simonne; Sung, Patrick; Xiong, Yong

    2014-12-01

    Human FANCD2-associated nuclease 1 (FAN1) is a DNA structure-specific nuclease involved in the processing of DNA interstrand crosslinks (ICLs). FAN1 maintains genomic stability and prevents tissue decline in multiple organs, yet it confers ICL-induced anti-cancer drug resistance in several cancer subtypes. Here we report three crystal structures of human FAN1 in complex with a 5‧ flap DNA substrate, showing that two FAN1 molecules form a head-to-tail dimer to locate the lesion, orient the DNA and unwind a 5‧ flap for subsequent incision. Biochemical experiments further validate our model for FAN1 action, as structure-informed mutations that disrupt protein dimerization, substrate orientation or flap unwinding impair the structure-specific nuclease activity. Our work elucidates essential aspects of FAN1-DNA lesion recognition and a unique mechanism of incision. These structural insights shed light on the cellular mechanisms underlying organ degeneration protection and cancer drug resistance mediated by FAN1.

  5. Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA

    SciTech Connect

    Verebová, Valéria; Adamcik, Jozef; Danko, Patrik; Podhradský, Dušan; Miškovský, Pavol; Staničová, Jana

    2014-01-31

    Highlights: • Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA. • Anthraquinones quinizarin and danthron lengthen linear DNA. • Anthraquinones quinizarin and danthron possess middle binding affinity to DNA. • Anthraquinones quinizarin and danthron interact with DNA by intercalating mode. - Abstract: The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode.

  6. POT1 stimulates RecQ helicases WRN and BLM to unwind telomeric DNA substrates.

    PubMed

    Opresko, Patricia L; Mason, Penelope A; Podell, Elaine R; Lei, Ming; Hickson, Ian D; Cech, Thomas R; Bohr, Vilhelm A

    2005-09-16

    Defects in human RecQ helicases WRN and BLM are responsible for the cancer-prone disorders Werner syndrome and Bloom syndrome. Cellular phenotypes of Werner syndrome and Bloom syndrome, including genomic instability and premature senescence, are consistent with telomere dysfunction. RecQ helicases are proposed to function in dissociating alternative DNA structures during recombination and/or replication at telomeric ends. Here we report that the telomeric single-strand DNA-binding protein, POT1, strongly stimulates WRN and BLM to unwind long telomeric forked duplexes and D-loop structures that are otherwise poor substrates for these helicases. This stimulation is dependent on the presence of telomeric sequence in the duplex regions of the substrates. In contrast, POT1 failed to stimulate a bacterial 3'-5'-helicase. We find that purified POT1 binds to WRN and BLM in vitro and that full-length POT1 (splice variant 1) precipitates a higher amount of endogenous WRN protein, compared with BLM, from the HeLa nuclear extract. We propose roles for the cooperation of POT1 with RecQ helicases WRN and BLM in resolving DNA structures at telomeric ends, in a manner that protects the telomeric 3' tail as it is exposed during unwinding.

  7. Intra-tRNA distance measurements for nucleocapsid proteindependent tRNA unwinding during priming of HIV reverse transcription.

    PubMed

    Chan, B; Weidemaier, K; Yip, W T; Barbara, P F; Musier-Forsyth, K

    1999-01-19

    We report here the direct measurement of intra-tRNA distances during annealing of the tRNA primer to the HIV RNA genome. This key step in the initiation of retroviral reverse transcription involves hybridization of one strand of the acceptor arm of a specific lysine tRNA to the primer binding site on the RNA genome. Although the mechanism of tRNA unwinding and annealing is not known, previous studies have shown that HIV nucleocapsid protein (NC) greatly accelerates primer/template binary complex formation in vitro. An open question is whether NC alone unwinds the primer or whether unwinding by NC requires the RNA genome. We monitored the annealing process in solution by using fluorescence resonance energy transfer (FRET). Distance measurements demonstrate unequivocally that the tRNA acceptor stem is not substantially unwound by NC in the absence of the RNA genome, that is, unwinding is not separable from hybridization. Moreover, FRET measurements show that both heat- and NC-mediated annealing result in an approximately 40-A increase in the separation of the two ends of the tRNA acceptor arm on binding to the template. This large increase in separation of the two ends suggests a complete displacement of the nonhybridized strand of the acceptor stem in the initiation complex.

  8. Impediment of E. coli UvrD by DNA-destabilizing force reveals a strained-inchworm mechanism of DNA unwinding.

    PubMed

    Sun, Bo; Wei, Kong-Ji; Zhang, Bo; Zhang, Xing-Hua; Dou, Shuo-Xing; Li, Ming; Xi, Xu Guang

    2008-12-17

    Escherichia coli UvrD is a non-ring-shaped model helicase, displaying a 3'-5' polarity in DNA unwinding. Using a transverse magnetic tweezer and DNA hairpins, we measured the unwinding kinetics of UvrD at various DNA-destabilizing forces. The multiform patterns of unwinding bursts and the distributions of the off-times favour the mechanism that UvrD unwinds DNA as a dimer. The two subunits of the dimer coordinate to unwind DNA processively. They can jointly switch strands and translocate backwards on the other strand to allow slow (approximately 40 bp/s) rewinding, or unbind simultaneously to allow quick rehybridization. Partial dissociation of the dimer results in pauses in the middle of the unwinding or increases the translocation rate from approximately 40 to approximately 150 nt/s in the middle of the rewinding. Moreover, the unwinding rate was surprisingly found to decrease from approximately 45 to approximately 10 bp/s when the force is increased from 2 to 12 pN. The results lead to a strained-inchworm mechanism in which a conformational change that bends and tenses the ssDNA is required to activate the dimer.

  9. Dda helicase tightly couples translocation on single-stranded DNA to unwinding of duplex DNA: Dda is an optimally active helicase.

    PubMed

    Byrd, Alicia K; Matlock, Dennis L; Bagchi, Debjani; Aarattuthodiyil, Suja; Harrison, David; Croquette, Vincent; Raney, Kevin D

    2012-07-13

    Helicases utilize the energy of ATP hydrolysis to unwind double-stranded DNA while translocating on the DNA. Mechanisms for melting the duplex have been characterized as active or passive, depending on whether the enzyme actively separates the base pairs or simply sequesters single-stranded DNA (ssDNA) that forms due to thermal fraying. Here, we show that Dda translocates unidirectionally on ssDNA at the same rate at which it unwinds double-stranded DNA in both ensemble and single-molecule experiments. Further, the unwinding rate is largely insensitive to the duplex stability and to the applied force. Thus, Dda transduces all of its translocase activity into DNA unwinding activity so that the rate of unwinding is limited by the rate of translocation and that the enzyme actively separates the duplex. Active and passive helicases have been characterized by dividing the velocity of DNA unwinding in base pairs per second (V(un)) by the velocity of translocation on ssDNA in nucleotides per second (V(trans)). If the resulting fraction is 0.25, then a helicase is considered to be at the lower end of the "active" range. In the case of Dda, the average DNA unwinding velocity was 257±42 bp/s, and the average translocation velocity was 267±15 nt/s. The V(un)/V(trans) value of 0.96 places Dda in a unique category of being an essentially "perfectly" active helicase.

  10. Structural basis for RNA-duplex recognition and unwinding by the DEAD-box helicase Mss116p.

    PubMed

    Mallam, Anna L; Del Campo, Mark; Gilman, Benjamin; Sidote, David J; Lambowitz, Alan M

    2012-10-04

    DEAD-box proteins are the largest family of nucleic acid helicases, and are crucial to RNA metabolism throughout all domains of life. They contain a conserved 'helicase core' of two RecA-like domains (domains (D)1 and D2), which uses ATP to catalyse the unwinding of short RNA duplexes by non-processive, local strand separation. This mode of action differs from that of translocating helicases and allows DEAD-box proteins to remodel large RNAs and RNA-protein complexes without globally disrupting RNA structure. However, the structural basis for this distinctive mode of RNA unwinding remains unclear. Here, structural, biochemical and genetic analyses of the yeast DEAD-box protein Mss116p indicate that the helicase core domains have modular functions that enable a novel mechanism for RNA-duplex recognition and unwinding. By investigating D1 and D2 individually and together, we find that D1 acts as an ATP-binding domain and D2 functions as an RNA-duplex recognition domain. D2 contains a nucleic-acid-binding pocket that is formed by conserved DEAD-box protein sequence motifs and accommodates A-form but not B-form duplexes, providing a basis for RNA substrate specificity. Upon a conformational change in which the two core domains join to form a 'closed state' with an ATPase active site, conserved motifs in D1 promote the unwinding of duplex substrates bound to D2 by excluding one RNA strand and bending the other. Our results provide a comprehensive structural model for how DEAD-box proteins recognize and unwind RNA duplexes. This model explains key features of DEAD-box protein function and affords a new perspective on how the evolutionarily related cores of other RNA and DNA helicases diverged to use different mechanisms.

  11. Differentiation of the DnaA-oriC subcomplex for DNA unwinding in a replication initiation complex.

    PubMed

    Ozaki, Shogo; Noguchi, Yasunori; Hayashi, Yasuhisa; Miyazaki, Erika; Katayama, Tsutomu

    2012-10-26

    In Escherichia coli, ATP-DnaA multimers formed on the replication origin oriC promote duplex unwinding, which leads to helicase loading. Based on a detailed functional analysis of the oriC sequence motifs, we previously proposed that the left half of oriC forms an ATP-DnaA subcomplex competent for oriC unwinding, whereas the right half of oriC forms a distinct ATP-DnaA subcomplex that facilitates helicase loading. However, the molecular basis for the functional difference between these ATP-DnaA subcomplexes remains unclear. By analyzing a series of novel DnaA mutants, we found that structurally distinct DnaA multimers form on each half of oriC. DnaA AAA+ domain residues Arg-227 and Leu-290 are specifically required for oriC unwinding. Notably, these residues are required for the ATP-DnaA-specific structure of DnaA multimers in complex with the left half of oriC but not for that with the right half. These results support the idea that the ATP-DnaA multimers formed on oriC are not uniform and that they can adopt different conformations. Based on a structural model, we propose that Arg-227 and Leu-290 play a crucial role in inter-ATP-DnaA interaction and are a prerequisite for the formation of unwinding-competent DnaA subcomplexes on the left half of oriC. These residues are not required for the interaction with DnaB, nucleotide binding, or regulatory DnaA-ATP hydrolysis, which further supports their important role in inter-DnaA interaction. The corresponding residues are evolutionarily conserved and are required for unwinding in the initial complexes of Thermotoga maritima, an ancient hyperthermophile. Therefore, our findings suggest a novel and common mechanism for ATP-DnaA-dependent activation of initial complexes.

  12. DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate.

    PubMed

    Liu, Bian; Baskin, Ronald J; Kowalczykowski, Stephen C

    2013-08-22

    Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing discrepancies, and has provided otherwise unattainable mechanistic insights into its enzymatic behaviour. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably, but the origin of this heterogeneity remains unknown. Here we investigate the physical basis for this behaviour. Although any individual RecBCD molecule unwound DNA at a constant rate for an average of approximately 30,000 steps, we discover that transiently halting a single enzyme-DNA complex by depleting Mg(2+)-ATP could change the subsequent rates of DNA unwinding by that enzyme after reintroduction to ligand. The proportion of molecules that changed rate increased exponentially with the duration of the interruption, with a half-life of approximately 1 second, suggesting that a conformational change occurred during the time that the molecule was arrested. The velocity after pausing an individual molecule was any velocity found in the starting distribution of the ensemble. We suggest that substrate binding stabilizes the enzyme in one of many equilibrium conformational sub-states that determine the rate-limiting translocation behaviour of each RecBCD molecule. Each stabilized sub-state can persist for the duration (approximately 1 minute) of processive unwinding of a DNA molecule, comprising tens of thousands of catalytic steps, each of which is much faster than the time needed for the conformational change required to alter kinetic behaviour. This ligand-dependent stabilization of rate-defining conformational sub-states results in seemingly static molecule-to-molecule variation in RecBCD helicase activity

  13. Juxta-terminal Helix Unwinding as a Stabilizing Factor to Modulate the Dynamics of Transmembrane Helices.

    PubMed

    Mortazavi, Armin; Rajagopalan, Venkatesan; Sparks, Kelsey A; Greathouse, Denise V; Koeppe, Roger E

    2016-03-15

    Transmembrane helices of integral membrane proteins often are flanked by interfacial aromatic residues that can serve as anchors to aid the stabilization of a tilted transmembrane orientation. Yet, physical factors that govern the orientation or dynamic averaging of individual transmembrane helices are not well understood and have not been adequately explained. Using solid-state (2) H NMR spectroscopy to examine lipid bilayer-incorporated model peptides of the GWALP23 (acetyl-GGALW(LA)6 LWLAGA-amide) family, we observed substantial unwinding at the terminals of several tilted helices spanning the membranes of DLPC, DMPC, or DOPC lipid bilayers. The fraying of helix ends might be vital for defining the dynamics and orientations of transmembrane helices in lipid bilayer membranes.

  14. Methods for detecting ATP hydrolysis and nucleic acid unwinding of Japanese encephalitis virus NS3 helicase.

    PubMed

    Fang, Jin'e; Li, Huan; Peng, Guiqing; Cao, Shengbo; Zhen, F Fu; Chen, Huanchun; Song, Yunfeng

    2013-12-01

    Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic pathogen that is prevalent in south-east Asia. Because there is no specific antiviral agent, JEV still causes a high rate of neurologic sequelae and mortality in humans. The helicase encoded by the NS3 gene of JEV has emerged recently as a novel antiviral target for treatment. In this study, a soluble recombinant JEV helicase protein was expressed and purified. Methods for detecting the ATP hydrolysis and nucleic acid unwinding activity were developed by luminescence and fluorescence resonance energy transfer (FRET). The concentrations of enzyme, substrate, capture strand, ATP, and divalent ions were optimised in the ATPase and helicase reactions. The feasibility of using these two methods for high-throughput screening of NS3 helicase inhibitors is discussed.

  15. Helix unwinding and base flipping enable human MTERF1 to terminate mitochondrial transcription.

    PubMed

    Yakubovskaya, Elena; Mejia, Edison; Byrnes, James; Hambardjieva, Elena; Garcia-Diaz, Miguel

    2010-06-11

    Defects in mitochondrial gene expression are associated with aging and disease. Mterf proteins have been implicated in modulating transcription, replication and protein synthesis. We have solved the structure of a member of this family, the human mitochondrial transcriptional terminator MTERF1, bound to dsDNA containing the termination sequence. The structure indicates that upon sequence recognition MTERF1 unwinds the DNA molecule, promoting eversion of three nucleotides. Base flipping is critical for stable binding and transcriptional termination. Additional structural and biochemical results provide insight into the DNA binding mechanism and explain how MTERF1 recognizes its target sequence. Finally, we have demonstrated that the mitochondrial pathogenic G3249A and G3244A mutations interfere with key interactions for sequence recognition, eliminating termination. Our results provide insight into the role of mterf proteins and suggest a link between mitochondrial disease and the regulation of mitochondrial transcription.

  16. Biochemical characterization of G4 quadruplex telomerase RNA unwinding by the RNA helicase RHAU.

    PubMed

    Booy, Evan P; McRae, Ewan K S; McKenna, Sean A

    2015-01-01

    G4 quadruplexes are stable secondary structures prevalent in DNA and RNA that exhibit diverse regulatory functions. Herein, we describe an in vitro technique using the purified RNA helicase RHAU to unwind a G4 quadruplex identified near the 5' end of the human telomerase RNA (hTR). A synthetic RNA corresponding to the quadruplex forming region of hTR (hTR10-43), as well as a predicted complementary strand (25P1), are combined in a reaction containing the purified helicase and ATP. Reaction products and appropriate controls are resolved by native gel electrophoresis. Gels can be stained using a combination of total RNA and quadruplex-specific dyes to observe the expected quadruplex to duplex conversion. This straightforward method can be extended to study structural changes in other inter- or intramolecular quadruplex containing DNA/RNA molecules with the RHAU helicase or other RNA/DNA remodeling enzymes.

  17. Dual DNA unwinding activities of the Rothmund-Thomson syndrome protein, RECQ4.

    PubMed

    Xu, Xiaohua; Liu, Yilun

    2009-03-04

    Human RECQ helicases have been linked to distinct clinical diseases with increased cancer rates and premature ageing. All RECQ proteins, except RECQ4, have been shown to be functional helicases. Mutations in RECQ4 lead to Rothmund-Thomson syndrome (RTS), and mouse models reveal that the conserved helicase motifs are required for avoidance of RTS. Furthermore, the amino (N) terminus of RECQ4 shares homology with yeast DNA replication initiation factor, Sld2, and is vital for embryonic development. Here, in contrast to previous reports, we show that RECQ4 exhibits DNA helicase activity. Importantly, two distinct regions of the protein, the conserved helicase motifs and the Sld2-like N-terminal domain, each independently promote ATP-dependent DNA unwinding. Taken together, our data provide the first biochemical clues underlying the molecular function of RECQ4 in DNA replication and genome maintenance.

  18. Transmembrane Helices Tilt, Bend, Slide, Torque, and Unwind between Functional States of Rhodopsin

    NASA Astrophysics Data System (ADS)

    Ren, Zhong; Ren, Peter X.; Balusu, Rohith; Yang, Xiaojing

    2016-09-01

    The seven-helical bundle of rhodopsin and other G-protein coupled receptors undergoes structural rearrangements as the transmembrane receptor protein is activated. These structural changes are known to involve tilting and bending of various transmembrane helices. However, the cause and effect relationship among structural events leading to a cytoplasmic crevasse for G-protein binding is less well defined. Here we present a mathematical model of the protein helix and a simple procedure to determine multiple parameters that offer precise depiction of a helical conformation. A comprehensive survey of bovine rhodopsin structures shows that the helical rearrangements during the activation of rhodopsin involve a variety of angular and linear motions such as torsion, unwinding, and sliding in addition to the previously reported tilting and bending. These hitherto undefined motion components unify the results obtained from different experimental approaches, and demonstrate conformational similarity between the active opsin structure and the photoactivated structures in crystallo near the retinal anchor despite their marked differences.

  19. Helix Unwinding and Base Flipping Enable Human MTERF1 to Terminate Mitochondrial Transcription

    SciTech Connect

    Yakubovskaya, E.; Mejia, E; Byrnes, J; Hambardjieva, E; Garcia-Diaz, M

    2010-01-01

    Defects in mitochondrial gene expression are associated with aging and disease. Mterf proteins have been implicated in modulating transcription, replication and protein synthesis. We have solved the structure of a member of this family, the human mitochondrial transcriptional terminator MTERF1, bound to dsDNA containing the termination sequence. The structure indicates that upon sequence recognition MTERF1 unwinds the DNA molecule, promoting eversion of three nucleotides. Base flipping is critical for stable binding and transcriptional termination. Additional structural and biochemical results provide insight into the DNA binding mechanism and explain how MTERF1 recognizes its target sequence. Finally, we have demonstrated that the mitochondrial pathogenic G3249A and G3244A mutations interfere with key interactions for sequence recognition, eliminating termination. Our results provide insight into the role of mterf proteins and suggest a link between mitochondrial disease and the regulation of mitochondrial transcription.

  20. Alkaline flooding injection strategy

    SciTech Connect

    French, T.R.; Josephson, C.B.

    1992-03-01

    The objective of this project is to improved alkali-surfactant flooding methods, and this includes determining the proper design of injection strategy. Several different injection strategies have been used or suggested for recovering heavy oils with surfactant-enhanced alkaline flooding methods. Oil recovery was compared for four different injection strategies: (1) surfactant followed by polymer, (2) surfactant followed by alkaline polymer, (3) alkaline surfactant followed by polymer, and (4) alkali, surfactant, and polymer mixed in a single formulation. The effect of alkaline preflush was also studied under two different conditions. All of the oil recovery experiments were conducted under optimal conditions with a viscous, non-acidic oil from Hepler (KS) oil field. The coreflood experiments were conducted with Berea sandstone cores since field core was not available in sufficient quantity for coreflood tests. The Tucker sand of Hepler field is a Class I fluvial dominated deltaic reservoir, as classified by the Department of Energy, which has been selected as the site of a DOE-sponsored field pilot test.

  1. Human Pif1 helicase is a G-quadruplex DNA-binding protein with G-quadruplex DNA-unwinding activity.

    PubMed

    Sanders, Cyril M

    2010-08-15

    Pif1 proteins are helicases that in yeast are implicated in the maintenance of genome stability. One activity of Saccharomyces cerevisiae Pif1 is to stabilize DNA sequences that could otherwise form deleterious G4 (G-quadruplex) structures by acting as a G4 resolvase. The present study shows that human Pif1 (hPif1, nuclear form) is a G4 DNA-binding and resolvase protein and that these activities are properties of the conserved helicase domain (amino acids 206-620 of 641, hPifHD). hPif1 preferentially bound synthetic G4 DNA relative to ssDNA (single-stranded DNA), dsDNA (double-stranded DNA) and a partially single-stranded duplex DNA helicase substrate. G4 DNA unwinding, but not binding, required an extended (>10 nucleotide) 5' ssDNA tail, and in competition assays, G4 DNA was an ineffective suppressor of helicase activity compared with ssDNA. These results suggest a distinction between the determinants of G4 DNA binding and the ssDNA interactions required for helicase action and that hPif1 may act on G4 substrates by binding alone or as a resolvase. Human Pif1 could therefore have a role in processing G4 structures that arise in the single-stranded nucleic acid intermediates formed during DNA replication and gene expression.

  2. In vitro mutagenicity and genotoxicity study of a number of short-chain chlorinated hydrocarbons using the micronucleus test and the alkaline single cell gel electrophoresis technique (Comet assay) in human lymphocytes: a structure-activity relationship (QSAR) analysis of the genotoxic and cytotoxic potential.

    PubMed

    Tafazoli, M; Baeten, A; Geerlings, P; Kirsch-Volders, M

    1998-03-01

    Using the micronucleus (MN) test and the alkaline single cell gel electrophoresis (Comet) assay, potential mutagenicity (MN formation), genotoxicity (DNA breakage capacity) and cytotoxicity (cell proliferation reduction) of five chlorinated hydrocarbons (carbon tetrachloride, hexachloroethane, 1,2-dichloroethane, 1-chlorohexane and 2,3-dichlorobutane) have been evaluated in isolated human lymphocytes. With the MN test a low but statistically significant mutagenic activity was detected for all tested substances (except 2,3-dichlorobutane) with one out of the two donors and in the presence or absence of an exogenous metabolic activation system (S9 mix). However, at the concentration ranges tested none of the positive compounds induced a clear dose-dependent mutagenic effect. The Comet assay detected a strong DNA damaging effect for 1-chlorohexane, 2,3-dichlorobutane and 1,2-dichloroethane, but not for carbon tetrachloride and hexachloroethane. The influence of metabolism on the genotoxic activity of the chemicals was more clear in the Comet assay than in the MN test. The experimental genotoxicity and cytotoxicity data obtained in this study, together with data on five more related chemicals previously investigated, and their physico-chemical descriptors or electronic parameters have been used for QSAR analysis. The QSAR analysis high-lighted that the toxicity of the tested compounds was influenced by different parameters, like lipophilicity (logP), electron donor ability (charge) and longest carbon-chlorine (LBC-Cl) bond length. In addition, steric parameters, like molar refractivity (MR) and LBC-Cl, and electronic parameters, like ELUMO (energy of the lowest unoccupied molecular orbital, indicating electrophilicity), were predominant factors discriminating genotoxins from non-genotoxins in the presence but not in the absence of S9 mix. Although a limited number of compounds have been examined and cytotoxicity and genotoxicity were identified in two different

  3. A conformational change in the helicase core is necessary but not sufficient for RNA unwinding by the DEAD box helicase YxiN.

    PubMed

    Karow, Anne R; Klostermeier, Dagmar

    2009-07-01

    Cooperative binding of ATP and RNA to DEAD-box helicases induces the closed conformation of their helicase core, with extensive interactions across the domain interface. The bound RNA is bent, and its distortion may constitute the first step towards RNA unwinding. To dissect the role of the conformational change in the helicase core for RNA unwinding, we characterized the RNA-stimulated ATPase activity, RNA unwinding and the propensity to form the closed conformer for mutants of the DEAD box helicase YxiN. The ATPase-deficient K52Q mutant forms a closed conformer upon binding of ATP and RNA, but is deficient in RNA unwinding. A mutation in motif III slows down the catalytic cycle, but neither affects the propensity for the closed conformer nor its global conformation. Hence, the closure of the cleft in the helicase core is necessary but not sufficient for RNA unwinding. In contrast, the G303A mutation in motif V prevents a complete closure of the inter-domain cleft, affecting ATP binding and hydrolysis and is detrimental to unwinding. Possibly, the K52Q and motif III mutants still introduce a kink into the backbone of bound RNA, whereas G303A fails to kink the RNA substrate.

  4. Electric-field-induced unwinding of ferroelectric helix in thin smectic C* layers with soft and rigid anchoring of molecules

    SciTech Connect

    Dolganov, P. V.; Zhilin, V. M. Dolganov, V. K.; Kats, E. I.

    2008-09-15

    The unwinding of a helical structure in thin films of a ferroelectric smectic liquid crystal (LC) by an external electric field has been theoretically studied using a discrete model in which every LC layer is characterized by a two-dimensional vector {xi}{sub i} (describing the orientation of molecules) and by the polarization P{sub i}. It is established that the unwinding of the LC helix in thin films significantly differs from the well-known behavior of thick samples. In particular, discrete intermediate states (differing by an integer or half-integer number of turns) are formed in thin films for both weak and strong anchoring of molecules to a substrate surface. The physical factor responsible for this behavior is the presence of near-surface regions with thicknesses below the helix pitch and the corresponding uncompensated polarization.

  5. Alkaline quinone flow battery.

    PubMed

    Lin, Kaixiang; Chen, Qing; Gerhardt, Michael R; Tong, Liuchuan; Kim, Sang Bok; Eisenach, Louise; Valle, Alvaro W; Hardee, David; Gordon, Roy G; Aziz, Michael J; Marshak, Michael P

    2015-09-25

    Storage of photovoltaic and wind electricity in batteries could solve the mismatch problem between the intermittent supply of these renewable resources and variable demand. Flow batteries permit more economical long-duration discharge than solid-electrode batteries by using liquid electrolytes stored outside of the battery. We report an alkaline flow battery based on redox-active organic molecules that are composed entirely of Earth-abundant elements and are nontoxic, nonflammable, and safe for use in residential and commercial environments. The battery operates efficiently with high power density near room temperature. These results demonstrate the stability and performance of redox-active organic molecules in alkaline flow batteries, potentially enabling cost-effective stationary storage of renewable energy.

  6. Advanced alkaline water electrolysis

    NASA Astrophysics Data System (ADS)

    Wakabayashi, N.; Torikai, E.; Kawami, Y.; Takenaka, H.

    Results are presented of experimental studies of possible separators and electrodes for use in advanced, high-temperature, high-pressure alkaline water electrolyzers. Material evaluations in alkaline water electrolyzers at temperatures from 100 to 120 C have shown a new type polytetrafluoroethylene membrane impregnated with potassium titanate to be the most promising when the separator is prepared by the hydrothermal treatment of a porous PFTE membrane impregnated with hydrated titanium oxide. Measurements of cell voltages in 30% KOH at current densities from 5 to 100 A/sq dm at temperatures up to 120 C with nickel electrodes of various structures have shown the foamed nickel electrode, with an average pore size of 1-1.5 mm, to have the best performance. When the foamed nickel is coated by fine powdered nickel, carbonyl nickel or Raney nickel to increase electrode surface areas, even lower cell voltages were found, indicating better performance.

  7. DNA unwinding produced by site-specific intrastrand cross-links of the antitumor drug cis-diamminedichloroplatinum(II).

    PubMed

    Bellon, S F; Coleman, J H; Lippard, S J

    1991-08-13

    The DNA unwinding produced by specific adducts of the antitumor drug cis-diamminedichloroplatinum(II) has been quantitatively determined. Synthetic DNA duplex oligonucleotides of varying lengths with two base pair cohesive ends were synthesized and characterized that contained site-specific intrastrand N7-purine/N7-purine cross-links. Included are cis-[Pt(NH3)2[d(GpG)

  8. Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil.

    PubMed

    Richardson, Tomas T; Wu, Xiaohua; Keith, Brian J; Heslop, Pauline; Jones, Anita C; Connolly, Bernard A

    2013-02-01

    Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3'-5' proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny. This publication uses both steady-state and time-resolved 2-aminopurine fluorescence to show pronounced unwinding of primer-templates with Pyrococcus furiosus (Pfu) polymerase-DNA complexes containing uracil at +2; much less strand separation is seen with uracil at +4. DNA unwinding has long been recognized as necessary for proof-reading exonuclease activity. The roles of M247 and Y261, amino acids suggested by structural studies to play a role in primer-template unwinding, have been probed. M247 appears to be unimportant, but 2-aminopurine fluorescence measurements show that Y261 plays a role in primer-template strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase.

  9. Conserved helicase domain of human RecQ4 is required for strand annealing-independent DNA unwinding.

    PubMed

    Rossi, Marie L; Ghosh, Avik K; Kulikowicz, Tomasz; Croteau, Deborah L; Bohr, Vilhelm A

    2010-07-01

    Humans have five members of the well conserved RecQ helicase family: RecQ1, Bloom syndrome protein (BLM), Werner syndrome protein (WRN), RecQ4, and RecQ5, which are all known for their roles in maintaining genome stability. BLM, WRN, and RecQ4 are associated with premature aging and cancer predisposition. Of the three, RecQ4's biological and cellular roles have been least thoroughly characterized. Here we tested the helicase activity of purified human RecQ4 on various substrates. Consistent with recent results, we detected ATP-dependent RecQ4 unwinding of forked duplexes. However, our results provide the first evidence that human RecQ4's unwinding is independent of strand annealing, and that it does not require the presence of excess ssDNA. Moreover, we demonstrate that a point mutation of the conserved lysine in the Walker A motif abolished helicase activity, implying that not the N-terminal portion, but the helicase domain is solely responsible for the enzyme's unwinding activity. In addition, we demonstrate a novel stimulation of RecQ4's helicase activity by replication protein A, similar to that of RecQ1, BLM, WRN, and RecQ5. Together, these data indicate that specific biochemical activities and protein partners of RecQ4 are conserved with those of the other RecQ helicases.

  10. Characterization of DbpA, an Escherichia coli DEAD box protein with ATP independent RNA unwinding activity.

    PubMed Central

    Böddeker, N; Stade, K; Franceschi, F

    1997-01-01

    DbpA is a putative Escherichia coli ATP dependent RNA helicase belonging to the family of DEAD box proteins. It hydrolyzes ATP in the presence of 23S ribosomal RNA and 93 bases in the peptidyl transferase center of 23S rRNA are sufficient to trigger 100% of the ATPase activity of DbpA. In the present study we characterized the ATPase and RNA unwinding activities of DbpA in more detail. We report that-in contrast to eIF-4A, the prototype of the DEAD box protein family-the ATPase and the helicase activities of DbpA are not coupled. Moreover, the RNA unwinding activity of DbpA is not specific for 23S rRNA, since DbpA is also able to unwind 16S rRNA hybrids. Furthermore, we determined that the ATPase activity of DbpA is triggered to a significant extent not only by the 93 bases of the 23S rRNA previously reported but also by other regions of the 23S rRNA molecule. Since all these regions of 23S rRNA are either part of the 'functional core' of the 50S ribosomal subunit or involved in the 50S assembly, DbpA may play an important role in the ribosomal assembly process. PMID:9016593

  11. The full-length Saccharomyces cerevisiae Sgs1 protein is a vigorous DNA helicase that preferentially unwinds holliday junctions.

    PubMed

    Cejka, Petr; Kowalczykowski, Stephen C

    2010-03-12

    The highly conserved RecQ family of DNA helicases has multiple roles in the maintenance of genome stability. Sgs1, the single RecQ homologue in Saccharomyces cerevisiae, acts both early and late during homologous recombination. Here we present the expression, purification, and biochemical analysis of full-length Sgs1. Unlike the truncated form of Sgs1 characterized previously, full-length Sgs1 binds diverse single-stranded and double-stranded DNA substrates, including DNA duplexes with 5'- and 3'-single-stranded DNA overhangs. Similarly, Sgs1 unwinds a variety of DNA substrates, including blunt-ended duplex DNA. Significantly, a substrate containing a Holliday junction is unwound most efficiently. DNA unwinding is catalytic, requires ATP, and is stimulated by replication protein A. Unlike RecQ homologues from multicellular organisms, Sgs1 is remarkably active at picomolar concentrations and can efficiently unwind duplex DNA molecules as long as 23,000 base pairs. Our analysis shows that Sgs1 resembles Escherichia coli RecQ protein more than any of the human RecQ homologues with regard to its helicase activity. The full-length recombinant protein will be invaluable for further investigation of Sgs1 biochemistry.

  12. Base methylations in the double-stranded RNA by a fused methyltransferase bearing unwinding activity

    PubMed Central

    Kimura, Satoshi; Ikeuchi, Yoshiho; Kitahara, Kei; Sakaguchi, Yuriko; Suzuki, Takeo; Suzuki, Tsutomu

    2012-01-01

    Modifications of rRNAs are clustered in functional regions of the ribosome. In Helix 74 of Escherichia coli 23S rRNA, guanosines at positions 2069 and 2445 are modified to 7-methylguanosine(m7G) and N2-methylguanosine(m2G), respectively. We searched for the gene responsible for m7G2069 formation, and identified rlmL, which encodes the methyltransferase for m2G2445, as responsible for the biogenesis of m7G2069. In vitro methylation of rRNA revealed that rlmL encodes a fused methyltransferase responsible for forming both m7G2069 and m2G2445. We renamed the gene rlmKL. The N-terminal RlmL activity for m2G2445 formation was significantly enhanced by the C-terminal RlmK. Moreover, RlmKL had an unwinding activity of Helix 74, facilitating cooperative methylations of m7G2069 and m2G2445 during biogenesis of 50S subunit. In fact, we observed that RlmKL was involved in the efficient assembly of 50S subunit in a mutant strain lacking an RNA helicase deaD. PMID:22210896

  13. Stimulation of the DNA unwinding activity of human DNA helicase II/Ku by phosphorylation.

    PubMed

    Ochem, Alexander E; Rechreche, Hocine; Skopac, Doris; Falaschi, Arturo

    2008-02-01

    The Ku autoantigen is a heterodimeric protein of 70- and 83-kDa subunits, endowed with duplex DNA end-binding capacity and DNA helicase activity (Human DNA Helicase II, HDH II). HDH II/Ku is well established as the DNA binding component, the regulatory subunit as well as a substrate for the DNA-dependent protein kinase DNA-PK, a complex involved in the repair of DNA double-strand breaks and in V(D)J recombination in eukaryotes. The effects of phosphorylation by this kinase on the helicase activity of Escherichia coli-produced HDH II/Ku were studied. The rate of DNA unwinding by recombinant HDH II/Ku heterodimer is stimulated at least fivefold upon phosphorylation by DNA-PK(cs). This stimulation is due to the effective transfer of phosphate residues to the helicase rather than the mere presence of the complex. In vitro dephosphorylation of HeLa cellular HDH II/Ku caused a significant decrease in the DNA helicase activity of this enzyme.

  14. Structural and Functional Insights into the Unwinding Mechanism of Bacteroides sp Pif1.

    PubMed

    Zhou, Xianglian; Ren, Wendan; Bharath, Sakshibeedu R; Tang, Xuhua; He, Yang; Chen, Chen; Liu, Zhou; Li, Dewang; Song, Haiwei

    2016-03-01

    Pif1 is a conserved SF1B DNA helicase involved in maintaining genome stability through unwinding double-stranded DNAs (dsDNAs), DNA/RNA hybrids, and G quadruplex (G4) structures. Here, we report the structures of the helicase domain of human Pif1 and Bacteroides sp Pif1 (BaPif1) in complex with ADP-AlF4(-) and two different single-stranded DNAs (ssDNAs). The wedge region equivalent to the β hairpin in other SF1B DNA helicases folds into an extended loop followed by an α helix. The Pif1 signature motif of BaPif1 interacts with the wedge region and a short helix in order to stabilize these ssDNA binding elements, therefore indirectly exerting its functional role. Domain 2B of BaPif1 undergoes a large conformational change upon concomitant binding of ATP and ssDNA, which is critical for Pif1's activities. BaPif1 cocrystallized with a tailed dsDNA and ADP-AlF4(-), resulting in a bound ssDNA bent nearly 90° at the ssDNA/dsDNA junction. The conformational snapshots of BaPif1 provide insights into the mechanism governing the helicase activity of Pif1.

  15. Transmembrane Helices Tilt, Bend, Slide, Torque, and Unwind between Functional States of Rhodopsin

    PubMed Central

    Ren, Zhong; Ren, Peter X.; Balusu, Rohith; Yang, Xiaojing

    2016-01-01

    The seven-helical bundle of rhodopsin and other G-protein coupled receptors undergoes structural rearrangements as the transmembrane receptor protein is activated. These structural changes are known to involve tilting and bending of various transmembrane helices. However, the cause and effect relationship among structural events leading to a cytoplasmic crevasse for G-protein binding is less well defined. Here we present a mathematical model of the protein helix and a simple procedure to determine multiple parameters that offer precise depiction of a helical conformation. A comprehensive survey of bovine rhodopsin structures shows that the helical rearrangements during the activation of rhodopsin involve a variety of angular and linear motions such as torsion, unwinding, and sliding in addition to the previously reported tilting and bending. These hitherto undefined motion components unify the results obtained from different experimental approaches, and demonstrate conformational similarity between the active opsin structure and the photoactivated structures in crystallo near the retinal anchor despite their marked differences. PMID:27658480

  16. Non-uniform helix unwinding of cholesteric liquid crystals in cells with interdigitated electrodes.

    PubMed

    Rumi, Mariacristina; Tondiglia, Vincent P; Natarajan, Lalgudi V; White, Timothy J; Bunning, Timothy J

    2014-05-19

    A microspectrophotometer was used to elucidate the local optical properties of cholesteric liquid crystals (CLCs) in cells with interdigitated electrodes as a function of applied voltage. The spectra collected from a spatially selective and micron-sized sampling area allow for new insights into the spectral properties of CLCs in the gaps between patterned electrodes. The microscopic electro-optic response is shown to be highly dependent on the cell thickness and the electrode periodicity. Specifically, the helix unwinding of the CLC superstructure does not always occur uniformly in the sample, as a result of field gradients through the cell thickness: for cells with relatively narrow gaps and electrodes, the redshift occurs initially only in the CLC layers closest to the substrate with the electrodes, leading to broad reflection spectra and different reflection colors depending on which side of the cell is illuminated. Theoretical estimates of the expected shift in the reflection band gap based on the critical field for a given CLC material and the spatial variation of electric field in the cell are found to be in good agreement with the complex behavior observed experimentally. In contrast, in thin cells with wider gaps, the pitch increase affects the whole CLC layer uniformly, because the electric field gradient is small.

  17. Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: quantitative analysis of the rate of the dsDNA unwinding, processivity and kinetic step-size of the Escherichia coli DnaB helicase using rapid quench-flow method.

    PubMed

    Galletto, Roberto; Jezewska, Maria J; Bujalowski, Wlodzimierz

    2004-10-08

    Kinetics of the double-stranded (ds) DNA unwinding by the Escherichia coli replicative helicase DnaB protein has been examined under single-turnover conditions using the chemical quench-flow technique. The unwinding reaction proceeds through an initial conformational transition followed by the unwinding catalytic steps and the release of the single-stranded (ss) DNA. Analyses of the reaction as a function of the number of base-pairs in the dsDNA reveal that the number of catalytic steps is not strictly proportional to the length of the dsDNA. As the helicase approaches the end of the substrate, the remaining approximately 11 bp of the DNA melts without catalytic participation of the enzyme. The kinetic step-size of the DnaB helicase, i.e. the number of the base-pairs unwound in a single catalytic step is only 1.4(+/- 0.2). The low value of the step-size indicates that the helicase unwinds a single base-pair in a single catalytic step. Thus, the DnaB helicase unzips the dsDNA in a reverse process to the zipping mechanism of the non-enzymatic double helix formation. The protein is a fast helicase that at 25 degrees C unwinds approximately 291 bp/s, much faster than previously thought, and the unwinding rate can be much higher at higher temperatures. However, the ATP-state of the enzyme has an increased dissociation rate, resulting in only a moderate unwinding processivity, P = 0.89(+/- 0.03), little dependent on the temperature. The conformational transition of the DnaB helicase-DNA complex, preceding the unwinding, is an intrinsic transition of the enzyme from the stationary conformation to the ATP-state of the helicase.

  18. Alkaline phosphatase of Physarum polycephalum is insoluble.

    PubMed

    Furuhashi, Kiyoshi

    2008-02-01

    The plasmodia of Physarum polycephalum grow as multinucleated cells in the presence of sufficient humidity and nutriment. Under non-illuminating conditions, stresses such as low temperature or high concentrations of salts transform the plasmodia into spherules whereas dehydration induces sclerotization. Some phosphatases including protein phosphatase and acid phosphatase have been purified from the plasmodia, but alkaline phosphatase remains to be elucidated. Phosphatase of the plasmodia, spherules and sclerotia was visualized by electrophoresis gel-staining assay using 5-bromo-4-chloro-3-indolyl phosphate. Insoluble fractions of the sclerotia were abundant in phosphatase activity. The phosphatase which was extracted by nonionic detergent was subjected to column chromatography and preparative electrophoresis. Purified phosphatase showed the highest activity at pH 8.8, indicating that this enzyme belongs to alkaline phosphatase. The apparent molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing condition was estimated to be 100 kDa whereas that under reducing was 105 kDa. An amount of 1% sodium dodecyl sulfate or 0.5 M NaCl had no effects on the activity although the phosphatase showed heat instability, Mg(2+)-dependency and sensitivity to 2-glycerophosphate or NaF. The extracting conditions and enzymatic properties suggest that this alkaline phosphatase which is in a membrane-bound form plays important roles in phosphate metabolism.

  19. G-quadruplex and G-rich sequence stimulate Pif1p-catalyzed downstream duplex DNA unwinding through reducing waiting time at ss/dsDNA junction.

    PubMed

    Zhang, Bo; Wu, Wen-Qiang; Liu, Na-Nv; Duan, Xiao-Lei; Li, Ming; Dou, Shuo-Xing; Hou, Xi-Miao; Xi, Xu-Guang

    2016-09-30

    Alternative DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by G-rich sequences that are widely distributed throughout the human genome. We have previously shown that Pif1p not only unfolds G4, but also unwinds the downstream duplex DNA in a G4-stimulated manner. In the present study, we further characterized the G4-stimulated duplex DNA unwinding phenomenon by means of single-molecule fluorescence resonance energy transfer. It was found that Pif1p did not unwind the partial duplex DNA immediately after unfolding the upstream G4 structure, but rather, it would dwell at the ss/dsDNA junction with a 'waiting time'. Further studies revealed that the waiting time was in fact related to a protein dimerization process that was sensitive to ssDNA sequence and would become rapid if the sequence is G-rich. Furthermore, we identified that the G-rich sequence, as the G4 structure, equally stimulates duplex DNA unwinding. The present work sheds new light on the molecular mechanism by which G4-unwinding helicase Pif1p resolves physiological G4/duplex DNA structures in cells.

  20. G-quadruplex and G-rich sequence stimulate Pif1p-catalyzed downstream duplex DNA unwinding through reducing waiting time at ss/dsDNA junction

    PubMed Central

    Zhang, Bo; Wu, Wen-Qiang; Liu, Na-Nv; Duan, Xiao-Lei; Li, Ming; Dou, Shuo-Xing; Hou, Xi-Miao; Xi, Xu-Guang

    2016-01-01

    Alternative DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by G-rich sequences that are widely distributed throughout the human genome. We have previously shown that Pif1p not only unfolds G4, but also unwinds the downstream duplex DNA in a G4-stimulated manner. In the present study, we further characterized the G4-stimulated duplex DNA unwinding phenomenon by means of single-molecule fluorescence resonance energy transfer. It was found that Pif1p did not unwind the partial duplex DNA immediately after unfolding the upstream G4 structure, but rather, it would dwell at the ss/dsDNA junction with a ‘waiting time’. Further studies revealed that the waiting time was in fact related to a protein dimerization process that was sensitive to ssDNA sequence and would become rapid if the sequence is G-rich. Furthermore, we identified that the G-rich sequence, as the G4 structure, equally stimulates duplex DNA unwinding. The present work sheds new light on the molecular mechanism by which G4-unwinding helicase Pif1p resolves physiological G4/duplex DNA structures in cells. PMID:27471032

  1. Supercoiled pseudocircular domains in single-twisted DNAs under tension: Elastic constants and unwinding dynamics in complexes with Topo I.

    PubMed

    Schurr, J Michael; Fujimoto, Bryant S

    2013-12-01

    Extension versus twist data of Koster et al. (Nature 2005, 434, 671-674) are analyzed to obtain C for the main-chain segments and the twist energy parameter (ET ) for the supercoiled pseudocircular (sp) domain(s) from which C is estimated via simulations. The torsional rigidity in the tension-free sp domain(s) (C = 163 fJ fm) is typical of the unstrained DNA and is less than half the value in the main-chain segments under tension (C = 350-410 fJ fm). Tension is suggested to induce a structural transition to a torsionally stiffer state. Data of Koster et al. for the rate of extension owing to unwinding of a covalent complex of DNA with human Topoisomerase Ib (H Topo I) are analyzed to determine the torque and rate of rotation from which an effective friction coefficient is obtained. A Langevin equation for the unwinding motion in a supercoiled DNA:H Topo I complex is solved to obtain the temporal trajectory of the average winding angle and the time-dependent distribution of winding angles. The mean rate constant for the religation reaction is estimated from the measured probability of reaction per turn. We predict that unwinding proceeds rather far during a single-cleavage and religation cycle, and is effectively completely equilibrated during the 3.2 cleavage and religation cycles that occur during each noncovalent binding and dissociation event. H Topo I is predicted to be completely processive as in accord with observations on calf-thymus Topo I (Brewood et al., Biochemistry 2010, 49, 3367-3380).

  2. DNA unwinding by ring-shaped T4 helicase gp41 is hindered by tension on the occluded strand.

    PubMed

    Ribeck, Noah; Saleh, Omar A

    2013-01-01

    The replicative helicase for bacteriophage T4 is gp41, which is a ring-shaped hexameric motor protein that achieves unwinding of dsDNA by translocating along one strand of ssDNA while forcing the opposite strand to the outside of the ring. While much study has been dedicated to the mechanism of binding and translocation along the ssDNA strand encircled by ring-shaped helicases, relatively little is known about the nature of the interaction with the opposite, 'occluded' strand. Here, we investigate the interplay between the bacteriophage T4 helicase gp41 and the ss/dsDNA fork by measuring, at the single-molecule level, DNA unwinding events on stretched DNA tethers in multiple geometries. We find that gp41 activity is significantly dependent on the geometry and tension of the occluded strand, suggesting an interaction between gp41 and the occluded strand that stimulates the helicase. However, the geometry dependence of gp41 activity is the opposite of that found previously for the E. coli hexameric helicase DnaB. Namely, tension applied between the occluded strand and dsDNA stem inhibits unwinding activity by gp41, while tension pulling apart the two ssDNA tails does not hinder its activity. This implies a distinct variation in helicase-occluded strand interactions among superfamily IV helicases, and we propose a speculative model for this interaction that is consistent with both the data presented here on gp41 and the data that had been previously reported for DnaB.

  3. Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

    NASA Technical Reports Server (NTRS)

    Karr, Laurel; Malone, Christine, C.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  4. Topoisomerase Assays

    PubMed Central

    Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

    2012-01-01

    Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro. PMID:22684721

  5. Alkaline battery, separator therefore

    NASA Technical Reports Server (NTRS)

    Schmidt, George F. (Inventor)

    1980-01-01

    An improved battery separator for alkaline battery cells has low resistance to electrolyte ion transfer and high resistance to electrode ion transfer. The separator is formed by applying an improved coating to an electrolyte absorber. The absorber, preferably, is a flexible, fibrous, and porous substrate that is resistant to strong alkali and oxidation. The coating composition includes an admixture of a polymeric binder, a hydrolyzable polymeric ester and inert fillers. The coating composition is substantially free of reactive fillers and plasticizers commonly employed as porosity promoting agents in separator coatings. When the separator is immersed in electrolyte, the polymeric ester of the film coating reacts with the electrolyte forming a salt and an alcohol. The alcohol goes into solution with the electrolyte while the salt imbibes electrolyte into the coating composition. When the salt is formed, it expands the polymeric chains of the binder to provide a film coating substantially permeable to electrolyte ion transfer but relatively impermeable to electrode ion transfer during use.

  6. Influence of DNA end structure on the mechanism of initiation of DNA unwinding by the Escherichia coli RecBCD and RecBC helicases.

    PubMed

    Wu, Colin G; Lohman, Timothy M

    2008-10-03

    Escherichia coli RecBCD is a bipolar DNA helicase possessing two motor subunits (RecB, a 3'-to-5' translocase, and RecD, a 5'-to-3' translocase) that is involved in the major pathway of recombinational repair. Previous studies indicated that the minimal kinetic mechanism needed to describe the ATP-dependent unwinding of blunt-ended DNA by RecBCD in vitro is a sequential n-step mechanism with two to three additional kinetic steps prior to initiating DNA unwinding. Since RecBCD can "melt out" approximately 6 bp upon binding to the end of a blunt-ended DNA duplex in a Mg(2+)-dependent but ATP-independent reaction, we investigated the effects of noncomplementary single-stranded (ss) DNA tails [3'-(dT)(6) and 5'-(dT)(6) or 5'-(dT)(10)] on the mechanism of RecBCD and RecBC unwinding of duplex DNA using rapid kinetic methods. As with blunt-ended DNA, RecBCD unwinding of DNA possessing 3'-(dT)(6) and 5'-(dT)(6) noncomplementary ssDNA tails is well described by a sequential n-step mechanism with the same unwinding rate (mk(U)=774+/-16 bp s(-1)) and kinetic step size (m=3.3+/-1.3 bp), yet two to three additional kinetic steps are still required prior to initiation of DNA unwinding (k(C)=45+/-2 s(-1)). However, when the noncomplementary 5' ssDNA tail is extended to 10 nt [5'-(dT)(10) and 3'-(dT)(6)], the DNA end structure for which RecBCD displays optimal binding affinity, the additional kinetic steps are no longer needed, although a slightly slower unwinding rate (mk(U)=538+/-24 bp s(-1)) is observed with a similar kinetic step size (m=3.9+/-0.5 bp). The RecBC DNA helicase (without the RecD subunit) does not initiate unwinding efficiently from a blunt DNA end. However, RecBC does initiate well from a DNA end possessing noncomplementary twin 5'-(dT)(6) and 3'-(dT)(6) tails, and unwinding can be described by a simple uniform n-step sequential scheme, without the need for the additional k(C) initiation steps, with a similar kinetic step size (m=4.4+/-1.7 bp) and unwinding rate

  7. Gallium nitrate inhibits alkaline phosphatase activity in a differentiating mesenchymal cell culture.

    PubMed

    Boskey, A L; Ziecheck, W; Guidon, P; Doty, S B

    1993-02-01

    The effect of gallium nitrate on alkaline phosphatase activity in a differentiating chick limb-bud mesenchymal cell culture was monitored in order to gain insight into the observation that rachitic rats treated with gallium nitrate failed to show the expected increase in serum alkaline phosphatase activity. Cultures maintained in media containing 15 microM gallium nitrate showed drastically decreased alkaline phosphatase activities in the absence of significant alterations in total protein synthesis and DNA content. However, addition of 15 microM gallium nitrate to cultures 18 h before assay for alkaline phosphatase activity had little effect. At the light microscopic and electron microscopic level, gallium-treated cultures differed morphologically from gallium-free cultures: with gallium present, there were fewer hypertrophic chondrocytes and cartilage nodules were flatter and further apart. Because of altered morphology, staining with an antibody against chick cartilage alkaline phosphatase appeared less extensive; however, all nodules stained equivalently relative to gallium-free controls. Histochemical staining for alkaline phosphatase activity was negative in gallium-treated cultures, demonstrating that the alkaline phosphatase protein present was not active. The defective alkaline phosphatase activity in cultures maintained in the presence of gallium was also evidenced when cultures were supplemented with the alkaline phosphatase substrate, beta-glycerophosphate (beta GP). The data presented suggest that gallium inhibits alkaline phosphatase activity in this culture system and that gallium causes alterations in the differentiation of mesenchymal cells into hypertrophic chondrocytes.

  8. Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases.

    PubMed

    Pinto, Cosimo; Kasaciunaite, Kristina; Seidel, Ralf; Cejka, Petr

    2016-09-09

    Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA.

  9. The DNA unwinding element binding protein DUE-B interacts with Cdc45 in preinitiation complex formation.

    PubMed

    Chowdhury, A; Liu, G; Kemp, M; Chen, X; Katrangi, N; Myers, S; Ghosh, M; Yao, J; Gao, Y; Bubulya, P; Leffak, M

    2010-03-01

    Template unwinding during DNA replication initiation requires the loading of the MCM helicase activator Cdc45 at replication origins. We show that Cdc45 interacts with the DNA unwinding element (DUE) binding protein DUE-B and that these proteins localize to the DUEs of active replication origins. DUE-B and Cdc45 are not bound at the inactive c-myc replicator in the absence of a functional DUE or at the recently identified ataxin 10 (ATX10) origin, which is silent before disease-related (ATTCT)(n) repeat length expansion of its DUE sequence, despite the presence of the origin recognition complex (ORC) and MCM proteins at these origins. Addition of a heterologous DUE to the ectopic c-myc origin, or expansion of the ATX10 DUE, leads to origin activation, DUE-B binding, and Cdc45 binding. DUE-B, Cdc45, and topoisomerase IIbeta binding protein 1 (TopBP1) form complexes in cell extracts and when expressed from baculovirus vectors. During replication in Xenopus egg extracts, DUE-B and Cdc45 bind to chromatin with similar kinetics, and DUE-B immunodepletion blocks replication and the loading of Cdc45 and a fraction of TopBP1. The coordinated binding of DUE-B and Cdc45 to origins and the physical interactions of DUE-B, Cdc45, and TopBP1 suggest that complexes of these proteins are necessary for replication initiation.

  10. Characterization of the DNA-unwinding activity of human RECQ1, a helicase specifically stimulated by human replication protein A.

    PubMed

    Cui, Sheng; Klima, Raffaella; Ochem, Alex; Arosio, Daniele; Falaschi, Arturo; Vindigni, Alessandro

    2003-01-17

    The RecQ helicases are involved in several aspects of DNA metabolism. Five members of the RecQ family have been found in humans, but only two of them have been carefully characterized, BLM and WRN. In this work, we describe the enzymatic characterization of RECQ1. The helicase has 3' to 5' polarity, cannot start the unwinding from a blunt-ended terminus, and needs a 3'-single-stranded DNA tail longer than 10 nucleotides to open the substrate. However, it was also able to unwind a blunt-ended duplex DNA with a "bubble" of 25 nucleotides in the middle, as previously observed for WRN and BLM. We show that only short DNA duplexes (<30 bp) can be unwound by RECQ1 alone, but the addition of human replication protein A (hRPA) increases the processivity of the enzyme (>100 bp). Our studies done with Escherichia coli single-strand binding protein (SSB) indicate that the helicase activity of RECQ1 is specifically stimulated by hRPA. This finding suggests that RECQ1 and hRPA may interact also in vivo and function together in DNA metabolism. Comparison of the present results with previous studies on WRN and BLM provides novel insight into the role of the N- and C-terminal domains of these helicases in determining their substrate specificity and in their interaction with hRPA.

  11. Structure and function of the c-myc DNA-unwinding element-binding protein DUE-B.

    PubMed

    Kemp, Michael; Bae, Brian; Yu, John Paul; Ghosh, Maloy; Leffak, Michael; Nair, Satish K

    2007-04-06

    Local zones of easily unwound DNA are characteristic of prokaryotic and eukaryotic replication origins. The DNA-unwinding element of the human c-myc replication origin is essential for replicator activity and is a target of the DNA-unwinding element-binding protein DUE-B in vivo. We present here the 2.0A crystal structure of DUE-B and complementary biochemical characterization of its biological activity. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA-editing enzymes. Consistent with this structural homology, the N-terminal core of DUE-B is shown to display both D-aminoacyl-tRNA deacylase activity and ATPase activity. We further demonstrate that the C-terminal portion of the enzyme is disordered and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA.

  12. Conserved XPB Core Structure and Motifs for DNA Unwinding:Implications for Pathway Selection of Transcription or ExcisionRepair

    SciTech Connect

    Fan, Li; Arval, Andrew S.; Cooper, Priscilla K.; Iwai, Shigenori; Hanaoka, Fumio; Tainer, John A.

    2005-04-01

    The human xeroderma pigmentosum group B (XPB) helicase is essential for transcription, nucleotide excision repair, and TFIIH functional assembly. Here, we determined crystal structures of an Archaeoglobus fulgidus XPB homolog (AfXPB) that characterize two RecA-like XPB helicase domains and discover a DNA damage recognition domain (DRD), a unique RED motif, a flexible thumb motif (ThM), and implied conformational changes within a conserved functional core. RED motif mutations dramatically reduce helicase activity, and the DRD and ThM, which flank the RED motif, appear structurally as well as functionally analogous to the MutS mismatch recognition and DNA polymerase thumb domains. Substrate specificity is altered by DNA damage, such that AfXPB unwinds dsDNA with 3' extensions, but not blunt-ended dsDNA, unless it contains a lesion, as shown for CPD or (6-4) photoproducts. Together, these results provide an unexpected mechanism of DNA unwinding with Implications for XPB damage verification in nucleotide excision repair.

  13. Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases

    PubMed Central

    Pinto, Cosimo; Kasaciunaite, Kristina; Seidel, Ralf; Cejka, Petr

    2016-01-01

    Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA. DOI: http://dx.doi.org/10.7554/eLife.18574.001 PMID:27612385

  14. The mechanism of adenosine to inosine conversion by the double-stranded RNA unwinding/modifying activity: A high-performance liquid chromatography-mass spectrometry analysis

    SciTech Connect

    Polson, A.G.; Crain, P.F.; Pomerantz, S.C.; McCloskey, J.A.; Bass, B.L. )

    1991-12-10

    The authors have used directly combined high-performance liquid chromatography-mass spectrometry (LC/MS) to examine the mechanism of the reaction catalyzed by the double-stranded RNA unwinding/modifying activity. A double-stranded RNA substrate in which all adenosines were uniformly labeled with {sup 13}C was synthesized. An LC/MS analysis of the nucleoside products from the modified, labeled substrate confirmed that adenosine is modified to inosine during the unwinding/modifying reaction. Most importantly, they found that no carbons are exchanged during the reaction. By including H{sub 2} {sup 18}O in the reaction, they showed that water serves efficiently as the oxygen donor in vitro. These results are consistent with a hydrolytic deamination mechanism and rule out a base replacement mechanism. Although the double-stranded RNA unwinding/modifying activity appears to utilize a catalytic mechanism similar to that of adenosine deaminase, coformycin, a transition-state analogue, will not inhibit the unwinding/modifying activity.

  15. Identification of human pulmonary alkaline phosphatase isoenzymes.

    PubMed

    Capelli, A; Cerutti, C G; Lusuardi, M; Donner, C F

    1997-04-01

    An increase of alkaline phosphatase (ALP) activity has been observed in the bronchoalveolar lavage fluid (BALF) of patients affected by pulmonary fibrosis in chronic interstitial lung disorders. To characterize the ALP isoenzymes in such cases, we used gel filtration, agarose gel electrophoresis, heat and amino acid inhibition assays, wheat-germ agglutinin (WGA) precipitation, and an immunoassay specific for the bone-isoform of ALP. Only one anodic band representing a high-molecular-weight isoform of ALP (Mr approximately 2,000 kDa) was observed on electrophoresis of BALF. The inhibition assay results were consistent for a tissue-nonspecific isoenzyme sensitive to a temperature of 56 degrees C (71.9 +/- 2.5% inhibition) and to homoarginine (65.7 +/- 1.9%), and resistant to L-phenylalanine and L-leucine. Less than 13% of ALP activity was heat-stable. After incubation of BALF specimens with glycosyl-phosphatidylinositol-phospholipase D plus Nonidet P-40, or with phosphatidylinositol-phospholipase C alone, an electrophoretic cathodic band (Mr approximately 220 kDa) appeared near the bone band of a standard serum. With the WGA assay, 84.4 +/- 3.3% of ALP precipitated and the band disappeared. After immunoassay for the bone isoform, a mean of less than 5% enzyme activity was measured. We conclude that the ALP found in BALF is a pulmonary isoform of a tissue nonspecific isoenzyme.

  16. Characterization of Human Bone Alkaline Phosphatase in Pichia Pastoris

    NASA Technical Reports Server (NTRS)

    Malone, Christine C.; Ciszak, Eva; Karr, Laurel J.

    1999-01-01

    A soluble form of human bone alkaline phosphatase has been expressed in a recombinant strain of the methylotrophic yeast Pichia pastoris. We constructed a plasmid containing cDNA encoding for human bone alkaline phosphatase, with the hydrophobic carboxyl terminal portion deleted. Alkaline phosphatase was secreted into the medium to a level of 32mg/L when cultured in shake flasks, and enzyme activity was 12U/mg, as measured by a spectrophotometric assay. By conversion to a fermentation system, a yield of 880mg/L has been achieved with an enzyme activity of 968U/mg. By gel electrophoresis analysis, it appears that greater than 50% of the total protein in the fermentation media is alkaline phosphatase. Although purification procedures are not yet completely optimized, they are expected to include filtration, ion exchange and affinity chromatography. Our presentation will focus on the purification and crystallization results up to the time of the conference. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  17. The alkaline and alkaline-carbonatite magmatism from Southern Brazil

    NASA Astrophysics Data System (ADS)

    Ruberti, E.; Gomes, C. D. B.; Comin-Chiaramonti, P.

    2015-12-01

    Early to Late Cretaceous lasting to Paleocene alkaline magmatism from southern Brazil is found associated with major extensional structural features in and around the Paraná Basin and grouped into various provinces on the basis of several data. Magmatism is variable in size, mode of occurrence and composition. The alkaline rocks are dominantly potassic, a few occurrences showing sodic affinity. The more abundant silicate rocks are evolved undersaturated to saturated in silica syenites, displaying large variation in igneous forms. Less evolved types are restricted to subvolcanic environments and outcrops of effusive suites occur rarely. Cumulatic mafic and ultramafic rock types are very common, particularly in the alkali-carbonatitic complexes. Carbonatite bodies are represented by Ca-carbonatites and Mg-carbonatites and more scarcely by Fe-carbonatites. Available radiometric ages for the alkaline rocks fit on three main chronological groups: around 130 Ma, subcoveal with the Early Cretaceous flood tholeiites of the Paraná Basin, 100-110 Ma and 80-90 Ma (Late Cretaceous). The alkaline magmatism also extends into Paleocene times, as indicated by ages from some volcanic lavas. Geochemically, alkaline potassic and sodic rock types are distinguished by their negative and positive Nb-Ta anomalies, respectively. Negative spikes in Nb-Ta are also a feature common to the associated tholeiitic rocks. Sr-Nd-Pb systematics confirm the contribution of both HIMU and EMI mantle components in the formation of the alkaline rocks. Notably, Early and Late Cretaceous carbonatites have the same isotopic Sr-Nd initial ratios of the associated alkaline rocks. C-O isotopic Sr-Nd isotopic ratios indicate typical mantle signature for some carbonatites and the influence of post-magmatic processes in others. Immiscibility of liquids of phonolitic composition, derived from mafic alkaline parental magmas, has been responsible for the origin of the carbonatites. Close association of alkaline

  18. Mycobacterium tuberculosis DinG is a structure-specific helicase that unwinds G4 DNA: implications for targeting G4 DNA as a novel therapeutic approach.

    PubMed

    Thakur, Roshan Singh; Desingu, Ambika; Basavaraju, Shivakumar; Subramanya, Shreelakshmi; Rao, Desirazu N; Nagaraju, Ganesh

    2014-09-05

    The significance of G-quadruplexes and the helicases that resolve G4 structures in prokaryotes is poorly understood. The Mycobacterium tuberculosis genome is GC-rich and contains >10,000 sequences that have the potential to form G4 structures. In Escherichia coli, RecQ helicase unwinds G4 structures. However, RecQ is absent in M. tuberculosis, and the helicase that participates in G4 resolution in M. tuberculosis is obscure. Here, we show that M. tuberculosis DinG (MtDinG) exhibits high affinity for ssDNA and ssDNA translocation with a 5' → 3' polarity. Interestingly, MtDinG unwinds overhangs, flap structures, and forked duplexes but fails to unwind linear duplex DNA. Our data with DNase I footprinting provide mechanistic insights and suggest that MtDinG is a 5' → 3' polarity helicase. Notably, in contrast to E. coli DinG, MtDinG catalyzes unwinding of replication fork and Holliday junction structures. Strikingly, we find that MtDinG resolves intermolecular G4 structures. These data suggest that MtDinG is a multifunctional structure-specific helicase that unwinds model structures of DNA replication, repair, and recombination as well as G4 structures. We finally demonstrate that promoter sequences of M. tuberculosis PE_PGRS2, mce1R, and moeB1 genes contain G4 structures, implying that G4 structures may regulate gene expression in M. tuberculosis. We discuss these data and implicate targeting G4 structures and DinG helicase in M. tuberculosis could be a novel therapeutic strategy for culminating the infection with this pathogen.

  19. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  20. Ferroplasma acidarmanus RPA2 facilitates efficient unwinding of forked DNA substrates by monomers of FacXPD helicase.

    PubMed

    Pugh, Robert A; Lin, Yuyen; Eller, Chelcie; Leesley, Haley; Cann, Isaac K O; Spies, Maria

    2008-11-28

    The strand-separation activity that is important for many cellular DNA processing machineries is provided by DNA helicases. In order to understand the physiological properties of a helicase acting in the context of its macromolecular machinery, it is imperative to identify the proteins that interact with the enzyme and to analyze how these proteins affect its helicase activities. The archaeal Rad3 helicase XPD (xeroderma pigmentosum group D protein) from Ferroplasma acidarmanus (FacXPD) is a superfamily II 5'-->3' DNA helicase. Similar to its mammalian homolog working as an integral part of the transcription factor IIH complex, FacXPD may play an important role in nucleotide excision repair (NER) and transcription initiation. Interaction between FacXPD and other archaeal NER proteins likely modulates their respective activities. Replication protein A (RPA), a single-stranded DNA (ssDNA)-binding protein, is one of the NER proteins that functionally interact with the human transcription factor IIH complex. There are two RPA proteins in F. acidarmanus: FacRPA1, a homodimer of two monomers consisting of two oligonucleotide/oligosaccharide binding folds, and FacRPA2, a monomer containing a single oligonucleotide/oligosaccharide binding fold. In this study, we analyzed the effect of these ssDNA-binding proteins on FacXPD helicase activity. We found that FacRPA2 stimulates DNA unwinding by FacXPD helicase through a novel mechanism by providing a helix-destabilizing function. In contrast, FacRPA1 fails to stimulate helicase activity to the same extent as FacRPA2 and competes with FacXPD for binding to the ssDNA-double-stranded DNA junction. We conclude that the FacRPA2-coated fork is a preferred and likely physiological substrate that a monomer of FacXPD can unwind with a processivity sufficient for expansion of the NER or transcription bubble. We also suggest that duplex melting by a cognate ssDNA-binding protein coordinated with translocation by a helicase may represent

  1. Visualization of local DNA unwinding by Mre11/Rad50/Nbs1 using single-molecule FRET.

    PubMed

    Cannon, Brian; Kuhnlein, Jeffrey; Yang, Soo-Hyun; Cheng, Anita; Schindler, Detlev; Stark, Jeremy M; Russell, Rick; Paull, Tanya T

    2013-11-19

    The Mre11/Rad50/Nbs1 (MRN) complex initiates and coordinates DNA repair and signaling events at double-strand breaks. The interaction between MRN and DNA ends is critical for the recruitment of DNA-processing enzymes, end tethering, and activation of the ATM protein kinase. Here we visualized MRN binding to duplex DNA molecules using single-molecule FRET, and found that MRN unwinds 15-20 base pairs at the end of the duplex, holding the branched structure open for minutes at a time in an ATP-dependent reaction. A Rad50 catalytic domain mutant that is specifically deficient in this ATP-dependent opening is impaired in DNA end resection in vitro and in resection-dependent repair of breaks in human cells, demonstrating the importance of MRN-generated single strands in the repair of DNA breaks.

  2. Processing of DNA structures via DNA unwinding and branch migration by the S. cerevisiae Mph1 protein.

    PubMed

    Zheng, Xiao-Feng; Prakash, Rohit; Saro, Dorina; Longerich, Simonne; Niu, Hengyao; Sung, Patrick

    2011-10-10

    The budding yeast Mph1 protein, the putative ortholog of human FANCM, possesses a 3' to 5' DNA helicase activity and is capable of disrupting the D-loop structure to suppress chromosome arm crossovers in mitotic homologous recombination. Similar to FANCM, genetic studies have implicated Mph1 in DNA replication fork repair. Consistent with this genetic finding, we show here that Mph1 is able to mediate replication fork reversal, and to process the Holliday junction via DNA branch migration. Moreover, Mph1 unwinds 3' and 5' DNA Flap structures that bear key features of the D-loop. These biochemical results not only provide validation for a role of Mph1 in the repair of damaged replication forks, but they also offer mechanistic insights as to its ability to efficiently disrupt the D-loop intermediate.

  3. Human coronavirus 229E nonstructural protein 13: characterization of duplex-unwinding, nucleoside triphosphatase, and RNA 5'-triphosphatase activities.

    PubMed

    Ivanov, Konstantin A; Ziebuhr, John

    2004-07-01

    The human coronavirus 229E (HCoV-229E) replicase gene-encoded nonstructural protein 13 (nsp13) contains an N-terminal zinc-binding domain and a C-terminal superfamily 1 helicase domain. A histidine-tagged form of nsp13, which was expressed in insect cells and purified, is reported to unwind efficiently both partial-duplex RNA and DNA of up to several hundred base pairs. Characterization of the nsp13-associated nucleoside triphosphatase (NTPase) activities revealed that all natural ribonucleotides and nucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed most efficiently. Using the NTPase active site, HCoV-229E nsp13 also mediates RNA 5'-triphosphatase activity, which may be involved in the capping of viral RNAs.

  4. Photo-induced morphological winding and unwinding motion of nanoscrolls composed of niobate nanosheets with a polyfluoroalkyl azobenzene derivative

    NASA Astrophysics Data System (ADS)

    Nabetani, Yu; Takamura, Hazuki; Uchikoshi, Akino; Hassan, Syed Zahid; Shimada, Tetsuya; Takagi, Shinsuke; Tachibana, Hiroshi; Masui, Dai; Tong, Zhiwei; Inoue, Haruo

    2016-06-01

    Photo-responsive nanoscrolls can be successfully fabricated by mixing a polyfluoroalkyl azobenzene derivative and a niobate nanosheet, which is exfoliated from potassium hexaniobate. In this study, we have found that the photo-responsive nanoscroll shows a morphological motion of winding and unwinding, which is basically due to the nanosheet sliding within the nanoscroll, by efficient photo-isomerization reactions of the intercalated azobenzene in addition to the interlayer distance change of the nanoscrolls. The relative nanosheet sliding of the nanoscroll is estimated to be ca. 280 nm from the AFM morphology analysis. The distance of the sliding motion is over 20 times that of the averaged nanosheet sliding in the azobenzene/niobate hybrid film reported previously. Photo-responsive nanoscrolls can be expected to be novel photo-activated actuators and artificial muscle model materials.Photo-responsive nanoscrolls can be successfully fabricated by mixing a polyfluoroalkyl azobenzene derivative and a niobate nanosheet, which is exfoliated from potassium hexaniobate. In this study, we have found that the photo-responsive nanoscroll shows a morphological motion of winding and unwinding, which is basically due to the nanosheet sliding within the nanoscroll, by efficient photo-isomerization reactions of the intercalated azobenzene in addition to the interlayer distance change of the nanoscrolls. The relative nanosheet sliding of the nanoscroll is estimated to be ca. 280 nm from the AFM morphology analysis. The distance of the sliding motion is over 20 times that of the averaged nanosheet sliding in the azobenzene/niobate hybrid film reported previously. Photo-responsive nanoscrolls can be expected to be novel photo-activated actuators and artificial muscle model materials. Electronic supplementary information (ESI) available: Fig. S1. Photo-isomerization reaction of nanoscrolls. See DOI: 10.1039/c6nr02177h

  5. Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding.

    PubMed Central

    Rinken, R; Thomas, B; Wackernagel, W

    1992-01-01

    Infection of Escherichia coli with phage T4 gene 2am was used to transport 3H-labeled linear duplex DNA into cells to follow its degradation in relation to the cellular genotype. In wild-type cells, 49% of the DNA was made acid soluble within 60 min; in recB or recC cells, only about 5% of the DNA was made acid soluble. Remarkably, in recD cells about 25% of the DNA was rendered acid soluble. The DNA degradation in recD cells depended on intact recB and recC genes. The degradation in recD cells was largely decreased by mutations in recJ (which eliminates the 5' single-strand-specific exonuclease coded by this gene) or xonA (which abolishes the 3' single-strand-specific exonuclease I). In a recD recJ xonA triple mutant, the degradation of linear duplex DNA was roughly at the level of a recB mutant. Results similar to those with the set of recD strains were also obtained with a recC++ mutant (in which the RecD protein is intact but does not function) and its recJ, xonA, and recJ xonA derivatives. The observations provide evidence for a recBC-dependent DNA-unwinding activity that renders unwound DNA susceptible to exonucleolytic degradation. It is proposed that the DNA-unwinding activity causes the efficient recombination, DNA repair, and SOS induction (after application of nalidixic acid) in recD mutants. The RecBC helicase indirectly detected here may have a central function in Chi-dependent recombination and in the recombinational repair of double-strand breaks by the RecBCD pathway. PMID:1322885

  6. KINEMATICS AND FINE STRUCTURE OF AN UNWINDING POLAR JET OBSERVED BY THE SOLAR DYNAMIC OBSERVATORY/ATMOSPHERIC IMAGING ASSEMBLY

    SciTech Connect

    Shen Yuandeng; Liu Yu; Ibrahim, Ahmed

    2011-07-10

    We present an observational study of the kinematics and fine structure of an unwinding polar jet, with high temporal and spatial observations taken by the Atmospheric Imaging Assembly on board the Solar Dynamic Observatory and the Solar Magnetic Activity Research Telescope. During the rising period, the shape of the jet resembled a cylinder with helical structures on the surface, while the mass of the jet was mainly distributed on the cylinder's shell. In the radial direction, the jet expanded successively at its western side and underwent three distinct phases: the gradually expanding phase, the fast expanding phase, and the steady phase. Each phase lasted for about 12 minutes. The angular speed of the unwinding motion of the jet and the twist transferred into the outer corona during the eruption are estimated to be 11.1 x 10{sup -3} rad s{sup -1} (period = 564 s) and 1.17-2.55 turns (or 2.34-5.1{pi}), respectively. On the other hand, by calculating the azimuthal component of the magnetic field in the jet and comparing the free energy stored in the non-potential magnetic field with the jet's total energy, we find that the non-potential magnetic field in the jet is enough to supply the energy for the ejection. These new observational results strongly support the scenario that the jets are driven by the magnetic twist, which is stored in the twisted closed field of a small bipole, and released through magnetic reconnection between the bipole and its ambient open field.

  7. Zinc electrode in alkaline electrolyte

    SciTech Connect

    McBreen, J.

    1995-12-31

    The zinc electrode in alkaline electrolyte is unusual in that supersaturated zincate solutions can form during discharge and spongy or mossy zinc deposits can form on charge at low overvoltages. The effect of additives on regular pasted ZnO electrodes and calcium zincate electrodes is discussed. The paper also reports on in situ x-ray absorption (XAS) results on mossy zinc deposits.

  8. Temperature dependence of the absorbance of alkaline solutions of 4-nitrophenyl phosphate--a potential source of error in the measurement of alkaline phosphatase activity.

    PubMed

    Burtis, C A; Seibert, L E; Baird, M A; Sampson, E J

    1977-09-01

    The absorbance of an alkaline solution of 4-nitrophenyl phosphate is a function of temperature. Quantitative evaluation of this phenomenon indicates that it (a) depends on the concentration of the compound and is independent of source, buffer concentration, and pH above 9.0; (b) is reversible; (c) is not a result of alkaline hydrolysis or 4-nitrophenol contamination; and (d) correlates with a temperature-induced shift of its absorbance spectrum. The phenomenon may represent a potential analytical problem in methods for alkaline phosphatase in which this compound is the substrate. If thermal equilibrium is not reached and maintained during an alkaline phosphatase assay, the thermochromic response will be included in the measured rate. The magnitude of this error depends on the thermal response and control characteristics of each particular instrument and the reaction conditions under which such an analysis is performed.

  9. Hexosaminidase assays.

    PubMed

    Wendeler, Michaela; Sandhoff, Konrad

    2009-11-01

    beta-Hexosaminidases (EC 3.2.1.52) are lysosomal enzymes that remove terminal beta-glycosidically bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited beta-hexosaminidase deficiency. More recently, aberrant hexosaminidase levels have also been found to be associated with a variety of inflammatory diseases. Apart from patient testing and carrier screening, practical in vitro assays are indispensable for the characterization of knock-out mice with potentially altered hexosaminidase activities, for detailed structure-function studies aimed at elucidating the enzymatic mechanism, and to characterize newly described enzyme variants from other organisms. The purpose of this article is to discuss convenient hexosaminidase assay procedures for these and other applications, using fluorogenic or chromogenic artificial substrates as well as the physiological glycolipid substrate GM2. Attempts are also made to provide an overview of less commonly used alternative techniques and to introduce recent developments enabling high-throughput screening for enzyme inhibitors.

  10. ATP-dependent unwinding of U4/U6 snRNAs by the Brr2 helicase requires the C terminus of Prp8.

    PubMed

    Maeder, Corina; Kutach, Alan K; Guthrie, Christine

    2009-01-01

    The spliceosome is a highly dynamic machine requiring multiple RNA-dependent ATPases of the DExD/H-box family. A fundamental unanswered question is how their activities are regulated. Brr2 function is necessary for unwinding the U4/U6 duplex, a step essential for catalytic activation of the spliceosome. Here we show that Brr2-dependent dissociation of U4/U6 snRNAs in vitro is activated by a fragment from the C terminus of the U5 snRNP protein Prp8. In contrast to its helicase-stimulating activity, this fragment inhibits Brr2 U4/U6-dependent ATPase activity. Notably, U4/U6 unwinding activity is not stimulated by fragments carrying alleles of prp8 that in humans confers an autosomal dominant form of retinitis pigmentosa. Because Brr2 activity must be restricted to prevent premature catalytic activation, our results have important implications for fidelity maintenance in the spliceosome.

  11. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  12. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

  13. A single-molecule view of the assembly pathway, subunit stoichiometry, and unwinding activity of the bacteriophage T4 primosome (helicase-primase) complex.

    PubMed

    Lee, Wonbae; Jose, Davis; Phelps, Carey; Marcus, Andrew H; von Hippel, Peter H

    2013-05-07

    Single-molecule fluorescence resonance energy transfer (smFRET) methods were used to study the assembly pathway and DNA unwinding activity of the bacteriophage T4 helicase-primase (primosome) complex. The helicase substrates used were surface-immobilized model DNA replication forks "internally" labeled in the duplex region with opposed donor/acceptor (iCy3/iCy5) chromophore pairs in the lagging and leading strands. The time dependence of the smFRET signals was monitored during the unwinding process, and helicase rates and processivities were measured as a function of GTP concentration. This smFRET approach was also used to investigate the subunit stoichiometry of the primosome and the assembly pathway required to form functional and fully active primosome-DNA complexes. We confirmed that gp41 helicase monomer subunits form stable hexameric helicases in the presence of GTP and that the resulting (gp41)(6) complexes bind only weakly at DNA fork junctions. The addition of a single subunit of gp61 primase stabilized the resulting primosome complex at the fork and resulted in fully active and processive primosome helicases with gp41:gp61 subunit ratios of 6:1, while higher and lower subunit ratios substantially reduced the primosome unwinding activity. The use of alternative assembly pathways resulted in a loss of helicase activity and the formation of metastable DNA-protein aggregates, which were easily detected in our smFRET experiments as intense light-scattering foci. These single-molecule experiments provide a detailed real-time visualization of the assembly pathway and duplex DNA unwinding activity of the T4 primosome and are consistent with more indirect equilibrium and steady state results obtained in bulk solution studies.

  14. Double strand break unwinding and resection by the mycobacterial helicase-nuclease AdnAB in the presence of single strand DNA-binding protein (SSB).

    PubMed

    Unciuleac, Mihaela-Carmen; Shuman, Stewart

    2010-11-05

    Mycobacterial AdnAB is a heterodimeric DNA helicase-nuclease and 3' to 5' DNA translocase implicated in the repair of double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Inclusion of mycobacterial single strand DNA-binding protein (SSB) in reactions containing linear plasmid dsDNA allowed us to study the AdnAB helicase under conditions in which the unwound single strands are coated by SSB and thereby prevented from reannealing or promoting ongoing ATP hydrolysis. We found that the AdnAB motor catalyzed processive unwinding of 2.7-11.2-kbp linear duplex DNAs at a rate of ∼250 bp s(-1), while hydrolyzing ∼5 ATPs per bp unwound. Crippling the AdnA phosphohydrolase active site did not affect the rate of unwinding but lowered energy consumption slightly, to ∼4.2 ATPs bp(-1). Mutation of the AdnB phosphohydrolase abolished duplex unwinding, consistent with a model in which the "leading" AdnB motor propagates a Y-fork by translocation along the 3' DNA strand, ahead of the "lagging" AdnA motor domain. By tracking the resection of the 5' and 3' strands at the DSB ends, we illuminated a division of labor among the AdnA and AdnB nuclease modules during dsDNA unwinding, whereby the AdnA nuclease processes the unwound 5' strand to liberate a short oligonucleotide product, and the AdnB nuclease incises the 3' strand on which the motor translocates. These results extend our understanding of presynaptic DSB processing by AdnAB and engender instructive comparisons with the RecBCD and AddAB clades of bacterial helicase-nuclease machines.

  15. When core competence is not enough: functional interplay of the DEAD-box helicase core with ancillary domains and auxiliary factors in RNA binding and unwinding.

    PubMed

    Rudolph, Markus G; Klostermeier, Dagmar

    2015-08-01

    DEAD-box helicases catalyze RNA duplex unwinding in an ATP-dependent reaction. Members of the DEAD-box helicase family consist of a common helicase core formed by two RecA-like domains. According to the current mechanistic model for DEAD-box mediated RNA unwinding, binding of RNA and ATP triggers a conformational change of the helicase core, and leads to formation of a compact, closed state. In the closed conformation, the two parts of the active site for ATP hydrolysis and of the RNA binding site, residing on the two RecA domains, become aligned. Closing of the helicase core is coupled to a deformation of the RNA backbone and destabilization of the RNA duplex, allowing for dissociation of one of the strands. The second strand remains bound to the helicase core until ATP hydrolysis and product release lead to re-opening of the core. The concomitant disruption of the RNA binding site causes dissociation of the second strand. The activity of the helicase core can be modulated by interaction partners, and by flanking N- and C-terminal domains. A number of C-terminal flanking regions have been implicated in RNA binding: RNA recognition motifs (RRM) typically mediate sequence-specific RNA binding, whereas positively charged, unstructured regions provide binding sites for structured RNA, without sequence-specificity. Interaction partners modulate RNA binding to the core, or bind to RNA regions emanating from the core. The functional interplay of the helicase core and ancillary domains or interaction partners in RNA binding and unwinding is not entirely understood. This review summarizes our current knowledge on RNA binding to the DEAD-box helicase core and the roles of ancillary domains and interaction partners in RNA binding and unwinding by DEAD-box proteins.

  16. Autosomal-dominant retinitis pigmentosa caused by a mutation in SNRNP200, a gene required for unwinding of U4/U6 snRNAs.

    PubMed

    Zhao, Chen; Bellur, Deepti L; Lu, Shasha; Zhao, Feng; Grassi, Michael A; Bowne, Sara J; Sullivan, Lori S; Daiger, Stephen P; Chen, Li Jia; Pang, Chi Pui; Zhao, Kanxing; Staley, Jonathan P; Larsson, Catharina

    2009-11-01

    Mutations in genes associated with the U4/U6-U5 small nuclear ribonucleoprotein (snRNP) complex of the spliceosome are implicated in autosomal-dominant retinitis pigmentosa (adRP), a group of progressive retinal degenerative disorders leading to visual impairment, loss of visual field, and even blindness. We recently assigned a locus (RP33) for adRP to 2cen-q12.1, a region that harbors the SNRNP200 gene encoding hBrr2, another U4/U6-U5 snRNP component that is required for unwinding of U4/U6 snRNAs during spliceosome activation and for disassembly of the spliceosome. Here, we report the identification of a missense mutation, c.3260C>T (p.S1087L), in exon 25 of the SNRNP200 gene in an RP33-linked family. The c.3260C>T substitution showed complete cosegregation with the retinitis pigmentosa (RP) phenotype over four generations, but was absent in a panel of 400 controls. The p.S1087L mutation and p.R1090L, another adRP-associated allele, reside in the "ratchet" helix of the first of two Sec63 domains implicated in the directionality and processivity of nucleic acid unwinding. Indeed, marked defects in U4/U6 unwinding, but not U4/U6-U5 snRNP assembly, were observed in budding yeast for the analogous mutations (N1104L and R1107L) of the corresponding Brr2p residues. The linkage of hBrr2 to adRP suggests that the mechanism of pathogenesis for splicing-factor-related RP may fundamentally derive from a defect in hBrr2-dependent RNA unwinding and a consequent defect in spliceosome activation.

  17. Neisseria gonorrhoeae RecQ helicase HRDC domains are essential for efficient binding and unwinding of the pilE guanine quartet structure required for pilin antigenic variation.

    PubMed

    Cahoon, Laty A; Manthei, Kelly A; Rotman, Ella; Keck, James L; Seifert, H Steven

    2013-05-01

    The strict human pathogen Neisseria gonorrhoeae utilizes homologous recombination to antigenically vary the pilus, thus evading the host immune response. High-frequency gene conversion reactions between many silent pilin loci and the expressed pilin locus (pilE) allow for numerous pilus variants per strain to be produced from a single strain. For pilin antigenic variation (Av) to occur, a guanine quartet (G4) structure must form upstream of pilE. The RecQ helicase is one of several recombination or repair enzymes required for efficient levels of pilin Av, and RecQ family members have been shown to bind to and unwind G4 structures. Additionally, the vast majority of RecQ helicase family members encode one "helicase and RNase D C-terminal" (HRDC) domain, whereas the N. gonorrhoeae RecQ helicase gene encodes three HRDC domains, which are critical for pilin Av. Here, we confirm that deletion of RecQ HRDC domains 2 and 3 causes a decrease in the frequency of pilin Av comparable to that obtained with a functional knockout. We demonstrate that the N. gonorrhoeae RecQ helicase can bind and unwind the pilE G4 structure. Deletion of the RecQ HRDC domains 2 and 3 resulted in a decrease in G4 structure binding and unwinding. These data suggest that the decrease in pilin Av observed in the RecQ HRDC domain 2 and 3 deletion mutant is a result of the enzyme's inability to efficiently bind and unwind the pilE G4 structure.

  18. Alkaline phosphatase revisited: hydrolysis of alkyl phosphates.

    PubMed

    O'Brien, Patrick J; Herschlag, Daniel

    2002-03-05

    Escherichia coli alkaline phosphatase (AP) is the prototypical two metal ion catalyst with two divalent zinc ions bound approximately 4 A apart in the active site. Studies spanning half a century have elucidated many structural and mechanistic features of this enzyme, rendering it an attractive model for investigating the potent catalytic power of bimetallic centers. Unfortunately, fundamental mechanistic features have been obscured by limitations with the standard assays. These assays generate concentrations of inorganic phosphate (P(i)) in excess of its inhibition constant (K(i) approximately 1 muM). This tight binding by P(i) has affected the majority of published kinetic constants. Furthermore, binding limits k(cat)/K(m) for reaction of p-nitrophenyl phosphate, the most commonly employed substrate. We describe a sensitive (32)P-based assay for hydrolysis of alkyl phosphates that avoids the complication of product inhibition. We have revisited basic mechanistic features of AP with these alkyl phosphate substrates. The results suggest that the chemical step for phosphorylation of the enzyme limits k(cat)/K(m). The pH-rate profile and additional results suggest that the serine nucleophile is active in its anionic form and has a pK(a) of < or = 5.5 in the free enzyme. An inactivating pK(a) of 8.0 is observed for binding of both substrates and inhibitors, and we suggest that this corresponds to ionization of a zinc-coordinated water molecule. Counter to previous suggestions, inorganic phosphate dianion appears to bind to the highly charged AP active site at least as strongly as the trianion. The dependence of k(cat)/K(m) on the pK(a) of the leaving group follows a Brønsted correlation with a slope of beta(lg) = -0.85 +/- 0.1, differing substantially from the previously reported value of -0.2 obtained from data with a less sensitive assay. This steep leaving group dependence is consistent with a largely dissociative transition state for AP-catalyzed hydrolysis of

  19. RecQ4 promotes the conversion of the pre-initiation complex at a site-specific origin for DNA unwinding in Xenopus egg extracts.

    PubMed

    Sanuki, Yosuke; Kubota, Yumiko; Kanemaki, Masato T; Takahashi, Tatsuro S; Mimura, Satoru; Takisawa, Haruhiko

    2015-01-01

    Eukaryotic DNA replication is initiated through stepwise assembly of evolutionarily conserved replication proteins onto replication origins, but how the origin DNA is unwound during the assembly process remains elusive. Here, we established a site-specific origin on a plasmid DNA, using in vitro replication systems derived from Xenopus egg extracts. We found that the pre-replicative complex (pre-RC) was preferentially assembled in the vicinity of GAL4 DNA-binding sites of the plasmid, depending on the binding of Cdc6 fused with a GAL4 DNA-binding domain in Cdc6-depleted extracts. Subsequent addition of nucleoplasmic S-phase extracts to the GAL4-dependent pre-RC promoted initiation of DNA replication from the origin, and components of the pre-initiation complex (pre-IC) and the replisome were recruited to the origin concomitant with origin unwinding. In this replication system, RecQ4 is dispensable for both recruitment of Cdc45 onto the origin and stable binding of Cdc45 and GINS to the pre-RC assembled plasmid. However, both origin binding of DNA polymerase α and unwinding of DNA were diminished upon depletion of RecQ4 from the extracts. These results suggest that RecQ4 plays an important role in the conversion of pre-ICs into active replisomes requiring the unwinding of origin DNA in vertebrates.

  20. The HRDC domain of E. coli RecQ helicase controls single-stranded DNA translocation and double-stranded DNA unwinding rates without affecting mechanoenzymatic coupling.

    PubMed

    Harami, Gábor M; Nagy, Nikolett T; Martina, Máté; Neuman, Keir C; Kovács, Mihály

    2015-06-11

    DNA-restructuring activities of RecQ-family helicases play key roles in genome maintenance. These activities, driven by two tandem RecA-like core domains, are thought to be controlled by accessory DNA-binding elements including the helicase-and-RnaseD-C-terminal (HRDC) domain. The HRDC domain of human Bloom's syndrome (BLM) helicase was shown to interact with the RecA core, raising the possibility that it may affect the coupling between ATP hydrolysis, translocation along single-stranded (ss)DNA and/or unwinding of double-stranded (ds)DNA. Here, we determined how these activities are affected by the abolition of the ssDNA interaction of the HRDC domain or the deletion of the entire domain in E. coli RecQ helicase. Our data show that the HRDC domain suppresses the rate of DNA-activated ATPase activity in parallel with those of ssDNA translocation and dsDNA unwinding, regardless of the ssDNA binding capability of this domain. The HRDC domain does not affect either the processivity of ssDNA translocation or the tight coupling between the ATPase, translocation, and unwinding activities. Thus, the mechanochemical coupling of E. coli RecQ appears to be independent of HRDC-ssDNA and HRDC-RecA core interactions, which may play roles in more specialized functions of the enzyme.

  1. A gating mechanism for Pi release governs the mRNA unwinding by eIF4AI during translation initiation.

    PubMed

    Lu, Junyan; Jiang, Chenxiao; Li, Xiaojing; Jiang, Lizhi; Li, Zengxia; Schneider-Poetsch, Tilman; Liu, Jianwei; Yu, Kunqian; Liu, Jun O; Jiang, Hualiang; Luo, Cheng; Dang, Yongjun

    2015-12-02

    Eukaryotic translation initiation factor eIF4AI, the founding member of DEAD-box helicases, undergoes ATP hydrolysis-coupled conformational changes to unwind mRNA secondary structures during translation initiation. However, the mechanism of its coupled enzymatic activities remains unclear. Here we report that a gating mechanism for Pi release controlled by the inter-domain linker of eIF4AI regulates the coupling between ATP hydrolysis and RNA unwinding. Molecular dynamic simulations and experimental results revealed that, through forming a hydrophobic core with the conserved SAT motif of the N-terminal domain and I357 from the C-terminal domain, the linker gated the release of Pi from the hydrolysis site, which avoided futile hydrolysis cycles of eIF4AI. Further mutagenesis studies suggested this linker also plays an auto-inhibitory role in the enzymatic activity of eIF4AI, which may be essential for its function during translation initiation. Overall, our results reveal a novel regulatory mechanism that controls eIF4AI-mediated mRNA unwinding and can guide further mechanistic studies on other DEAD-box helicases.

  2. A gating mechanism for Pi release governs the mRNA unwinding by eIF4AI during translation initiation

    PubMed Central

    Lu, Junyan; Jiang, Chenxiao; Li, Xiaojing; Jiang, Lizhi; Li, Zengxia; Schneider-Poetsch, Tilman; Liu, Jianwei; Yu, Kunqian; Liu, Jun O.; Jiang, Hualiang; Luo, Cheng; Dang, Yongjun

    2015-01-01

    Eukaryotic translation initiation factor eIF4AI, the founding member of DEAD-box helicases, undergoes ATP hydrolysis-coupled conformational changes to unwind mRNA secondary structures during translation initiation. However, the mechanism of its coupled enzymatic activities remains unclear. Here we report that a gating mechanism for Pi release controlled by the inter-domain linker of eIF4AI regulates the coupling between ATP hydrolysis and RNA unwinding. Molecular dynamic simulations and experimental results revealed that, through forming a hydrophobic core with the conserved SAT motif of the N-terminal domain and I357 from the C-terminal domain, the linker gated the release of Pi from the hydrolysis site, which avoided futile hydrolysis cycles of eIF4AI. Further mutagenesis studies suggested this linker also plays an auto-inhibitory role in the enzymatic activity of eIF4AI, which may be essential for its function during translation initiation. Overall, our results reveal a novel regulatory mechanism that controls eIF4AI-mediated mRNA unwinding and can guide further mechanistic studies on other DEAD-box helicases. PMID:26464436

  3. The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes.

    PubMed Central

    Wagner, J D; Jankowsky, E; Company, M; Pyle, A M; Abelson, J N

    1998-01-01

    Of the proteins required for pre-mRNA splicing, at least four, the DEAH-box proteins, are closely related due to the presence of a central 'RNA helicase-like' region, and extended homology through a large portion of the protein. A major unresolved question is the function of these proteins. Indirect evidence suggests that several of these proteins are catalysts for important structural rearrangements in the spliceosome. However, the mechanism for the proposed alterations is presently unknown. We present evidence that PRP22, a DEAH-box protein required for mRNA release from the spliceosome, unwinds RNA duplexes in a concentration- and ATP-dependent manner. This demonstrates that PRP22 can modify RNA structure directly. We also show that the PRP22-dependent release of mRNA from the spliceosome is an ATP-dependent process and that recombinant PRP22 is an ATPase. Non-hydrolyzable ATP analogs did not substitute for ATP in the RNA-unwinding reaction, suggesting that ATP hydrolysis is required for this reaction. Specific mutation of a putative ATP phosphate-binding motif in the recombinant protein eliminated the ATPase and RNA-unwinding capacity. Significantly, these data suggest that the DEAH-box proteins act directly on RNA substrates within the spliceosome. PMID:9582286

  4. The secondary alkaline zinc electrode

    NASA Astrophysics Data System (ADS)

    McLarnon, Frank R.; Cairns, Elton J.

    1991-02-01

    The worldwide studies conducted between 1975 and 1990 with the aim of improving cell lifetimes of secondary alkaline zinc electrodes are overviewed. Attention is given the design features and characteristics of various secondary alkaline zinc cells, including four types of zinc/nickel oxide cell designs (vented static-electrolyte, sealed static-electrolyte, vibrating-electrode, and flowing-electrolyte); two types of zinc/air cells (mechanically rechargeable consolidated-electrode and mechanically rechargeable particulate-electrode); zinc/silver oxide battery; zinc/manganese dioxide cell; and zinc/ferric cyanide battery. Particular consideration is given to recent research in the fields of cell thermodynamics, zinc electrodeposition, zinc electrodissolution, zinc corrosion, electrolyte properties, mathematical and phenomenological models, osmotic pumping, nonuniform current distribution, and cell cycle-life perforamnce.

  5. Development of alkaline fuel cells.

    SciTech Connect

    Hibbs, Michael R.; Jenkins, Janelle E.; Alam, Todd Michael; Janarthanan, Rajeswari; Horan, James L.; Caire, Benjamin R.; Ziegler, Zachary C.; Herring, Andrew M.; Yang, Yuan; Zuo, Xiaobing; Robson, Michael H.; Artyushkova, Kateryna; Patterson, Wendy; Atanassov, Plamen Borissov

    2013-09-01

    This project focuses on the development and demonstration of anion exchange membrane (AEM) fuel cells for portable power applications. Novel polymeric anion exchange membranes and ionomers with high chemical stabilities were prepared characterized by researchers at Sandia National Laboratories. Durable, non-precious metal catalysts were prepared by Dr. Plamen Atanassovs research group at the University of New Mexico by utilizing an aerosol-based process to prepare templated nano-structures. Dr. Andy Herrings group at the Colorado School of Mines combined all of these materials to fabricate and test membrane electrode assemblies for single cell testing in a methanol-fueled alkaline system. The highest power density achieved in this study was 54 mW/cm2 which was 90% of the project target and the highest reported power density for a direct methanol alkaline fuel cell.

  6. Investigation of translocation, DNA unwinding, and protein displacement by NS3h, the helicase domain from the hepatitis C virus helicase.

    PubMed

    Matlock, Dennis L; Yeruva, Laxmi; Byrd, Alicia K; Mackintosh, Samuel G; Langston, Clint; Brown, Carrie; Cameron, Craig E; Fischer, Christopher J; Raney, Kevin D

    2010-03-16

    Helicases are motor proteins that are involved in DNA and RNA metabolism, replication, recombination, transcription, and repair. The motors are powered by ATP binding and hydrolysis. Hepatitis C virus encodes a helicase called nonstructural protein (NS3). NS3 possesses protease and helicase activities on its N-terminal and C-terminal domains, respectively. The helicase domain of NS3 is termed NS3h. In vitro, NS3h catalyzes RNA and DNA unwinding in a 3'-5' direction. The directionality of unwinding is thought to arise in part from the enzyme's ability to translocate along DNA, but translocation has not been shown explicitly. We examined the DNA translocase activity of NS3h by using single-stranded oligonucleotide substrates containing a fluorescent probe on the 5' end. NS3h can bind to the ssDNA and in the presence of ATP move toward the 5' end. When the enzyme encounters the fluorescent probe, a fluorescence change is observed that allows translocation to be characterized. Under conditions that favor binding of one NS3h per DNA substrate (100 nM NS3h and 200 nM oligonucleotide), we find that NS3h translocates on ssDNA at a rate of 46 +/- 5 nucleotides/s, and that it can move for 230 +/- 60 nucleotides before dissociating from the DNA. The translocase activity of some helicases is responsible for displacing proteins that are bound to DNA. We studied protein displacement by using a ssDNA oligonucleotide covalently linked to biotin on the 5' end. Upon addition of streptavidin, a "protein block" was placed in the pathway of the helicase. Interestingly, NS3h was unable to displace streptavidin from the end of the oligonucleotide, despite its ability to translocate along the DNA. The DNA unwinding activity of NS3h was examined using a 22 bp duplex DNA substrate under conditions that were identical to those used to study translocation. NS3h exhibited little or no DNA unwinding under single-cycle conditions, supporting the conclusion that NS3h is a relatively poor helicase

  7. Investigation of translocation, DNA unwinding, and protein displacement by NS3h, the helicase domain from the Hepatitis C virus helicase†

    PubMed Central

    Matlock, Dennis L.; Yeruva, Laxmi; Byrd, Alicia K.; Mackintosh, Samuel G.; Langston, Clint; Brown, Carrie; Cameron, Craig E.; Fischer, Christopher J.; Raney, Kevin D.

    2010-01-01

    Helicases are motor proteins that are involved in DNA and RNA metabolism, replication, recombination, transcription and repair. The motors are powered by ATP binding and hydrolysis. Hepatitis C virus encodes a helicase called non-structural protein (NS3). NS3 possesses protease and helicase activities on its N-terminal and C-terminal domains respectively. The helicase domain of NS3 protein is referred as NS3h. In vitro, NS3h catalyzes RNA and DNA unwinding in a 3’ to -5’ direction. The directionality for unwinding is thought to arise in part from the enzyme's ability to translocate along DNA, but translocation has not been shown explicitly. We examined the DNA translocase activity of NS3h by using single-stranded oligonucleotide substrates containing a fluorescent probe on the 5’ end. NS3h can bind to the ssDNA and in the presence of ATP, move towards the 5’-end. When the enzyme encounters the fluorescent probe, a fluorescence change is observed that allows translocation to be characterized. Under conditions that favor binding of one NS3h per DNA substrate (100 nM NS3h, 200 nM oligonucleotide) we find that NS3h translocates on ssDNA at a rate of 46 ± 5 nt s−1 and that it can move for 230 ± 60 nt before dissociating from the DNA. The translocase activity of some helicases is responsible for displacing proteins that are bound to DNA. We studied protein displacement by using a ssDNA oligonucleotide covalently linked to biotin on the 5’-end. Upon addition of streptavidin, a ‘protein-block’ was placed in the pathway of the helicase. Interestingly, NS3h was unable to displace streptavidin from the end of the oligonucleotide, despite its ability to translocate along the DNA. The DNA unwinding activity of NS3h was examined using a 22 bp duplex DNA substrate under conditions that were identical to those used to study translocation. NS3h exhibited little or no DNA unwinding under single cycle conditions, supporting the conclusion that NS3h is a relatively

  8. Rewinding Frankenstein and the body-machine: organ transplantation in the dystopian young adult fiction series Unwind.

    PubMed

    Wohlmann, Anita; Steinberg, Ruth

    2016-12-01

    While the separation of body and mind (and the entailing metaphor of the body as a machine) has been a cornerstone of Western medicine for a long time, reactions to organ transplantation among others challenge this clear-cut dichotomy. The limits of the machine-body have been negotiated in science fiction, most canonically in Mary Shelley's Frankenstein (1818). Since then, Frankenstein's monster itself has become a motif that permeates both medical and fictional discourses. Neal Shusterman's contemporary dystology for young adults, Unwind, draws on traditional concepts of the machine-body and the Frankenstein myth. This article follows one of the young protagonists in the series, who is entirely constructed from donated tissue, and analyses how Shusterman explores the complicated relationship between body and mind and between self and other as the teenager matures into an adult. It will be shown that, by framing the story of a transplanted individual along the lines of a coming-of-age narrative, Shusterman inter-relates the acceptance of a donor organ with the transitional space of adolescence and positions the quest for embodied selfhood at the centre of both developments. By highlighting the interconnections between medical discourse and a literary tradition, the potential contribution of the series to the treatment and understanding of post-transplant patients will be addressed.

  9. MODULATION BY IONIC STRENGTH AND SUPERHELICITY OF BENZO[a]PYRENE DIOL EPOXIDE INDUCED DNA ALKYLATION AND UNWINDING

    SciTech Connect

    Gamper, Howard B.; Straub, Kenneth; Calvin, Melvin; Bartholomew, James C.

    1980-01-01

    Superhelical and partially relaxed SV40 DNA were reacted in vitro with (+)7{beta}, 8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP diol epoxide). The modified DNA contained N{sup 2} guanine and N{sup 6} adeninte hydrocarbon adducts in the ratio 86:14. Superhelical SV40 DNA was approximately 6% more susceptible to modification than partially relaxed viral DNA. Counterions inhibited DNA alkylation by up to 90%, Mg{sup 2+} being 50-fold more effective than Na{sup +}. The sensitivity of covalent binding to helix stability is consistent with a reaction complex in which BaP diol epoxide is intercalated. The superhelical density of the modified DNA substrates was determined electrophoretically relative to partially relaxed standards and an unwinding angle for the hydrocarbon adducts was calculated. The angle was dependent upon the superhelicity of the DNA molecule and ranged from 330{sup o} to 30{sup o}. This data indicates that the modified base pairs are disrupted and, in the presence of torsional strain, act as centers for the further denaturation of up to 8 adjacent base pairs. In the absence of such strain the alkylation sites have an ordered structure with the attached hydrocarbon probably oriented in the minor or major groove of the helix.

  10. Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha.

    PubMed

    Weber, Janine; Bao, Han; Hartlmüller, Christoph; Wang, Zhiqin; Windhager, Almut; Janowski, Robert; Madl, Tobias; Jin, Peng; Niessing, Dierk

    2016-01-08

    The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.

  11. Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration: insights from DNA complex structures.

    PubMed

    Pike, Ashley C W; Gomathinayagam, Shivasankari; Swuec, Paolo; Berti, Matteo; Zhang, Ying; Schnecke, Christina; Marino, Francesca; von Delft, Frank; Renault, Ludovic; Costa, Alessandro; Gileadi, Opher; Vindigni, Alessandro

    2015-04-07

    RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration.

  12. Analysis of the unwinding activity of the dimeric RECQ1 helicase in the presence of human replication protein A.

    PubMed

    Cui, Sheng; Arosio, Daniele; Doherty, Kevin M; Brosh, Robert M; Falaschi, Arturo; Vindigni, Alessandro

    2004-01-01

    RecQ helicases are required for the maintenance of genome stability. Characterization of the substrate specificity and identification of the binding partners of the five human RecQ helicases are essential for understanding their function. In the present study, we have developed an efficient baculovirus expression system that allows us to obtain milligram quantities of recombinant RECQ1. Our gel filtration and dynamic light scattering experiments show that RECQ1 has an apparent molecular mass of 158 kDa and a hydrodynamic radius of 5.4 +/- 0.6 nm, suggesting that RECQ1 forms dimers in solution. The oligomeric state of RECQ1 remains unchanged upon binding to a single-stranded (ss)DNA fragment of 50 nt. We show that RECQ1 alone is able to unwind short DNA duplexes (<110 bp), whereas considerably longer substrates (501 bp) can be unwound only in the presence of human replication protein A (hRPA). The same experiments with Escherichia coli SSB show that RECQ1 is specifically stimulated by hRPA. However, hRPA does not affect the ssDNA-dependent ATPase activity of RECQ1. In addition, our far western, ELISA and co-immunoprecipitation experiments demonstrate that RECQ1 physically interacts with the 70 kDa subunit of hRPA and that this interaction is not mediated by DNA.

  13. Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration: Insights from DNA complex structures

    PubMed Central

    Pike, Ashley C. W.; Gomathinayagam, Shivasankari; Swuec, Paolo; Berti, Matteo; Schnecke, Christina; Marino, Francesca; von Delft, Frank; Renault, Ludovic; Costa, Alessandro; Vindigni, Alessandro

    2015-01-01

    RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration. PMID:25831490

  14. Functional analysis of DNA bending and unwinding by the high mobility group domain of LEF-1

    PubMed Central

    Giese, Klaus; Pagel, John; Grosschedl, Rudolf

    1997-01-01

    LEF-1 (lymphoid enhancer-binding factor 1) is a cell type-specific member of the family of high mobility group (HMG) domain proteins that recognizes a specific nucleotide sequence in the T cell receptor (TCR) α enhancer. In this study, we extend the analysis of the DNA-binding properties of LEF-1 and examine their contributions to the regulation of gene expression. We find that LEF-1, like nonspecific HMG-domain proteins, can interact with irregular DNA structures such as four-way junctions, albeit with lower efficiency than with specific duplex DNA. We also show by a phasing analysis that the LEF-induced DNA bend is directed toward the major groove. In addition, we find that the interaction of LEF-1 with a specific binding site in circular DNA changes the linking number of DNA and unwinds the double helix. Finally, we identified two nucleotides in the LEF-1-binding site that are important for protein-induced DNA bending. Mutations of these nucleotides decrease both the extent of DNA bending and the transactivation of the TCRα enhancer by LEF-1, suggesting a contribution of protein-induced DNA bending to the function of TCRα enhancer. PMID:9371763

  15. Trypanosoma brucei 20 S Editosomes Have One RNA Substrate-binding Site and Execute RNA Unwinding Activity*

    PubMed Central

    Böhm, Cordula; Katari, Venkata Subbaraju; Brecht, Michael; Göringer, H. Ulrich

    2012-01-01

    Editing of mitochondrial pre-mRNAs in African trypanosomes generates full-length transcripts by the site-specific insertion and deletion of uridylate nucleotides. The reaction is catalyzed by a 0.8 MDa multienzyme complex, the editosome. Although the binding of substrate pre-edited mRNAs and cognate guide RNAs (gRNAs) represents the first step in the reaction cycle, the biochemical and biophysical details of the editosome/RNA interaction are not understood. Here we show that editosomes bind full-length substrate mRNAs with nanomolar affinity in a nonselective fashion. The complexes do not discriminate–neither kinetically nor thermodynamically–between different mitochondrial pre-mRNAs or between edited and unedited versions of the same transcript. They also bind gRNAs and gRNA/pre-mRNA hybrid RNAs with similar affinities and association rate constants. Gold labeling of editosome-bound RNA in combination with transmission electron microscopy identified a single RNA-binding site per editosome. However, atomic force microscopy of individual pre-mRNA-editosome complexes revealed that multiple editosomes can interact with one pre-mRNA. Lastly, we demonstrate a so far unknown activity of the editing machinery: editosome-bound RNA becomes unfolded by a chaperone-type RNA unwinding activity. PMID:22661715

  16. The Barley stripe mosaic virus γb protein promotes chloroplast-targeted replication by enhancing unwinding of RNA duplexes.

    PubMed

    Zhang, Kun; Zhang, Yongliang; Yang, Meng; Liu, Songyu; Li, Zhenggang; Wang, Xianbing; Han, Chenggui; Yu, Jialin; Li, Dawei

    2017-04-07

    RNA viruses encode various RNA binding proteins that function in many steps of viral infection cycles. These proteins function as RNA helicases, methyltransferases, RNA-dependent RNA polymerases, RNA silencing suppressors, RNA chaperones, movement proteins, and so on. Although many of the proteins bind the viral RNA genome during different stages of infection, our knowledge about the coordination of their functions is limited. In this study, we describe a novel role for the Barley stripe mosaic virus (BSMV) γb as an enhancer of αa RNA helicase activity, and we show that the γb protein is recruited by the αa viral replication protein to chloroplast membrane sites of BSMV replication. Mutagenesis or deletion of γb from BSMV resulted in reduced positive strand (+) RNAα accumulation, but γb mutations abolishing viral suppressor of RNA silencing (VSR) activity did not completely eliminate genomic RNA replication. In addition, cis- or trans-expression of the Tomato bushy stunt virus p19 VSR protein failed to complement the γb replication functions, indicating that the direct involvement of γb in BSMV RNA replication is independent of VSR functions. These data support a model whereby two BSMV-encoded RNA-binding proteins act coordinately to regulate viral genome replication and provide new insights into strategies whereby double-stranded viral RNA unwinding is regulated, as well as formation of viral replication complexes.

  17. Distortion and unwinding of the helical structure in polymer-stabilized short-pitch ferroelectric liquid crystal.

    PubMed

    Petit, M; Daoudi, A; Ismaili, M; Buisine, J M

    2006-07-01

    We report the effect of an anisotropic polymer network formed from an achiral photoreactive monomer in a short-pitch chiral SmC* phase on the distortion and the unwinding of the helical structure of the ferroelectric phase. The electro-optical behaviour and ferroelectric properties were experimentally determined for films containing various polymer concentrations. The critical field, E(u), for the transition from the distorted structure to the homogeneous state was measured as a function of polymer concentration. A linear increase of E(u) versus polymer concentration was observed, showing that the helical structure of the short-pitch SmC* phase was stabilized by the polymer network. This behaviour was expected to be a consequence of the increase of the apparent elastic constants of the ferroelectric liquid crystal stabilized by the anisotropic polymer network films. The polymer network morphology was investigated using atomic-force microscopy, revealing a twisted structure of the polymer fibers. This twisted structure was transferred onto a polymer network during the polymerization process within a short-pitch SmC* phase. The increase of the apparent elasticity can then be interpreted by a strong interaction between polymer network and the liquid-crystal molecules. From our experimental data, the coupling coefficient, W(p), characterizing this interaction was evaluated for all studied polymer concentrations.

  18. Diclofenac salts. III. Alkaline and earth alkaline salts.

    PubMed

    Fini, Adamo; Fazio, Giuseppe; Rosetti, Francesca; Angeles Holgado, M; Iruín, Ana; Alvarez-Fuentes, Josefa

    2005-11-01

    Diclofenac salts containing the alkaline and two earth alkaline cations have been prepared and characterized by scanning electron microscopy (SEM) and EDAX spectroscopy; and by thermal and thermogravimetric analysis (TGA): all of them crystallize as hydrate when precipitated from water. The salts dehydrate at room temperature and more easily on heating, but recovery the hydration, when placed in a humid environment. X-ray diffraction spectra suggest that on dehydration new peaks appear on diffractograms and the lattice of the salts partially looses crystallinity. This phenomenon is readily visible in the case of the calcium and magnesium salts, whose thermograms display a crystallization exotherm, before melting or decomposing at temperatures near or above 200 degrees C; these last salts appear to form solvates, when prepared from methanol. The thermogram of each salt shows a complex endotherm of dehydration about 100 degrees C; the calcium salt displays two endotherms, well separated at about 120 and 160 degrees C, which disappear after prolonged heating. Decomposition exotherms, before or soon after the melting, appear below 300 degrees C. The ammonium salt is thermally unstable and, when heated to start dehydration, dissociates and leaves acidic diclofenac.

  19. Unwinding fibril formation of medin, the peptide of the most common form of human amyloid.

    PubMed

    Larsson, Annika; Söderberg, Linda; Westermark, Gunilla T; Sletten, Knut; Engström, Ulla; Tjernberg, Lars O; Näslund, Jan; Westermark, Per

    2007-10-05

    Medin amyloid affects the medial layer of the thoracic aorta of most people above 50 years of age. The consequences of this amyloid are not completely known but the deposits may contribute to diseases such as thoracic aortic aneurysm and dissection or to the general diminished elasticity of blood vessels seen in elderly people. We show that the 50-amino acid residue peptide medin forms amyloid-like fibrils in vitro. With the use of Congo red staining, Thioflavin T fluorescence, electron microscopy, and a solid-phase binding assay on different synthetic peptides, we identified the last 18-19 amino acid residues to constitute the amyloid-promoting region of medin. We also demonstrate that the two C-terminal phenylalanines, previously suggested to be of importance for amyloid formation, are not required for medin amyloid formation.

  20. Unwinding fibril formation of medin, the peptide of the most common form of human amyloid

    SciTech Connect

    Larsson, Annika; Soederberg, Linda; Westermark, Gunilla T.; Sletten, Knut; Engstroem, Ulla; Tjernberg, Lars O.; Naeslund, Jan; Westermark, Per

    2007-10-05

    Medin amyloid affects the medial layer of the thoracic aorta of most people above 50 years of age. The consequences of this amyloid are not completely known but the deposits may contribute to diseases such as thoracic aortic aneurysm and dissection or to the general diminished elasticity of blood vessels seen in elderly people. We show that the 50-amino acid residue peptide medin forms amyloid-like fibrils in vitro. With the use of Congo red staining, Thioflavin T fluorescence, electron microscopy, and a solid-phase binding assay on different synthetic peptides, we identified the last 18-19 amino acid residues to constitute the amyloid-promoting region of medin. We also demonstrate that the two C-terminal phenylalanines, previously suggested to be of importance for amyloid formation, are not required for medin amyloid formation.

  1. Alkaline fuel cell performance investigation

    NASA Technical Reports Server (NTRS)

    Martin, R. E.; Manzo, M. A.

    1988-01-01

    An exploratory experimental fuel cell test program was conducted to investigate the performance characteristics of alkaline laboratory research electrodes. The objective of this work was to establish the effect of temperature, pressure, and concentration upon performance and evaluate candidate cathode configurations having the potential for improved performance. The performance characterization tests provided data to empirically establish the effect of temperature, pressure, and concentration upon performance for cell temperatures up to 300 F and reactant pressures up to 200 psia. Evaluation of five gold alloy cathode catalysts revealed that three doped gold alloys had more that two times the surface areas of reference cathodes and therefore offered the best potential for improved performance.

  2. Application of Intracellular Alkaline Phosphatase Activity Measurement in Detection of Neutrophil Adherence In Vitro

    PubMed Central

    Bednarska, Katarzyna; Klink, Magdalena; Sulowska, Zofia

    2006-01-01

    We have proposed the use of the fluorimetric method with 4-methylumbelliferyl phosphate (4-MUP) specific substrate for the alkaline phosphatase determination in the neutrophil adhesion assay. We provide evidence that the endogenous neutrophil alkaline phosphatase (NAP) activity evaluation is reliable to quantify neutrophil adhesion at a wide range of cell numbers (104−106). The results obtained by fluorimetric NAP activity test correlate to the results of adherence evaluated using the MTT reduction assay. The fluorimetric NAP activity test may be applied for resting as well as activated neutrophils without the risk of the activators interferences into the test. The alkaline phosphatase survey with the use of 4-MUP substrate is recommended herein as a sensitive, repeatable, simple, and reliable method of the neutrophil adherence determination in vitro. PMID:17047286

  3. Bactericidal activity of alkaline salts of fatty acids towards bacteria associated with poultry processing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibacterial activity of alkaline salts of caproic, caprylic, capric, lauric, and myristic acids were determined using the agar diffusion assay. A 0.5M concentration of each fatty acid (FA) was dissolved in 1.0 M potassium hydroxide (KOH), and pH of the mixtures was adjusted to 10.5 with citric aci...

  4. Alkaline detergent recycling via ultrafiltration

    SciTech Connect

    Steffani, C.; Meltzer, M.

    1995-06-01

    The metal finishing industry uses alkaline cleaners and detergents to remove oils and dirt from manufactured parts, often before they are painted or plated. The use of these cleaners has grown because environmental regulations are phasing out ozone depleting substances and placing restrictions on the use and disposal of many hazardous solvents. Lawrence Livermore National Laboratory is examining ultrafiltration as a cleaning approach that reclaims the cleaning solutions and minimizes wastes. The ultrafiltration membrane is made from sheets of polymerized organic film. The sheets are rolled onto a supporting frame and installed in a tube. Spent cleaning solution is pumped into a filter chamber and filtered through the membrane that captures oils and dirt and allows water and detergent to pass. The membrane is monitored and when pressure builds from oil and dirt, an automatic system cleans the surface to maintain solution flow and filtration quality. The results show that the ultrafiltration does not disturb the detergent concentration or alkalinity but removed almost all the oils and dirt leaving the solution in condition to be reused.

  5. Analysis of the DNA unwinding activity of RecQ family helicases.

    PubMed

    Bachrati, Csanád Z; Hickson, Ian D

    2006-01-01

    The RecQ family of DNA helicases is highly conserved in evolution from bacteria to mammals. There are five human RecQ family members (RECQ1, BLM, WRN, RECQ4 and RECQ5), defects, three of which give rise to inherited human disorders. Mutations of BLM have been identified in patients with Bloom's syndrome, WRN has been shown to be mutated in Werner's syndrome, while mutations of RECQ4 have been associated with at least a subset of cases of both Rothmund-Thomson syndrome and RAPADILINO. The most characteristic features of these diseases are a predisposition to the development of malignancies of different types (particularly in Bloom's syndrome), some aspects of premature aging (particularly in Werner's syndrome), and on the cellular level, genome instability. In order to gain understanding of the molecular defects underlying these diseases, many laboratories have focused their research on a study of the biochemical properties of human RecQ helicases, particularly those associated with disease, and of RecQ proteins from other organisms (e.g., Sgs1p of budding yeast, Rqh1p of fission yeast, and RecQ of E.coli). In this chapter, we summarize the assay systems that we employ to analyze the catalytic properties of the BLM helicase. We have successfully used these methods for the study of other RecQ and non-RecQ helicases, indicating that they are likely to be applicable to all helicases.

  6. Alkaline and alkaline earth metal phosphate halides and phosphors

    DOEpatents

    Lyons, Robert Joseph; Setlur, Anant Achyut; Cleaver, Robert John

    2012-11-13

    Compounds, phosphor materials and apparatus related to nacaphite family of materials are presented. Potassium and rubidium based nacaphite family compounds and phosphors designed by doping divalent rare earth elements in the sites of alkaline earth metals in the nacaphite material families are descried. An apparatus comprising the phosphors based on the nacaphite family materials are presented herein. The compounds presented is of formula A.sub.2B.sub.1-yR.sub.yPO.sub.4X where the elements A, B, R, X and suffix y are defined such that A is potassium, rubidium, or a combination of potassium and rubidium and B is calcium, strontium, barium, or a combination of any of calcium, strontium and barium. X is fluorine, chlorine, or a combination of fluorine and chlorine, R is europium, samarium, ytterbium, or a combination of any of europium, samarium, and ytterbium, and y ranges from 0 to about 0.1.

  7. Protein phosphatase 2A and Cdc7 kinase regulate the DNA unwinding element-binding protein in replication initiation.

    PubMed

    Gao, Yanzhe; Yao, Jianhong; Poudel, Sumeet; Romer, Eric; Abu-Niaaj, Lubna; Leffak, Michael

    2014-12-26

    The DNA unwinding element (DUE)-binding protein (DUE-B) binds to replication origins coordinately with the minichromosome maintenance (MCM) helicase and the helicase activator Cdc45 in vivo, and loads Cdc45 onto chromatin in Xenopus egg extracts. Human DUE-B also retains the aminoacyl-tRNA proofreading function of its shorter orthologs in lower organisms. Here we report that phosphorylation of the DUE-B unstructured C-terminal domain unique to higher organisms regulates DUE-B intermolecular binding. Gel filtration analyses show that unphosphorylated DUE-B forms multiple high molecular weight (HMW) complexes. Several aminoacyl-tRNA synthetases and Mcm2-7 proteins were identified by mass spectrometry of the HMW complexes. Aminoacyl-tRNA synthetase binding is RNase A sensitive, whereas interaction with Mcm2-7 is nuclease resistant. Unphosphorylated DUE-B HMW complex formation is decreased by PP2A inhibition or direct DUE-B phosphorylation, and increased by inhibition of Cdc7. These results indicate that the state of DUE-B phosphorylation is maintained by the equilibrium between Cdc7-dependent phosphorylation and PP2A-dependent dephosphorylation, each previously shown to regulate replication initiation. Alanine mutation of the DUE-B C-terminal phosphorylation target sites increases MCM binding but blocks Cdc45 loading in vivo and inhibits cell division. In egg extracts alanine mutation of the DUE-B C-terminal phosphorylation sites blocks Cdc45 loading and inhibits DNA replication. The effects of DUE-B C-terminal phosphorylation reveal a novel S phase kinase regulatory mechanism for Cdc45 loading and MCM helicase activation.

  8. Unwinding protein specific for mRNA translation fractionated together with rabbit reticulocyte initiation factor 3 complex

    PubMed Central

    Ilan, Joseph; Ilan, Judith

    1977-01-01

    Experiments with a rabbit reticulocyte cell-free system dependent on the addition of initiation factor 3 (eIF-3) and mRNA were carried out. In this system, using ribosomal subunits, AUG(U)n can direct polyphenylalanine synthesis in the absence of eIF-3 at 3 mM MgCl2. Globin mRNA was not translated under similar conditions; its translation requires the addition of eIF-3. Moreover, the maximal rate of globin synthesis was achieved when the molar ratio of eIF-3 to ribosomes was approximately 1. This was taken to indicate that some ribosomal proteins were fractionated with eIF-3 and functioned in reconstitution of salt-washed ribosomes. In our system, almost all ribosomes were active, as evident from the fact that all were found in polysomes when analyzed at the time of linear incorporation, and the molar ratio of ribosomes to mRNA was maintained at 4:1. When AUG(U)n was hybridized with poly(A), it could not direct polyphenylalanine synthesis with or without eIF-3 and was a potent inhibitor of the translation of globin mRNA in the presence of eIF-3. When poly(A) containing 10% U was hybridized with AUG(U)n and added to the cell-free system, addition of eIF-3 promoted polyphenylalanine synthesis to about 80% of control. Moreover, eIF-3 was seen to shift significantly the melting temperature of globin and synthetic double-stranded RNA. These observations suggest that extraction of ribosomes with 0.5 M KCl may release a ribosomal protein that fractionates with eIF-3. This protein may function in unwinding or melting the secondary structure of mRNA and thus facilitate translation. PMID:267926

  9. Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction.

    PubMed

    Patil, Ajay Vitthal; Hsieh, Tao-Shih

    2017-03-10

    Human RecQ-like helicase 4 (RECQL4) plays crucial roles in replication initiation and DNA repair; however, the contextual regulation of its unwinding activity is not fully described. Mutations in RECQL4 have been linked to three diseases including Rothmund-Thomson syndrome, which is characterized by osteoskeletal deformities, photosensitivity, and increased osteosarcoma susceptibility. Understanding regulation of RECQL4 helicase activity by interaction partners will allow deciphering its role as an enzyme and a signaling cofactor in different cellular contexts. We became interested in studying the interaction of RECQL4 with ribosomal protein S3 (RPS3) because previous studies have shown that RPS3 activity is sometimes associated with phenotypes mimicking those of mutated RECQL4. RPS3 is a small ribosomal protein that also has extraribosomal functions, including apurnic-apyrimidinic endonuclease-like activity suggested to be important during DNA repair. Here, we report a functional and physical interaction between RPS3 and RECQL4 and show that this interaction may be enhanced during cellular stress. We show that RPS3 inhibits ATPase, DNA binding, and helicase activities of RECQL4 through their direct interaction. Further domain analysis shows that N-terminal 1-320 amino acids of RECQL4 directly interact with the C-terminal 94-244 amino acids of RPS3 (C-RPS3). Biochemical analysis of C-RPS3 revealed that it comprises a standalone apurnic-apyrimidinic endonuclease-like domain. We used U2OS cells to show that oxidative stress and UV exposure could enhance the interaction between nuclear RPS3 and RECQL4. Regulation of RECQL4 biochemical activities by RPS3 along with nuclear interaction during UV and oxidative stress may serve to modulate active DNA repair.

  10. Unwinding the differences of the mammalian PERIOD clock proteins from crystal structure to cellular function.

    PubMed

    Kucera, Nicole; Schmalen, Ira; Hennig, Sven; Öllinger, Rupert; Strauss, Holger M; Grudziecki, Astrid; Wieczorek, Caroline; Kramer, Achim; Wolf, Eva

    2012-02-28

    The three PERIOD homologues mPER1, mPER2, and mPER3 constitute central components of the mammalian circadian clock. They contain two PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B), which mediate homo- and heterodimeric mPER-mPER interactions as well as interactions with transcription factors and kinases. Here we present crystal structures of PAS domain fragments of mPER1 and mPER3 and compare them with the previously reported mPER2 structure. The structures reveal homodimers, which are mediated by interactions of the PAS-B β-sheet surface including a highly conserved tryptophan (Trp448(mPER1), Trp419(mPER2), Trp359(mPER3)). mPER1 homodimers are additionally stabilized by interactions between the PAS-A domains and mPER3 homodimers by an N-terminal region including a predicted helix-loop-helix motive. We have verified the existence of these homodimer interfaces in solution and inside cells using analytical gel filtration and luciferase complementation assays and quantified their contributions to homodimer stability by analytical ultracentrifugation. We also show by fluorescence recovery after photobleaching analyses that destabilization of the PAS-B/tryptophan dimer interface leads to a faster mobility of mPER2 containing complexes in human U2OS cells. Our study reveals structural and quantitative differences between the homodimeric interactions of the three mouse PERIOD homologues, which are likely to contribute to their distinct clock functions.

  11. Single-molecule FRET reveals nucleotide-driven conformational changes in molecular machines and their link to RNA unwinding and DNA supercoiling.

    PubMed

    Klostermeier, Dagmar

    2011-04-01

    Many complex cellular processes in the cell are catalysed at the expense of ATP hydrolysis. The enzymes involved bind and hydrolyse ATP and couple ATP hydrolysis to the catalysed process via cycles of nucleotide-driven conformational changes. In this review, I illustrate how smFRET (single-molecule fluorescence resonance energy transfer) can define the underlying conformational changes that drive ATP-dependent molecular machines. The first example is a DEAD-box helicase that alternates between two different conformations in its catalytic cycle during RNA unwinding, and the second is DNA gyrase, a topoisomerase that undergoes a set of concerted conformational changes during negative supercoiling of DNA.

  12. DNA double helix unwinding triggers transcription block-dependent apoptosis: a semiquantitative probe of the response of ATM, RNAPII, and p53 to two DNA intercalators.

    PubMed

    Zhang, Zhichao; Wang, Yuanyuan; Song, Ting; Gao, Jin; Wu, Guiye; Zhang, Jing; Qian, Xuhong

    2009-03-16

    We have previously shown the binding modes of two DNA interacting analogues (1)a {3-(4-methyl-piperazin)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile} and (3)a {3-(3-dimethylamino-propylamino)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile} with the DNA double helix. In this study, we have determined the notably different DNA damage signal pathway elicited by (1)a and (3)a due to the different extents to which they unwind the DNA double helix. First, we have identified that ataxia-telangiectasia-mutated (ATM) protein kinase can respond to DNA double helix unwinding caused by both (1)a and (3)a. In addition, the amount of ATM activation is consistent with the degree to which the DNA double helix was unwound. Consequently, we used (1)a and (3)a to semiquantitatively probe the response of RNA polymerase II (RNAPII) and p53 toward DNA double helix unwinding in vivo. By means of flow cytometry, immunocytochemistry, ChIP, quantitative real-time polymerase chain reaction, and Western blot analyses, we measured the level of p53 and RNAPII phosphorylation, in addition to the dynamics of the RNAPII distribution along the c-Myc gene. These results provided novel evidence for the impact of subtle DNA structural changes on the activity of RNAPII and p53. Moreover, DNA double helix conformational damage-dependent apoptosis was studied for the first time. These results indicated that (1)a can induce transcriptional blockage following a shift of the unphosphorylated IIa form of RNAPII to the phosphorylated IIo form, while (3)a is unable to induce the same effect. Subsequently, p53 accumulation and phosphorylation events occur that lead to apoptosis in the case of (1)a exposure. This suggests that the transcriptional blockage is also correlated to the degree of double helix unwinding. Furthermore, we found that the degree of DNA conformational damage determines whether or not apoptosis occurs through transcriptional blockage. Under our experimental conditions, ATM does not

  13. Closed type alkaline storage battery

    SciTech Connect

    Hayama, H.

    1980-06-10

    The alkaline storage battery employs a metallic hat shaped terminal closure which has a piercing needle as well as a puncturable metallic diaphragm positioned below the piercing needle. The needle is fixed by caulking at its peripheral edge portion to a edge of the closure. A comparatively thick and hard metal plate is placed on the inner surface of the diaphragm and is applied to an open portion of a tubular metallic container which has a battery element. A peripheral edge portion of the closure, the diaphragm and the metallic plate are clamped in airtight relationship through a packing between the caulked end portion and an inner annular step portion of the metallic container of the battery. A lead wire extends from one polarity electrode of the battery element and is connected to a central portion of the metallic plate.

  14. Evolution of alkaline phosphatases in primates.

    PubMed Central

    Goldstein, D J; Rogers, C; Harris, H

    1982-01-01

    Alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] in placenta, intestine, liver, kidney, bone, and lung from a variety of primate species has been characterized by quantitative inhibition, thermostability, and immunological studies. Characteristic human placental-type alkaline phosphatase occurs in placentas of great apes (chimpanzee and orangutan) but not in placentas of other primates, including gibbon. It is also present in trace amounts in human lung but not in lung or other tissues of various Old and New World monkeys. However, a distinctive alkaline phosphatase resembling it occurs in substantial amounts in lungs from Old World monkeys but not New World monkeys. It appears that duplication of alkaline phosphatase genes and mutations of genetic elements controlling their tissue expression have occurred relatively recently in mammalian evolution. Images PMID:6950431

  15. Alkaline pH sensor molecules.

    PubMed

    Murayama, Takashi; Maruyama, Ichiro N

    2015-11-01

    Animals can survive only within a narrow pH range. This requires continual monitoring of environmental and body-fluid pH. Although a variety of acidic pH sensor molecules have been reported, alkaline pH sensor function is not well understood. This Review describes neuronal alkaline pH sensors, grouped according to whether they monitor extracellular or intracellular alkaline pH. Extracellular sensors include the receptor-type guanylyl cyclase, the insulin receptor-related receptor, ligand-gated Cl- channels, connexin hemichannels, two-pore-domain K+ channels, and transient receptor potential (TRP) channels. Intracellular sensors include TRP channels and gap junction channels. Identification of molecular mechanisms underlying alkaline pH sensing is crucial for understanding how animals respond to environmental alkaline pH and how body-fluid pH is maintained within a narrow range.

  16. Alkaline phosphatase activity of Escherichia coli starved in sterile lake water microcosms.

    PubMed

    Ozkanca, R; Flint, K P

    1996-03-01

    Escherichia coli grown in high or low phosphate medium was inoculated into a lake water starvation medium. The viable count decreased at 37 degrees C but not at the lower temperatures over 70 d. Alkaline phosphatase was monitored using a colorimetric assay with pNPP as the substrate. Derepression of the enzyme occurred in cultures starved for > 30 d in the lake water and within 5 d in lake water microcosms supplemented with carbon and nitrogen sources where there was rarely an increase in viable count. Chloramphenicol prevented the synthesis of alkaline phosphatase suggesting that, even under starvation conditions, de novo synthesis of the enzyme occurs.

  17. Crystal structures of the BsPif1 helicase reveal that a major movement of the 2B SH3 domain is required for DNA unwinding.

    PubMed

    Chen, Wei-Fei; Dai, Yang-Xue; Duan, Xiao-Lei; Liu, Na-Nv; Shi, Wei; Li, Na; Li, Ming; Dou, Shou-Xing; Dong, Yu-Hui; Rety, Stephane; Xi, Xu-Guang

    2016-04-07

    Pif1 helicases are ubiquitous members of the SF1B family and are essential for maintaining genome stability. It was speculated that Pif1-specific motifs may fold in specific structures, conferring distinct activities upon it. Here, we report the crystal structures of the Pif1 helicase from Bacteroides spp with and without adenosine triphosphate (ATP) analog/ssDNA. BsPif1 shares structural similarities with RecD2 and Dda helicases but has specific features in the 1B and 2B domains. The highly conserved Pif1 family specific sequence motif interacts with and constraints a putative pin-loop in domain 1B in a precise conformation. More importantly, we found that the 2B domain which contains a specific extended hairpin undergoes a significant rotation and/or movement upon ATP and DNA binding, which is absolutely required for DNA unwinding. We therefore propose a mechanism for DNA unwinding in which the 2B domain plays a predominant role. The fact that the conformational change regulates Pif1 activity may provide insight into the puzzling observation that Pif1 becomes highly processive during break-induced replication in association with Polδ, while the isolated Pif1 has low processivity.

  18. A prominent β-hairpin structure in the winged-helix domain of RECQ1 is required for DNA unwinding and oligomer formation.

    PubMed

    Lucic, Bojana; Zhang, Ying; King, Oliver; Mendoza-Maldonado, Ramiro; Berti, Matteo; Niesen, Frank H; Burgess-Brown, Nicola A; Pike, Ashley C W; Cooper, Christopher D O; Gileadi, Opher; Vindigni, Alessandro

    2011-03-01

    RecQ helicases have attracted considerable interest in recent years due to their role in the suppression of genome instability and human diseases. These atypical helicases exert their function by resolving a number of highly specific DNA structures. The crystal structure of a truncated catalytic core of the human RECQ1 helicase (RECQ1(49-616)) shows a prominent β-hairpin, with an aromatic residue (Y564) at the tip, located in the C-terminal winged-helix domain. Here, we show that the β-hairpin is required for the DNA unwinding and Holliday junction (HJ) resolution activity of full-length RECQ1, confirming that it represents an important determinant for the distinct substrate specificity of the five human RecQ helicases. In addition, we found that the β-hairpin is required for dimer formation in RECQ1(49-616) and tetramer formation in full-length RECQ1. We confirmed the presence of stable RECQ1(49-616) dimers in solution and demonstrated that dimer formation favours DNA unwinding; even though RECQ1 monomers are still active. Tetramers are instead necessary for more specialized activities such as HJ resolution and strand annealing. Interestingly, two independent protein-protein contacts are required for tetramer formation, one involves the β-hairpin and the other the N-terminus of RECQ1, suggesting a non-hierarchical mechanism of tetramer assembly.

  19. Process for extracting technetium from alkaline solutions

    DOEpatents

    Moyer, Bruce A.; Sachleben, Richard A.; Bonnesen, Peter V.

    1995-01-01

    A process for extracting technetium values from an aqueous alkaline solution containing at least one alkali metal hydroxide and at least one alkali metal nitrate, the at least one alkali metal nitrate having a concentration of from about 0.1 to 6 molar. The solution is contacted with a solvent consisting of a crown ether in a diluent for a period of time sufficient to selectively extract the technetium values from the aqueous alkaline solution. The solvent containing the technetium values is separated from the aqueous alkaline solution and the technetium values are stripped from the solvent.

  20. Alkaline sorbent injection for mercury control

    DOEpatents

    Madden, Deborah A.; Holmes, Michael J.

    2002-01-01

    A mercury removal system for removing mercury from combustion flue gases is provided in which alkaline sorbents at generally extremely low stoichiometric molar ratios of alkaline earth or an alkali metal to sulfur of less than 1.0 are injected into a power plant system at one or more locations to remove at least between about 40% and 60% of the mercury content from combustion flue gases. Small amounts of alkaline sorbents are injected into the flue gas stream at a relatively low rate. A particulate filter is used to remove mercury-containing particles downstream of each injection point used in the power plant system.

  1. Alkaline sorbent injection for mercury control

    DOEpatents

    Madden, Deborah A.; Holmes, Michael J.

    2003-01-01

    A mercury removal system for removing mercury from combustion flue gases is provided in which alkaline sorbents at generally extremely low stoichiometric molar ratios of alkaline earth or an alkali metal to sulfur of less than 1.0 are injected into a power plant system at one or more locations to remove at least between about 40% and 60% of the mercury content from combustion flue gases. Small amounts of alkaline sorbents are injected into the flue gas stream at a relatively low rate. A particulate filter is used to remove mercury-containing particles downstream of each injection point used in the power plant system.

  2. Dairy products and the French paradox: Could alkaline phosphatases play a role?

    PubMed

    Lallès, Jean-Paul

    2016-07-01

    intestine of model animals. Furthermore, stool residual IAP, a possible early marker of diabetes, should be assayed in human cohorts. If confirmed, this "alkaline phosphatase" hypothesis will highlight the protective effects of milk alkaline phosphatase and promote the consumption of (microbiologically safe) raw milk and dairy products. Microorganisms secreting alkaline phosphatases may be privileged as ferments in dairy products.

  3. Alkaline tolerant dextranase from streptomyces anulatus

    DOEpatents

    Decker, Stephen R.; Adney, William S.; Vinzant, Todd B.; Himmel, Michael E.

    2003-01-01

    A process for production of an alkaline tolerant dextranase enzyme comprises culturing a dextran-producing microorganism Streptomyces anulatus having accession no. ATCC PTA-3866 to produce an alkaline tolerant dextranase, Dex 1 wherein the protein in said enzyme is characterized by a MW of 63.3 kDa and Dex 2 wherein its protein is characterized by a MW of 81.8 kDa.

  4. Technetium recovery from high alkaline solution

    DOEpatents

    Nash, Charles A.

    2016-07-12

    Disclosed are methods for recovering technetium from a highly alkaline solution. The highly alkaline solution can be a liquid waste solution from a nuclear waste processing system. Methods can include combining the solution with a reductant capable of reducing technetium at the high pH of the solution and adding to or forming in the solution an adsorbent capable of adsorbing the precipitated technetium at the high pH of the solution.

  5. Toxicity of alkalinity to Hyalella azteca

    USGS Publications Warehouse

    Lasier, P.J.; Winger, P.V.; Reinert, R.E.

    1997-01-01

    Toxicity testing and chemical analyses of sediment pore water have been suggested for use in sediment quality assessments and sediment toxicity identification evaluations. However, caution should be exercised in interpreting pore-water chemistry and toxicity due to inherent chemical characteristics and confounding relationships. High concentrations of alkalinity, which are typical of sediment pore waters from many regions, have been shown to be toxic to test animals. A series of tests were conducted to assess the significance of elevated alkalinity concentrations to Hyalella azteca, an amphipod commonly used for sediment and pore-water toxicity testing. Toxicity tests with 14-d old and 7-d old animals were conducted in serial dilutions of sodium bicarbonate (NaHCO3) solutions producing alkalinities ranging between 250 to 2000 mg/L as CaCO3. A sodium chloride (NaCl) toxicity test was also conducted to verify that toxicity was due to bicarbonate and not sodium. Alkalinity was toxic at concentrations frequently encountered in sediment pore water. There was also a significant difference in the toxicity of alkalinity between 14-d old and 7-d old animals. The average 96-h LC50 for alkalinity was 1212 mg/L (as CaCO3) for 14-d old animals and 662 mg/L for the younger animals. Sodium was not toxic at levels present in the NaHCO3 toxicity tests. Alkalinity should be routinely measured in pore-water toxicity tests, and interpretation of toxicity should consider alkalinity concentration and test-organism tolerance.

  6. Purification and Comparative Assay of the Human Mitochondrial Replicative DNA Helicase

    PubMed Central

    Rosado-Ruiz, Fernando A.; So, Minyoung; Kaguni, Laurie S.

    2015-01-01

    The replicative mitochondrial DNA (mtDNA) helicase is essential for mtDNA replication and maintenance of the mitochondrial genome. Despite substantial advances that have been made in its characterization, there is still much to be understood about the functional roles of its domains and its interactions with the other components of the minimal mitochondrial DNA replisome. Critical to achieving this is the ability to isolate the enzyme in a stable, active form. In this chapter we describe a modified, streamlined purification strategy for recombinant forms of the enzyme. We also present assays to assess its helix unwinding activity and the stimulatory effects of the mitochondrial single-stranded DNA-binding protein (mtSSB). Finally, we describe a concentration/buffer exchange method that we have employed to achieve greater enzyme stability and appropriate conditions for biochemical and biophysical studies. PMID:26530683

  7. Alkaline Water and Longevity: A Murine Study

    PubMed Central

    Magro, Massimiliano; Corain, Livio; Ferro, Silvia; Baratella, Davide; Bonaiuto, Emanuela; Terzo, Milo; Corraducci, Vittorino; Salmaso, Luigi; Vianello, Fabio

    2016-01-01

    The biological effect of alkaline water consumption is object of controversy. The present paper presents a 3-year survival study on a population of 150 mice, and the data were analyzed with accelerated failure time (AFT) model. Starting from the second year of life, nonparametric survival plots suggest that mice watered with alkaline water showed a better survival than control mice. Interestingly, statistical analysis revealed that alkaline water provides higher longevity in terms of “deceleration aging factor” as it increases the survival functions when compared with control group; namely, animals belonging to the population treated with alkaline water resulted in a longer lifespan. Histological examination of mice kidneys, intestine, heart, liver, and brain revealed that no significant differences emerged among the three groups indicating that no specific pathology resulted correlated with the consumption of alkaline water. These results provide an informative and quantitative summary of survival data as a function of watering with alkaline water of long-lived mouse models. PMID:27340414

  8. Performed surfactant-optimized aqueous alkaline flood

    SciTech Connect

    Thigpen, D.R.; Lawson, J.B.; Nelson, R.C.

    1991-11-26

    This paper describes improvement in a process for recovering oil from an acidic oil reservoir by injecting an aqueous alkaline solution comprising water, sodium chloride, and alkaline material for reacting with the reservoir oil forming a petroleum acid soap to form an in-situ surfactant system. The improvement comprises: selecting a preformed cosurfactant which is soluble in both the aqueous solution and the reservoir oil and has a solubility ratio which is grater than the solubility ratio of the petroleum acid soap where the solubility ratio is the ratio of solubility in the aqueous alkaline solution to the solubility in the reservoir oil; combining with the alkaline solution an amount of the preformed cosurfactant which will result in the in-situ surfacant system having a salinity about equal to a salinity which results in minimal interfacial tension between the oil in the reservoir and the in-situ surfactant system at reservoir temperature, wherein the amount of the preformed cosurfactant is about 0.3 percent by weight in the aqueous alkaline solution; and injecting the cosurfactant-aqueous alkaline solution mixture into the reservoir to displace oil toward a fluid production location.

  9. The Prp8 RNase H-like domain inhibits Brr2-mediated U4/U6 snRNA unwinding by blocking Brr2 loading onto the U4 snRNA.

    PubMed

    Mozaffari-Jovin, Sina; Santos, Karine F; Hsiao, He-Hsuan; Will, Cindy L; Urlaub, Henning; Wahl, Markus C; Lührmann, Reinhard

    2012-11-01

    The spliceosomal RNA helicase Brr2 catalyzes unwinding of the U4/U6 snRNA duplex, an essential step for spliceosome catalytic activation. Brr2 is regulated in part by the spliceosomal Prp8 protein by an unknown mechanism. We demonstrate that the RNase H (RH) domain of yeast Prp8 binds U4/U6 small nuclear RNA (snRNA) with the single-stranded regions of U4 and U6 preceding U4/U6 stem I, contributing to its binding. Via cross-linking coupled with mass spectrometry, we identify RH domain residues that contact the U4/U6 snRNA. We further demonstrate that the same single-stranded region of U4 preceding U4/U6 stem I is recognized by Brr2, indicating that it translocates along U4 and first unwinds stem I of the U4/U6 duplex. Finally, we show that the RH domain of Prp8 interferes with U4/U6 unwinding by blocking Brr2's interaction with the U4 snRNA. Our data reveal a novel mechanism whereby Prp8 negatively regulates Brr2 and potentially prevents premature U4/U6 unwinding during splicing. They also support the idea that the RH domain acts as a platform for the exchange of U6 snRNA for U1 at the 5' splice site. Our results provide insights into the mechanism whereby Brr2 unwinds U4/U6 and show how this activity is potentially regulated prior to spliceosome activation.

  10. [Alkaline phosphatase in Amoeba proteus].

    PubMed

    Sopina, V A

    2005-01-01

    In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.

  11. Biochemical Localization of Alkaline Phosphatase in the Cell Wall of a Marine Pseudomonad

    PubMed Central

    Thompson, Linda M. M.; MacLeod, Robert A

    1974-01-01

    The various layers of the cell envelope of marine pseudomonad B-16 (ATCC 19855) have been separated from the cells and assayed directly for alkaline phosphatase activity under conditions established previously to be optimum for maintenance of the activity of the enzyme. Under conditions known to lead to the release of the contents of the periplasmic space from the cells, over 90% of the alkaline phosphatase was released into the medium. Neither the loosely bound outer layer nor the outer double-track layer (cell wall membrane) showed significant activity. A small amount of the alkaline phosphatase activity of the cells remained associated with the mureinoplasts when the outer layers of the cell wall were removed. Upon treatment of the mureinoplasts with lysozyme, some alkaline phosphatase was released into the medium and some remained with the protoplasts formed. Cells washed and suspended in 0.5 M NaCl were lysed by treatment with 2% toluene, and 95% of the alkaline phosphatase in the cells was released into the medium. Cells washed and suspended in complete salts solution (0.3 M NaCl, 0.05 M MgSO4, and 0.01 M KCl) or 0.05 M MgSO4 appeared intact after treatment with toluene but lost 50 and 10%, respectively, of their alkaline phosphatase. The results suggest that the presence of Mg2+ in the cell wall is necessary to prevent disruption of the cells by toluene and may also be required to prevent the release of alkaline phosphatase by toluene when disruption of the cells by toluene does not take place. PMID:4811547

  12. Heat stable alkaline phosphatase from thermophiles. Final report, March-October 1993

    SciTech Connect

    Combie, J.D.; Runnion, K.N.; Williamson, M.L.

    1994-07-01

    Alkaline phosphatase has been the most widely used enzyme for colorimetric immunoassays. The current potential for this enzyme lies in biosensors, fieldable assay kits, biotechnology applications, degradation of certain nerve agents and pesticides and detoxification of heavy metal waste streams. While the commercial source of this enzyme is predominantly from mammalian tissues, expanded commercial application is restricted by the enzyme's instability at elevated temperatures. Although alkaline phosphatases are ubiquitous in nature, two isolates out of 44 alkaline phosphatase producing isolates occurring in habitats at 50 deg C and above have been isolated possessing extremely stable enzymes. One enzyme retained 98% of original activity following boiling for 1 hr. The secretion of the enzyme by the organism is an added benefit promoting efficient and economical production capability. Procedures for the screening, isolation, and optimal growth and fermentation of organisms acquired from geothermal sources located in Yellowstone National Park, WY are described. Purification was most effectively achieved using size exclusion chromatography where 101% of the activity and 33% of the crude mother liquor protein were recovered. Although the presence of manganese in the assay buffer was observed to significantly elevate the enzyme's catalytic activity, a precipitate incompatibility with calcium chloride, a requirement for high temperature stability, prohibits its use. Bacteria, Fermentation, Alkaline phosphatase, Biosensors, Biotechnology, Heat stable enzymes, Biochemistry, Bioremediation, Thermophilic microorganisms.

  13. Production of alkaline cellulase by fungi isolated from an undisturbed rain forest of peru.

    PubMed

    Vega, Karin; Villena, Gretty K; Sarmiento, Victor H; Ludeña, Yvette; Vera, Nadia; Gutiérrez-Correa, Marcel

    2012-01-01

    Alkaline cellulase producing fungi were isolated from soils of an undisturbed rain forest of Peru. The soil dilution plate method was used for the enumeration and isolation of fast growing cellulolytic fungi on an enriched selective medium. Eleven out of 50 different morphological colonies were finally selected by using the plate clearing assay with CMC as substrate at different pH values. All 11 strains produced cellulases in liquid culture with activities at alkaline pH values without an apparent decrease of them indicating that they are true alkaline cellulase producers. Aspergillus sp. LM-HP32, Penicillium sp. LM-HP33, and Penicillium sp. LM-HP37 were the best producers of FP cellulase (>3 U mL(-1)) with higher specific productivities (>30 U g(-1) h(-1)). Three strains have been found suitable for developing processes for alkaline cellulase production. Soils from Amazonian rain forests are good sources of industrial fungi with particular characteristics. The results of the present study are of commercial and biological interest. Alkaline cellulases may be used in the polishing and washing of denim processing of the textile industry.

  14. Chemiluminescent assay of enzymes using proenhancers and pro-anti-enhancers.

    PubMed

    Kricka, L J; Schmerfeld-Pruss, D; Edwards, B

    1991-01-01

    Enhanced chemiluminescent assays for hydrolase enzymes have been developed using proenhancer and pro-anti-enhancer substrates. Alkaline phosphatase is measured using disodium para-iodophenyl phosphate (proenhancer) which is converted to para-iodophenol and this in turn enhances the light emission from the horseradish peroxidase catalysed chemiluminescent oxidation of luminol by peroxide. An alternative strategy uses para-nitrophenyl phosphate which is converted by alkaline phosphatase to para-nitrophenol which inhibits the enhanced chemiluminescent reaction. The detection limit for the enzyme using the proenhancer and pro-anti-enhancer assays was 100 attomoles and 1 picomole, respectively. The proenhancer strategy was effective in assays for beta-D-galactosidase, beta-D-glucosidase and aryl sulfatase. A limited comparison of the proenhancer and a conventional colorimetric assay for an alkaline phosphatase label in an enzyme immunoassay for alpha-fetoprotein showed good agreement.

  15. De Novo Transcriptional Analysis of Alfalfa in Response to Saline-Alkaline Stress

    PubMed Central

    An, Yi-Min; Song, Li-Li; Liu, Ying-Rui; Shu, Yong-Jun; Guo, Chang-Hong

    2016-01-01

    Saline-alkaline stress, caused by high levels of harmful carbonate salts and high soil pH, is a major abiotic stress that affects crop productivity. Alfalfa is a widely cultivated perennial forage legume with some tolerance to biotic and abiotic stresses, especially to saline-alkaline stress. To elucidate the mechanism underlying plant saline-alkaline tolerance, we conducted transcriptome analysis of whole alfalfa seedlings treated with saline-alkaline solutions for 0 day (control), 1 day (short-term treatment), and 7 days (long-term treatment) using ion torrent sequencing technology. A transcriptome database dataset of 53,853 unigenes was generated, and 2,286 and 2,233 genes were differentially expressed in the short-term and long-term treatment, respectively. Gene ontology analysis revealed 14 highly enriched pathways and demonstrated the differential response of metabolic pathways between the short-term and long-term treatment. The expression levels of 109 and 96 transcription factors were significantly altered significantly after 1 day and 7 days of treatment, respectively. Specific responses of peroxidase, flavonoids, and the light pathway component indicated that the antioxidant capacity was one of the central mechanisms of saline-alkaline stress tolerance response in alfalfa. Among the 18 differentially expressed genes examined by real time PCR, the expression levels of eight genes, including inositol transporter, DNA binding protein, raffinose synthase, ferritin, aldo/keto reductase, glutathione S-transferase, xyloglucan endotrans glucosylase, and a NAC transcription factor, exhibited different patterns in response to saline and alkaline stress. The expression levels of the NAC transcription factor and glutathione S-transferase were altered significantly under saline stress and saline-alkaline stress; they were upregulated under saline-alkaline stress and downregulated under salt stress. Physiology assays showed an increased concentration of reactive oxygen

  16. In Vitro Enzyme Comparative Kinetics: Unwinding of Surface-Bound DNA Nanostructures by RecQ and RecQ1.

    PubMed

    Parisse, Pietro; Vindigni, Alessandro; Scoles, Giacinto; Casalis, Loredana

    2012-12-06

    Many cellular processes entail the separation of nucleic acid strands. Helicases are involved in the separation of the double-stranded DNA, a process fueled by ATP hydrolysis. We investigated the reaction mechanism of two homologous helicases, the bacterial RecQ and the human RECQ1, in vitro, that is, within confined DNA monolayers. We generate arrays of engineered DNA sequences by atomic force microscopy (AFM) nanografting and monitor the enzyme activity on the surface by means of differential, highly precise AFM measurements of the DNA height variation. The latter is associated with the unwinding action of the enzyme onto the surface-bound DNAs because it arises from the different mechanical properties of single- versus double-stranded DNA that are sensibly detected by AFM. Our results highlight different kinetic behaviors for these enzymes under the same experimental conditions.

  17. Residues in the RecQ C-terminal Domain of the Human Werner Syndrome Helicase Are Involved in Unwinding G-quadruplex DNA.

    PubMed

    Ketkar, Amit; Voehler, Markus; Mukiza, Tresor; Eoff, Robert L

    2017-02-24

    The structural and biophysical properties typically associated with G-quadruplex (G4) structures render them a significant block for DNA replication, which must be overcome for cell division to occur. The Werner syndrome protein (WRN) is a RecQ family helicase that has been implicated in the efficient processing of G4 DNA structures. The aim of this study was to identify the residues of WRN involved in the binding and ATPase-driven unwinding of G4 DNA. Using a c-Myc G4 DNA model sequence and recombinant WRN, we have determined that the RecQ-C-terminal (RQC) domain of WRN imparts a 2-fold preference for binding to G4 DNA relative to non-G4 DNA substrates. NMR studies identified residues involved specifically in interactions with G4 DNA. Three of the amino acids in the WRN RQC domain that exhibited the largest G4-specific changes in NMR signal were then mutated alone or in combination. Mutating individual residues implicated in G4 binding had a modest effect on WRN binding to DNA, decreasing the preference for G4 substrates by ∼25%. Mutating two G4-interacting residues (T1024G and T1086G) abrogated preferential binding of WRN to G4 DNA. Very modest decreases in G4 DNA-stimulated ATPase activity were observed for the mutant enzymes. Most strikingly, G4 unwinding by WRN was inhibited ∼50% for all three point mutants and >90% for the WRN double mutant (T1024G/T1086G) relative to normal B-form dsDNA substrates. Our work has helped to identify residues in the WRN RQC domain that are involved specifically in the interaction with G4 DNA.

  18. Effect of pH alkaline salts of fatty acids on the inhibition of bacteria associated with poultry processing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The agar diffusion assay was used to examine the effect of pH on the ability of alkaline salts of three fatty acids (FA) to inhibit growth of bacteria associated with poultry processing. FA solutions were prepared by dissolving 0.5 M concentrations of caprylic, capric, or lauric acid in separate ali...

  19. Intermediate range order in alkaline borate glasses

    NASA Astrophysics Data System (ADS)

    Crupi, C.; Carini, G.; Ruello, G.; D'Angelo, G.

    2016-03-01

    We describe the neutron diffraction patterns of a series of alkaline borate glasses at different metal oxide content. Strong differences are observed in the intermediate range order as a function of the specific alkaline ion and of its concentration. On these results, we propose that the first sharp diffraction peak arises from correlations of atoms of voids and show that the compositional variation of this peak intensity in alkaline borate glasses is due to changes in the distribution of void sizes within the three-dimensional network. We argue that our interpretation in terms of interstitial (empty and/or filled) voids, having different sizes, provides a general explanation for all anomalous behaviours revealed for the first sharp diffraction peak.

  20. Laser direct write of planar alkaline microbatteries

    NASA Astrophysics Data System (ADS)

    Arnold, C. B.; Kim, H.; Piqué, A.

    We are developing a laser engineering approach to fabricate and optimize alkaline microbatteries in planar geometries. The laser direct-write technique enables multicapability for adding, removing and processing material and provides the ability to pattern complicated structures needed for fabricating complete microbattery assemblies. In this paper, we demonstrate the production of planar zinc-silver oxide alkaline cells under ambient conditions. The microbattery cells exhibit 1.55-V open-circuit potentials, as expected for the battery chemistry, and show a flat discharge behavior under constant-current loads. High capacities of over 450 μAhcm-2 are obtained for 5-mm2 microbatteries.

  1. Alkaline earth filled nickel skutterudite antimonide thermoelectrics

    DOEpatents

    Singh, David Joseph

    2013-07-16

    A thermoelectric material including a body centered cubic filled skutterudite having the formula A.sub.xFe.sub.yNi.sub.zSb.sub.12, where A is an alkaline earth element, x is no more than approximately 1.0, and the sum of y and z is approximately equal to 4.0. The alkaline earth element includes guest atoms selected from the group consisting of Be, Mb, Ca, Sr, Ba, Ra and combinations thereof. The filled skutterudite is shown to have properties suitable for a wide variety of thermoelectric applications.

  2. Alkaline Capacitors Based on Nitride Nanoparticles

    NASA Technical Reports Server (NTRS)

    Aldissi, Matt

    2003-01-01

    High-energy-density alkaline electrochemical capacitors based on electrodes made of transition-metal nitride nanoparticles are undergoing development. Transition- metal nitrides (in particular, Fe3N and TiN) offer a desirable combination of high electrical conductivity and electrochemical stability in aqueous alkaline electrolytes like KOH. The high energy densities of these capacitors are attributable mainly to their high capacitance densities, which, in turn, are attributable mainly to the large specific surface areas of the electrode nanoparticles. Capacitors of this type could be useful as energy-storage components in such diverse equipment as digital communication systems, implanted medical devices, computers, portable consumer electronic devices, and electric vehicles.

  3. RAS - Screens & Assays

    Cancer.gov

    A primary goal of the RAS Initiative is to develop assays for RAS activity, localization, and signaling and adapt those assays so they can be used for finding new drug candidates. Explore the work leading to highly validated screening protocols.

  4. Assays of Serum Testosterone.

    PubMed

    Herati, Amin S; Cengiz, Cenk; Lamb, Dolores J

    2016-05-01

    The diagnosis of male hypogonadism depends on an assessment of the clinical signs and symptoms of hypogonadism and serum testosterone level. Current clinical laboratory testosterone assay platforms include immunoassays and mass spectrometry. Despite significant advances to improve the accuracy and precision of the currently available assays, limited comparability exists between assays at the lower and upper extremes of the testosterone range. Because of this lack of comparability, there is no current gold standard assay for the assessment of total testosterone levels.

  5. The Alkaline Diet: Is There Evidence That an Alkaline pH Diet Benefits Health?

    PubMed Central

    Schwalfenberg, Gerry K.

    2012-01-01

    This review looks at the role of an alkaline diet in health. Pubmed was searched looking for articles on pH, potential renal acid loads, bone health, muscle, growth hormone, back pain, vitamin D and chemotherapy. Many books written in the lay literature on the alkaline diet were also reviewed and evaluated in light of the published medical literature. There may be some value in considering an alkaline diet in reducing morbidity and mortality from chronic diseases and further studies are warranted in this area of medicine. PMID:22013455

  6. The alkaline diet: is there evidence that an alkaline pH diet benefits health?

    PubMed

    Schwalfenberg, Gerry K

    2012-01-01

    This review looks at the role of an alkaline diet in health. Pubmed was searched looking for articles on pH, potential renal acid loads, bone health, muscle, growth hormone, back pain, vitamin D and chemotherapy. Many books written in the lay literature on the alkaline diet were also reviewed and evaluated in light of the published medical literature. There may be some value in considering an alkaline diet in reducing morbidity and mortality from chronic diseases and further studies are warranted in this area of medicine.

  7. Alkaline phosphatase in stallion semen: characterization and clinical applications.

    PubMed

    Turner, R M O; McDonnell, S M

    2003-06-01

    Significant amounts of alkaline phosphatase (AP) activity have been found in semen plasma from numerous species. In species in which the majority of semen plasma AP (SPAP) activity originates from the epididymis and testicle, SPAP activity can be used clinically as a marker to differentiate testicular origin azoospermia or oligospermia from ejaculatory failure. Information on SPAP activity in stallions to date has been limited. In this study, a standard clinical chemistry analyzer was used to determine AP activity in pre-ejaculatory fluid and ejaculates from groups of normal stallions. Additionally, accessory glands, epididymides, testicles and other components of the urogenital tract of normal stallions were assayed to determine which tissues contain SPAP activity. The results indicated that levels of AP activity are low in pre-ejaculatory fluid, but significantly higher in ejaculatory fluid from normal stallions. Spermatozoa were not a significant source of SPAP activity. High levels of SPAP activity were found in the testes and epididymides. These findings suggest that SPAP activity is a candidate for a sperm-independent marker for ejaculation in the stallion. Finally, AP activity was determined in ejaculatory fluid from a stallion with bilaterally blocked ampullae, both before and after relief of the blockage. While the blockage was present, AP activity in ejaculatory fluid was low. However, following relief of the blockage, AP activity in ejaculatory fluid rose dramatically, thus suggesting that AP activity will be useful as an inexpensive, simple clinical assay for differentiating ejaculatory failure or excurrent duct blockages from testicular origin azoospermia and oligospermia.

  8. Negative Electrode For An Alkaline Cell

    DOEpatents

    Coco, Isabelle; Cocciantelli, Jean-Michel; Villenave, Jean-Jacques

    1998-07-14

    The present invention concerns a negative electrode for an alkaline cell, comprising a current collector supporting a paste containing an electrochemically active material and a binder, characterized in that said binder is a polymer containing hydrophilic and hydrophobic groups, said polymer being selected from an acrylic homopolymer, copolymer and terpolymer, an unsaturated organic acid copolymer and an unsaturated acid anhydride copolymer.

  9. Alkaline electrochemical cells and method of making

    NASA Technical Reports Server (NTRS)

    Hoyt, H. E.; Pfluger, H. L. (Inventor)

    1970-01-01

    Equilibrated cellulose ether membranes of increased electrolytic conductivity for use as separators in concentrated alkaline electrochemical cells are investigated. The method of making such membranes by equilibration to the degree desired in an aqueous alkali solution mantained at a temperature below about 10 C is described.

  10. Kinetics of the alkaline hydrolysis of nitrocellulose.

    PubMed

    Christodoulatos, C; Su, T L; Koutsospyros, A

    2001-01-01

    Cellulose nitrate (nitrocellulose) is an explosive solid substance used in large quantities in various formulations of rocket and gun propellants. Safe destruction of nitrocellulose can be achieved by alkaline hydrolysis, which converts it to biodegradable products that can then be treated by conventional biological processes. The kinetics of the alkaline hydrolysis of munitions-grade nitrocellulose in sodium hydroxide solutions were investigated in completely mixed batch reactors. Experiments were conducted using solutions of alkaline strength ranging from 0.1 to 15% by mass and temperatures in the range of 30 to 90 degrees C. Regression analysis of the kinetic data revealed that alkaline hydrolysis of nitrocellulose is of the order 1.0 and 1.5 with respect to nitrocellulose and hydroxide concentration, respectively. The activation energy of the hydrolysis reaction was found to be 100.9 kJ/mol with a preexponential Arrhenius constant of 4.73 x 10(13). Nitrite and nitrate, in a 3:1 ratio, were the primary nitrogen species present in the posthydrolysis solution. The kinetic information is pertinent to the development and optimization of nitrocellulose chemical-biological treatment systems.

  11. MERCURIC CHLORIDE CAPTURE BY ALKALINE SORBENTS

    EPA Science Inventory

    The paper gives results of bench-scale mechanistic studies of mercury/sorbent reactions that showed that mercuric chloride (HgC12) is readily adsorbed by alkaline sorbents, which may offers a less expensive alternative to the use of activated carbons. A laboratory-scale, fixed-b...

  12. ISSUES WITH ALKALINE TREATMENT OF SLUDGE

    EPA Science Inventory

    This presentation begins with a discussion of the use of lime and other alkaline materials from the very earliest times to the present for killing bacteria, viruses and parasites and for controlling odors in wastewaters and sludge. It answers the question "How did EPA arrive at i...

  13. Alkaline earth metal catalysts for asymmetric reactions.

    PubMed

    Kobayashi, Shū; Yamashita, Yasuhiro

    2011-01-18

    The group 2 alkaline earth metals calcium (Ca), strontium (Sr), and barium (Ba) are among the most common elements on Earth, abundant in both the sea and the Earth's crust. Although they are familiar in our daily lives, their application to organic synthesis has, so far, been limited. Some particularly useful properties of these elements include (i) low electronegativity, (ii) a stable oxidation state of +2, meaning that they can potentially form two covalent bonds with anions, and (iii) the ability to occupy a variety of coordination sites due to their large ionic radius. Furthermore, the alkaline earth metals, found between the group 1 and group 3 elements, show mild but significant Lewis acidity, which can be harnessed to control coordinative molecules via a Lewis acid-base interaction. Taken together, these characteristics make the metals Ca, Sr, and Ba very promising components of highly functionalized acid-base catalysts. In this Account, we describe the development of chiral alkaline earth metal catalysts for asymmetric carbon-carbon bond-forming reactions. Recently prepared chiral alkaline earth metal complexes have shown high diastereo- and enantioselectivities in fundamental and important chemical transformations. We chose chiral bisoxazoline (Box) derivatives bearing a methylene tether as a ligand for chiral modification. These molecules are very useful because they can covalently coordinate to alkaline earth metals in a bidentate fashion through deprotonation of the tether portion. It was found that chiral calcium-Box complexes could successfully promote catalytic asymmetric 1,4-addition and [3 + 2] cycloaddition reactions with high diastereo- and enantioselectivities. Both the calcium-Box complexes and chiral strontium-bis-sulfonamide and chiral barium-BINOLate complexes could catalyze asymmetric 1,4-addition reactions with high enantioselectivities. Furthermore, we designed a calcium-neutral coordinative ligand complex as a new type of chiral alkaline

  14. Crystal structure of alkaline cellulase K: insight into the alkaline adaptation of an industrial enzyme.

    PubMed

    Shirai, T; Ishida, H; Noda, J; Yamane, T; Ozaki, K; Hakamada, Y; Ito, S

    2001-07-27

    The crystal structure of the catalytic domain of alkaline cellulase K was determined at 1.9 A resolution. Because of the most alkaliphilic nature and it's highest activity at pH 9.5, it is used commercially in laundry detergents. An analysis of the structural bases of the alkaliphilic character of the enzyme suggested a mechanism similar to that previously proposed for alkaline proteases, that is, an increase in the number of Arg, His, and Gln residues, and a decrease in Asp and Lys residues. Some ion pairs were formed by the gained Arg residues, which is similar to what has been found in the alkaline proteases. Lys-Asp ion pairs are disfavored and partly replaced with Arg-Asp ion pairs. The alkaline adaptation appeared to be a remodeling of ion pairs so that the charge balance is kept in the high pH range.

  15. Bioprecipitation of uranium from alkaline waste solutions using recombinant Deinococcus radiodurans.

    PubMed

    Kulkarni, Sayali; Ballal, Anand; Apte, Shree Kumar

    2013-11-15

    Bioremediation of uranium (U) from alkaline waste solutions remains inadequately explored. We engineered the phoK gene (encoding a novel alkaline phosphatase, PhoK) from Sphingomonas sp. for overexpression in the radioresistant bacterium Deinococcus radiodurans. The recombinant strain thus obtained (Deino-PhoK) exhibited remarkably high alkaline phosphatase activity as evidenced by zymographic and enzyme activity assays. Deino-PhoK cells could efficiently precipitate uranium over a wide range of input U concentrations. At low uranyl concentrations (1 mM), the strain precipitated >90% of uranium within 2h while a high loading capacity of around 10.7 g U/g of dry weight of cells was achieved at 10 mM U concentration. Uranium bioprecipitation by Deino-PhoK cells was not affected in the presence of Cs and Sr, commonly present in intermediate and low level liquid radioactive waste, or after exposure to very high doses of ionizing radiation. Transmission electron micrographs revealed the extracellular nature of bioprecipitated U, while X-ray diffraction and fluorescence analysis identified the precipitated uranyl phosphate species as chernikovite. When immobilized into calcium alginate beads, Deino-PhoK cells efficiently removed uranium, which remained trapped in beads, thus accomplishing physical separation of precipitated uranyl phosphate from solutions. The data demonstrate superior ability of Deino-PhoK, over earlier reported strains, in removal of uranium from alkaline solutions and its potential use in bioremediation of nuclear and other waste.

  16. Colorimetric protein assay techniques.

    PubMed

    Sapan, C V; Lundblad, R L; Price, N C

    1999-04-01

    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  17. eIF4B stimulates eIF4A ATPase and unwinding activities by direct interaction through its 7-repeats region.

    PubMed

    Andreou, Alexandra Zoi; Harms, Ulf; Klostermeier, Dagmar

    2017-01-02

    Eukaryotic translation initiation starts with binding of the eIF4F complex to the 5'-m(7)G cap of the mRNA. Recruitment of the 43S pre-initiation complex (PIC), formed by the 40S ribosomal subunit and other translation initiation factors, leads to formation of the 48S PIC that then scans the 5'-untranslated region (5'-UTR) toward the start codon. The eIF4F complex consists of eIF4E, the cap binding protein, eIF4A, a DEAD-box RNA helicase that is believed to unwind secondary structures in the 5'-UTR during scanning, and eIF4G, a scaffold protein that binds to both eIF4E and eIF4A. The ATPase and helicase activities of eIF4A are jointly stimulated by eIF4G and the translation initiation factor eIF4B. Yeast eIF4B mediates recruitment of the 43S PIC to the cap-bound eIF4F complex by interacting with the 40S subunit and possibly with eIF4A. However, a direct interaction between yeast eIF4A and eIF4B has not been demonstrated yet. Here we show that eIF4B binds to eIF4A in the presence of RNA and ADPNP, independent of the presence of eIF4G. A stretch of seven moderately conserved repeats, the r1-7 region, is responsible for complex formation, for modulation of the conformational energy landscape of eIF4A by eIF4B, and for stimulating the RNA-dependent ATPase- and ATP-dependent RNA unwinding activities of eIF4A. The isolated r1-7 region only slightly stimulates eIF4A conformational changes and activities, suggesting that communication of the repeats with other regions of eIF4B is required for full stimulation of eIF4A activity, for recruitment of the PIC to the mRNA and for translation initiation.

  18. The sulphate-reduction alkalinity pump tested

    NASA Astrophysics Data System (ADS)

    Meister, Patrick; Petrishcheva, Elena

    2016-04-01

    Carbonate precipitation has been suggested to be induced by alkalinity increase during sulphate reduction under anoxic conditions. This mechanism may explain the formation of carbonate deposits in shallow marine environments, either within a redox stratified sediment inhabited by phototrophic microbial mats or in shallow water within the photic zone where sulphidic water is upwelling onto the shelf. The alkalinity pump may work as long as the sulphide is not reoxidized to sulphate, a process that would acidify the surrounding. The alkalinity effect of sulphate reduction was recently tested by Aloisi (2008) for microbial mats using a model approach. He found that sulphate reduction does not significantly increase or even decrease carbonate saturation and is unlikely to have played a significant role through Earth history. The model considers many environmental factors, including the effect of carbonate precipitation itself on the carbonate equilbrium and on the alkalinity. We used a modified version of Aloisi's (2008) model to simulate the saturation states of aragonite, calcite and dolomite without the effects of carbonate precipitation. This is necessary to evaluate the effect of microbial metabolisms exclusively on carbonate saturation, since carbonate precipitation is only the consequence, but not the cause of oversaturation. First results show that the saturation state is increased in the zone of phototrophic CO2 uptake. In contrast, the saturation state is strongly decreased in the zone where dissolved oxygen overlaps with dissolved sulphide. Aerobic sulphide oxidation consumes most of the HS- and dissipates most of the alkalinity produced in the sulphate reduction zone below. Hence, our results are consistent with the findings of Aloisi (2008), and they even more clearly show that sulphate reduction does not induce carbonate precipitation nor contributes to carbonate precipitation in combination with phototrophic CO2 uptake. The alkalinity effect of sulphate

  19. [Leucocyte alkaline phosphatase in normal and pathological pregnancy (author's transl)].

    PubMed

    Stark, K H; Zaki, I; Sobolewski, K

    1981-01-01

    The activities of leucocyte alkaline phosphatase were determined in 511 patients with normal and pathological pregnancy. Mean values were compared and the enzyme followed up, and the conclusion was drawn that leucocyte alkaline phosphatase was no safe indicator of foetal condition. No direct relationship were found to exist between leucocyte alkaline phosphatase, total oestrogens, HSAP, HLAP, HPL, and oxytocinase.

  20. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2012-05-15

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  1. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K.; Snyderman, Neal J.; Rowland, Mark S.

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  2. Alkaline Phosphatase Revisited:  Hydrolysis of Alkyl Phosphates (†).

    PubMed

    O'Brien, Patrick J; Herschlag, Daniel

    2002-03-05

    Escherichia coli alkaline phosphatase (AP) is the prototypical two metal ion catalyst with two divalent zinc ions bound ∼4 Å apart in the active site. Studies spanning half a century have elucidated many structural and mechanistic features of this enzyme, rendering it an attractive model for investigating the potent catalytic power of bimetallic centers. Unfortunately, fundamental mechanistic features have been obscured by limitations with the standard assays. These assays generate concentrations of inorganic phosphate (Pi) in excess of its inhibition constant (K i ≈ 1 μM). This tight binding by Pi has affected the majority of published kinetic constants. Furthermore, binding limits k cat/K m for reaction of p-nitrophenyl phosphate, the most commonly employed substrate. We describe a sensitive (32)P-based assay for hydrolysis of alkyl phosphates that avoids the complication of product inhibition. We have revisited basic mechanistic features of AP with these alkyl phosphate substrates. The results suggest that the chemical step for phosphorylation of the enzyme limits k cat/K m. The pH-rate profile and additional results suggest that the serine nucleophile is active in its anionic form and has a pK a of ≤5.5 in the free enzyme. An inactivating pK a of 8.0 is observed for binding of both substrates and inhibitors, and we suggest that this corresponds to ionization of a zinc-coordinated water molecule. Counter to previous suggestions, inorganic phosphate dianion appears to bind to the highly charged AP active site at least as strongly as the trianion. The dependence of k cat/K m on the pK a of the leaving group follows a Brønsted correlation with a slope of βlg = -0.85 ± 0.1, differing substantially from the previously reported value of -0.2 obtained from data with a less sensitive assay. This steep leaving group dependence is consistent with a largely dissociative transition state for AP-catalyzed hydrolysis of phosphate monoesters. The new (32)P

  3. Surfactant-enhanced alkaline flooding field project

    SciTech Connect

    French, T.R.

    1991-10-01

    The Tucker sand of Helper (KS) field is a candidate for surfactant-enhanced alkaline flooding. The geology of the Helper site is typical of many DOE Class I reservoirs. The Tucker sand of Helper field was deposited in a fluvial dominated deltaic environment. Helper oil can be mobilized with either chemical system 2 or chemical system 3, as described in this report. Oil fields in the Gulf Coast region are also good candidates for surfactant-enhanced alkaline flooding. The results from laboratory tests conducted in Berea sandstone cores with oil brine from Helper (KS) field are encouraging. The crude oil is viscous and non-acidic and, yet, was mobilized by the chemical formulations described in this report. Significant amounts of the oil were mobilized under simulated reservoir conditions. The results in Berea sandstone cores were encouraging and should be verified by tests with field core. Consumption of alkali, measured with field core, was very low. Surfactant loss appeared to be acceptable. Despite the good potential for mobilization of Helper oil, certain reservoir characteristics such as low permeability, compartmentalization, and shallow depth place constraints on applications of any chemical system in the Tucker sand. These constraints are typical of many DOE Class I reservoirs. Although Hepler field is not a perfect reservoir in which to apply surfactant- enhanced alkaline flooding, Hepler oil is particularly amenable to mobilization by surfactant-enhanced alkaline systems. A field test is recommended, dependent upon final evaluation of well logs and cores from the proposed pilot area. 14 refs., 21 figs., 10 tabs.

  4. The alkaline earth intercalates of molybdenum disulfide

    NASA Technical Reports Server (NTRS)

    Somoano, R. B.; Hadek, V.; Rembaum, A.; Samson, S.; Woollam, J. A.

    1975-01-01

    Molybdenum disulfide has been intercalated with calcium and strontium by means of the liquid ammonia technique. Chemical, X-ray, and superconductivity data are presented. The X-ray data reveal a lowering of crystal symmetry and increase of complexity of the structure upon intercalation with the alkaline earth metals. The Ca and Sr intercalates start to superconduct at 4 and 5.6 K, respectively, and show considerable anisotropy regarding the critical magnetic field.

  5. Alkaline earth cation extraction from acid solution

    DOEpatents

    Dietz, Mark; Horwitz, E. Philip

    2003-01-01

    An extractant medium for extracting alkaline earth cations from an aqueous acidic sample solution is described as are a method and apparatus for using the same. The separation medium is free of diluent, free-flowing and particulate, and comprises a Crown ether that is a 4,4'(5')[C.sub.4 -C.sub.8 -alkylcyclohexano]18-Crown-6 dispersed on an inert substrate material.

  6. Oxidation catalysts on alkaline earth supports

    DOEpatents

    Mohajeri, Nahid

    2017-03-21

    An oxidation catalyst includes a support including particles of an alkaline earth salt, and first particles including a palladium compound on the support. The oxidation catalyst can also include precious metal group (PMG) metal particles in addition to the first particles intermixed together on the support. A gas permeable polymer that provides a continuous phase can completely encapsulate the particles and the support. The oxidation catalyst may be used as a gas sensor, where the first particles are chemochromic particles.

  7. Inhibition of Alkaline Phosphatase by Several Diuretics

    DTIC Science & Technology

    1980-01-01

    August 20th, 1979) . . Summary , . Acetazolamide, furosemide, ethacrynic acid and chlorothiazide, diuretics of considerable structural diversity, inhibit...Ki is calculated to be 8.4, 7.0, 2.8 and 0.1 mmol/l for acetazolamide, furosemide, ethacrynic acid and chlorothiazide, respectively. Chlorothiazide...is a much more potent inhibitor of alkaline phos- phatase than the other three diuretics. The combination of ethacrynic acid and cysteine, itself an

  8. Alkaline flooding for enhanced oil recovery

    SciTech Connect

    Gittler, W.E.

    1983-09-01

    There are over 12 active projects of varying size using one of 3 major types of alkaline agents. These include sodium silicate, caustic soda, and soda ash. Among the largest pilots currently is the THUMS project in the Wilmington field, California. Plans called for the injection of a 4% weight concentration of sodium orthosilicate over a 60% PV. Through the first 3 yr, over 27 million bbl of chemicals have been injected. Gulf Oil is operating several alkaline floods, one of which is located off shore in the Quarantine Bay field, Louisiana. In this pilot, sodium hydroxide in a weight concentration of 5 to 12% is being injected. Belco Petroleum Corp. has reported that their pilot operating in the Isenhour Unit in Wyoming is using a .5% weight concentration of soda ash in conjunction with a polymer. Other uses for alkaline agents in chemical flooding include the use of silicate as a preflush or sacrificial agent in micellar/polymer and surfactant recovery systems. In addition, caustic has been tested in the surface-mixed caustic emulsion process while orthosilicate has been tested in a recovery method known as mobility-controlled caustic floods.

  9. A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

    PubMed

    Garbelli, Anna; Beermann, Sandra; Di Cicco, Giulia; Dietrich, Ursula; Maga, Giovanni

    2011-05-12

    DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

  10. Structure of p53 binding to the BAX response element reveals DNA unwinding and compression to accommodate base-pair insertion

    SciTech Connect

    Chen, Y.; Zhang, X.; Dantas Machado, A. C.; Ding, Y.; Chen, Z.; Qin, P. Z.; Rohs, R.; Chen, L.

    2013-07-08

    The p53 core domain binds to response elements (REs) that contain two continuous half-sites as a cooperative tetramer, but how p53 recognizes discontinuous REs is not well understood. Here we describe the crystal structure of the p53 core domain bound to a naturally occurring RE located at the promoter of the Bcl-2-associated X protein (BAX) gene, which contains a one base-pair insertion between the two half-sites. Surprisingly, p53 forms a tetramer on the BAX-RE that is nearly identical to what has been reported on other REs with a 0-bp spacer. Each p53 dimer of the tetramer binds in register to a half-site and maintains the same protein–DNA interactions as previously observed, and the two dimers retain all the protein–protein contacts without undergoing rotation or translation. To accommodate the additional base pair, the DNA is deformed and partially disordered around the spacer region, resulting in an apparent unwinding and compression, such that the interactions between the dimers are maintained. Furthermore, DNA deformation within the p53-bound BAX-RE is confirmed in solution by site-directed spin labeling measurements. Our results provide a structural insight into the mechanism by which p53 binds to discontinuous sites with one base-pair spacer.

  11. Expression of a human placental alkaline phosphatase gene in transfected cells: Use as a reporter for studies of gene expression

    SciTech Connect

    Henthorn, P.; Zervos, P.; Raducha, M.; Harris, H.; Kadesch, T.

    1988-09-01

    The human placental alkaline phosphatase gene has been cloned and reintroduced into mammalian cells. When a plasmid carrying the gene under control of the simian virus 40 early promoter (pSV2Apap) is transfected into a variety of different cell types, placental alkaline phosphatase activity can readily be detected by using whole cell suspensions or cell lysates. Alkaline phosphatase activity can also be visualized directly in individual transfected cells by histochemical staining. The gene is appropriate for use as a reporter in studies of gene regulation since its expression is dependent on the presence of exogenous transcription control elements. The overall assay to detect the expression of the gene is quantitative, very rapid, and inexpensive. Cotransfections of cells with pSV2Apap and a related plasmid carrying the bacterial chloramphenicol acetyltransferase gene (pSV2Acat) indicate that transcription of these two genes is detected with roughly the same sensitivity.

  12. Mesozoic mafic alkaline magmatism of southern Scandinavia

    NASA Astrophysics Data System (ADS)

    Tappe, Sebastian

    2004-11-01

    More than 100 volcanic necks in central Scania (southern Sweden) are the product of Jurassic continental rift-related mafic alkaline magmatism at the southwest margin of the Baltic Shield. They are mainly basanites, with rarer melanephelinites. Both rock groups display overlapping primitive Mg-numbers, Cr and Ni contents, steep chondrite-normalized rare earth element patterns (LaN /YbN = 17 27) and an overall enrichment in incompatible elements. However, the melanephelinites are more alkaline and have stronger high field strength element enrichment than the basanites. The existence of distinct primary magmas is also indicated by heterogeneity in highly incompatible element ratios (e.g. Zr/Nb, La/Nb). Trace element modelling indicates that the magmas were generated by comparably low degrees of melting of a heterogeneous mantle source. Such a source can best be explained by a metasomatic overprint of the mantle lithosphere by percolating evolved melts. The former existence of such alkaline trace element-enriched melts can be demonstrated by inversion of the trace element content of green-core clinopyroxenes and anorthoclase which occur as xenocrysts in the melanephelinites and are interpreted as being derived from crystallization of evolved mantle melts. Jurassic magmatic activity in Scania was coeval with the generation of nephelinites in the nearby Egersund Basin (Norwegian North Sea). Both Scanian and North Sea alkaline magmas share similar trace element characteristics. Mantle enrichment processes at the southwest margin of the Baltic Shield and the North Sea Basin generated trace element signatures similar to those of ocean island basalts (e.g. low Zr/Nb and La/Nb) but there are no indications of plume activity during the Mesozoic in this area. On the contrary, the short duration of rifting, absence of extensive lithospheric thinning, and low magma volumes argue against a Mesozoic mantle plume. It seems likely that the metasomatic imprint resulted from the

  13. Alkaline and ultrasound assisted alkaline pretreatment for intensification of delignification process from sustainable raw-material.

    PubMed

    Subhedar, Preeti B; Gogate, Parag R

    2014-01-01

    Alkaline and ultrasound-assisted alkaline pretreatment under mild operating conditions have been investigated for intensification of delignification. The effect of NaOH concentration, biomass loading, temperature, ultrasonic power and duty cycle on the delignification has been studied. Most favorable conditions for only alkaline pretreatment were alkali concentration of 1.75 N, solid loading of 0.8% (w/v), temperature of 353 K and pretreatment time of 6 h and under these conditions, 40.2% delignification was obtained. In case of ultrasound-assisted alkaline approach, most favorable conditions obtained were alkali concentration of 1N, paper loading of 0.5% (w/v), sonication power of 100 W, duty cycle of 80% and pretreatment time of 70 min and the delignification obtained in ultrasound-assisted alkaline approach under these conditions was 80%. The material samples were characterized by FTIR, SEM, XRD and TGA technique. The lignin was recovered from solution by precipitation method and was characterized by FTIR, GPC and TGA technique.

  14. Alkaline solution/binder ratio as a determining factor in the alkaline activation of aluminosilicates

    SciTech Connect

    Ruiz-Santaquiteria, C.; Fernandez-Jimenez, A.; Palomo, A.

    2012-09-15

    This study investigates the effect of the alkaline solution/binder (S/B) ratio on the composition and nanostructure of the reaction products generated in the alkaline activation of aluminosilicates. The experiments used two mixtures of fly ash and dehydroxylated white clay and for each of these, varying proportions of the solution components. The alkali activator was an 8 M NaOH solution (with and without sodium silicate) used at three S/B ratios: 0.50, 0.75 and 1.25. The {sup 29}Si, {sup 27}Al MAS NMR and XRD characterisation of the reaction products reveal that for ratios nearest the value delivering suitable paste workability, the reaction-product composition and structure depend primarily on the nature and composition of the starting materials and the alkaline activator used. However, when an excess alkaline activator is present in the system, the reaction products tend to exhibit SiO{sub 2}/Al{sub 2}O{sub 3} ratios of approximately 1, irrespective of the composition of the starting binder or the alkaline activator.

  15. Trypanosoma rangeli: an alkaline ecto-phosphatase activity is involved with survival and growth of the parasite.

    PubMed

    Dos-Santos, André L A; Dick, Claudia F; Silveira, Thaís S; Fonseca-de-Souza, André L; Meyer-Fernandes, José R

    2013-10-01

    The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using β-glycerophosphate (β-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of β-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of β-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca(2+) present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. β-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (Pi). Levamisole at the IC50 spared significantly parasite growth when β-GP was the sole source of Pi and stopped it in the absence of β-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.

  16. Cascade enzyme-linked immunosorbent assay (CELISA).

    PubMed

    Lee, Young-mi; Jeong, Yujin; Kang, Hyo Jin; Chung, Sang J; Chung, Bong Hyun

    2009-10-15

    Immunoassays are representative biochemical detection methods. Among them, sandwich-type immunoassays, typified by sandwich ELISA, have used in disease diagnosis or biochemical detection with high target selectivity. Horseradish peroxidase and alkaline phosphatase have been typically used for signal amplification in ELISA. Recently developed sandwich-type immunoassays such as biobarcode immunoassays, immuno-PCR, and immuno-RCA have improved sensitivity by changing mainly the signal amplification method. To develop a novel amplification method in ELISA, an enzyme-cascading system was incorporated into an ELISA, and the new assay is termed a cascading enzyme-linked immunosorbent assay (CELISA). This CELISA includes a trypsinogen-enterokinase combination as the cascading enzyme system, and was used to detect alpha-fetoprotein (AFP), which is a liver cancer marker, and prostate-specific antigen (PSA). Using a colorimetric reagent for signal generation, CELISA had 0.1-10pM limits-of-detection for AFP and PSA in whole human serum and assay buffers, depending on the platform, well plate, or microbead type used. This study represents the first example that incorporated an enzyme cascading step in an ELISA system, resulting in successful signal amplification with sensitive detection of pathogenic antigens in serum.

  17. Downscaling Alkaline Phosphatase Activity in a Subtropical Reservoir

    NASA Astrophysics Data System (ADS)

    Tseng, Y.

    2011-12-01

    This research was conducted by downscaling study to understand phosphorus (P)-deficient status of different plankton and the role of alkaline phosphatase activity (APA) in subtropical Feitsui Reservoir. Results from field survey showed that bulk APA (1.6~95.2 nM h-1) was widely observed in the epilimnion (0~20 m) with an apparent seasonal variations, suggesting that plankton in the system were subjected to P-deficient seasonally. Mixed layer depth (an index of phosphate availability) is the major factor influencing the variation of bulk APA and specific APA (124~1,253 nmol mg C-1 h-1), based on multiple linear regression analysis. Size-fractionated APA assays showed that picoplankton (size 0.2~3 um) contributed most of the bulk APA in the system. In addition, single-cell APA detected by enzyme-labeled fluorescence (ELF) assay indicated that heterotrophic bacteria are the major contributors of APA. Thus, we can infer that bacteria play an important role in accelerating P-cycle within P-deficient systems. Light/nutrient manipulation bioassays showed that bacterial growth was directly controlled by phosphate, while picocyanobacterial growth is controlled by light and can out-compete bacteria under P-limited condition with the aid of light. Further analysis revealed that the strength of summer typhoon is a factor responsible for the inter-annual variability of bulk and specific APA. APA study demonstrated the episodic events (e.g. strong typhoon and extreme precipitation) had significant influence on APA variability in sub-tropical to tropical aquatic ecosystems. Hence, the results herein will allow future studies on monitoring typhoon disturbance (intensity and frequency) as well as the APA of plankton during summer-to-autumn in subtropical systems.

  18. CPTAC Assay Portal: a repository of targeted proteomic assays

    SciTech Connect

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Rodriguez, Henry; Abbateillo, Susan E.; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Paulovich, Amanda G.

    2014-06-27

    To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigators to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.

  19. An overview on fermentation, downstream processing and properties of microbial alkaline proteases.

    PubMed

    Gupta, R; Beg, Q K; Khan, S; Chauhan, B

    2002-12-01

    Microbial alkaline proteases dominate the worldwide enzyme market, accounting for a two-thirds share of the detergent industry. Although protease production is an inherent property of all organisms, only those microbes that produce a substantial amount of extracellular protease have been exploited commercially. Of these, strains of Bacillus sp. dominate the industrial sector. To develop an efficient enzyme-based process for the industry, prior knowledge of various fermentation parameters, purification strategies and properties of the biocatalyst is of utmost importance. Besides these, the method of measurement of proteolytic potential, the selection of the substrate and the assay protocol depends upon the ultimate industrial application. A large array of assay protocols are available in the literature; however, with the predominance of molecular approaches for the generation of better biocatalysts, the search for newer substrates and assay protocols that can be conducted at micro/nano-scale are becoming important. Fermentation of proteases is regulated by varying the C/N ratio and can be scaled-up using fed-batch, continuous or chemostat approaches by prolonging the stationary phase of the culture. The conventional purification strategy employed, involving e.g., concentration, chromatographic steps, or aqueous two-phase systems, depends on the properties of the protease in question. Alkaline proteases useful for detergent applications are mostly active in the pH range 8-12 and at temperatures between 50 and 70 degrees C, with a few exceptions of extreme pH optima up to pH 13 and activity at temperatures up to 80-90 degrees C. Alkaline proteases mostly have their isoelectric points near to their pH optimum in the range of 8-11. Several industrially important proteases have been subjected to crystallization to extensively study their molecular homology and three-dimensional structures.

  20. Lateral flow assays

    PubMed Central

    Koczula, Katarzyna M.

    2016-01-01

    Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored. PMID:27365041

  1. Cell viability assays: introduction.

    PubMed

    Stoddart, Martin J

    2011-01-01

    The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

  2. Tube-Forming Assays.

    PubMed

    Brown, Ryan M; Meah, Christopher J; Heath, Victoria L; Styles, Iain B; Bicknell, Roy

    2016-01-01

    Angiogenesis involves the generation of new blood vessels from the existing vasculature and is dependent on many growth factors and signaling events. In vivo angiogenesis is dynamic and complex, meaning assays are commonly utilized to explore specific targets for research into this area. Tube-forming assays offer an excellent overview of the molecular processes in angiogenesis. The Matrigel tube forming assay is a simple-to-implement but powerful tool for identifying biomolecules involved in angiogenesis. A detailed experimental protocol on the implementation of the assay is described in conjunction with an in-depth review of methods that can be applied to the analysis of the tube formation. In addition, an ImageJ plug-in is presented which allows automatic quantification of tube images reducing analysis times while removing user bias and subjectivity.

  3. Desialylated alkaline phosphatase: activation by 4-nitrophenol.

    PubMed

    Nayudu, P R

    1984-01-01

    Mouse ileal alkaline phosphatase is a sialyl enzyme (12-14 moles per mole of enzyme). When partially desialylated by treatment with neuraminidase, the enzyme loses most of its activity, associated with reduced apparent Vmax and Km. Part of that loss, however, is recovered as the product 4-nitrophenol's concentration builds up in the cuvette. Experimental results are presented to demonstrate that the activation is due to the binding of 4-nitrophenol as a ligand by the partially desialylated enzyme and that both the loss of activity by sialic acid removal and activation by ligand-binding are correlated with changes in protein conformation.

  4. Doped colorimetric assay liposomes

    DOEpatents

    Charych, Deborah; Stevens, Raymond C.

    2001-01-01

    The present invention provides compositions comprising colorimetric assay liposomes. The present invention also provides methods for producing colorimetric liposomes and calorimetric liposome assay systems. In preferred embodiments, these calorimetric liposome systems provide high levels of sensitivity through the use of dopant molecules. As these dopants allow the controlled destabilization of the liposome structure, upon exposure of the doped liposomes to analyte(s) of interest, the indicator color change is facilitated and more easily recognized.

  5. Multiple log potash assay

    NASA Astrophysics Data System (ADS)

    Hill, D. G.

    1993-10-01

    A five-mineral multiple-log potash assay technique has been successfully applied to evaluate potash-rich intervals in evaporite sequences. The technique is able to distinguish economic potash minerals from non-economic potash minerals and from other non-potash radioactive minerals. It can be applied on location, using a programmable calculator or microcomputer, providing near real-time logs of potash mineral concentrations. Log assay values show good agreement with core wet chemistry analyses.

  6. SNAP Assay Technology.

    PubMed

    O'Connor, Thomas P

    2015-12-01

    The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described.

  7. Serum selenium assay following serum ferritin assay

    SciTech Connect

    Stevens, R.G.; Morris, J.S.; Hann, H.L.; Pulsipher, B.; Stahlhut, M.W.

    1986-08-01

    Stored serum samples can be an important research resource into the etiology of cancer. These sera cannot be replaced and should therefore be used to best advantage. In previous epidemiologic studies, only single serum constituents have been assayed in individual serum samples. For example, serum ferritin has been examined in samples stored for as long as 10 years at -20C for a possible relation with general mortality (1) and cancer death (2). Ferritin is the tissue iron-storage protein and is therefore subject to denaturation. Serum selenium has also been examined in relation to cancer risk in a prospective manner by using stored frozen serum samples (3, 4). The interactions of a variety of serum factors in relation to cancer risk would be a desirable research goal, except that the amounts of serum typically available in frozen serum banks are less than 1 ml. It was the purpose of this investigation to determine if a radioimmunoassay for ferritin affected a subsequent neutron activation assay for selenium on the same 0.1 ml serum sample.

  8. Alkaline chemistry of transuranium elements and technetium and the treatment of alkaline radioactive wastes

    SciTech Connect

    Delegard, C.H.; Peretrukhin, V.F.; Shilov, V.P.; Pikaev, A.K.

    1995-05-01

    Goal of this survey is to generalize the known data on fundamental physical-chemical properties of TRUs and Tc, methods for their isolation, and to provide recommendations that will be useful for partitioning them from alkaline high-level wastes.

  9. Comparative structural effects of HIV-1 Gag and nucleocapsid proteins in binding to and unwinding of the viral RNA packaging signal.

    PubMed

    Bell, Neil M; Kenyon, Julia C; Balasubramanian, Shankar; Lever, Andrew M L

    2012-04-17

    The major RNA binding region of the HIV-1 Gag polyprotein is the nucleocapsid (NC) domain, which is responsible for the specific capture of the genomic RNA genome during viral assembly. The Gag polyprotein has other RNA chaperone functions, which are mirrored by the isolated NC protein after physiological cleavage from Gag. Gag, however, is suggested to have superior nucleic acid chaperone activity. Here we investigate the interaction of Gag and NC with the core RNA structure of the HIV-1 packaging signal (Ψ), using 2-aminopurine substitution to create a series of modified RNAs based on the Ψ helix loop structure. The effects of 2-aminopurine substitution on the physical and structural properties of the viral Ψ were characterized. The fluorescence properties of the 2-aminopurine substitutions showed features consistent with the native GNAR tetraloop. Dissociation constants (K(d)) of the two viral proteins, measured by fluorescence polarization (FP), were similar, and both NC and Gag affected the 2-aminopurine fluorescence of bases close to the loop binding region in a similar fashion. However, the influence of Gag on the fluorescence of the 2-aminopurine nucleotides at the base of the helix implied a much more potent helix destabilizing action on the RNA stem loop (SL) versus that seen with NC. This was further supported when the viral Ψ SL was tagged with a 5' fluorophore and 3' quencher. In the absence of any viral protein, minimal fluorescence was detected; addition of NC yielded a slight increase in fluorescence, while addition of the Gag protein yielded a large change in fluorescence, further suggesting that, compared to NC, the Gag protein has a greater propensity to affect RNA structure and that Ψ helix unwinding may be an intrinsic step in RNA encapsidation.

  10. DNA Unwinding Functions of Minute Virus of Mice NS1 Protein Are Modulated Specifically by the Lambda Isoform of Protein Kinase C

    PubMed Central

    Dettwiler, Sabine; Rommelaere, Jean; Nüesch, Jürg P. F.

    1999-01-01

    The parvovirus minute virus of mice NS1 protein is a multifunctional protein involved in a variety of processes during virus propagation, ranging from viral DNA replication to promoter regulation and cytotoxic action to the host cell. Since NS1 becomes phosphorylated during infection, it was proposed that the different tasks of this protein might be regulated in a coordinated manner by phosphorylation. Indeed, comparing biochemical functions of native NS1 with its dephosphorylated counterpart showed that site-specific nicking of the origin and the helicase and ATPase activities are remarkably reduced upon NS1 dephosphorylation while site-specific affinity of the protein to the origin became enhanced. As a consequence, the dephosphorylated polypeptide is deficient for initiation of DNA replication. By adding fractionated cell extracts to a kinase-free in vitro replication system, the combination of two protein components containing members of the protein kinase C (PKC) family was found to rescue the replication activity of the dephosphorylated NS1 protein upon addition of PKC cofactors. One of these components, termed HA-1, also stimulated NS1 helicase function in response to acidic lipids but not phorbol esters, indicating the involvement of atypical PKC isoforms in the modulation of this NS1 function (J. P. F. Nüesch, S. Dettwiler, R. Corbau, and J. Rommelaere, J. Virol. 72:9966–9977, 1998). The present study led to the identification of atypical PKCλ/ι as the active component of HA-1 responsible for the regulation of NS1 DNA unwinding and replicative functions. Moreover, a target PKCλ phosphorylation site was localized at S473 of NS1. By site-directed mutagenesis, we showed that this residue is essential for NS1 helicase activity but not promoter regulation, suggesting a possible modulation of NS1 functions by PKCλ phosphorylation at residue S473. PMID:10438831

  11. Response of Desulfovibrio vulgaris to alkaline stress.

    PubMed

    Stolyar, Sergey; He, Qiang; Joachimiak, Marcin P; He, Zhili; Yang, Zamin Koo; Borglin, Sharon E; Joyner, Dominique C; Huang, Katherine; Alm, Eric; Hazen, Terry C; Zhou, Jizhong; Wall, Judy D; Arkin, Adam P; Stahl, David A

    2007-12-01

    The response of exponentially growing Desulfovibrio vulgaris Hildenborough to pH 10 stress was studied using oligonucleotide microarrays and a study set of mutants with genes suggested by microarray data to be involved in the alkaline stress response deleted. The data showed that the response of D. vulgaris to increased pH is generally similar to that of Escherichia coli but is apparently controlled by unique regulatory circuits since the alternative sigma factors (sigma S and sigma E) contributing to this stress response in E. coli appear to be absent in D. vulgaris. Genes previously reported to be up-regulated in E. coli were up-regulated in D. vulgaris; these genes included three ATPase genes and a tryptophan synthase gene. Transcription of chaperone and protease genes (encoding ATP-dependent Clp and La proteases and DnaK) was also elevated in D. vulgaris. As in E. coli, genes involved in flagellum synthesis were down-regulated. The transcriptional data also identified regulators, distinct from sigma S and sigma E, that are likely part of a D. vulgaris Hildenborough-specific stress response system. Characterization of a study set of mutants with genes implicated in alkaline stress response deleted confirmed that there was protective involvement of the sodium/proton antiporter NhaC-2, tryptophanase A, and two putative regulators/histidine kinases (DVU0331 and DVU2580).

  12. Autonomous in situ measurements of seawater alkalinity.

    PubMed

    Spaulding, Reggie S; DeGrandpre, Michael D; Beck, James C; Hart, Robert D; Peterson, Brittany; De Carlo, Eric H; Drupp, Patrick S; Hammar, Terry R

    2014-08-19

    Total alkalinity (AT) is an important parameter for describing the marine inorganic carbon system and understanding the effects of atmospheric CO2 on the oceans. Measurements of AT are limited, however, because of the laborious process of collecting and analyzing samples. In this work we evaluate the performance of an autonomous instrument for high temporal resolution measurements of seawater AT. The Submersible Autonomous Moored Instrument for alkalinity (SAMI-alk) uses a novel tracer monitored titration method where a colorimetric pH indicator quantifies both pH and relative volumes of sample and titrant, circumventing the need for gravimetric or volumetric measurements. The SAMI-alk performance was validated in the laboratory and in situ during two field studies. Overall in situ accuracy was -2.2 ± 13.1 μmol kg(-1) (n = 86), on the basis of comparison to discrete samples. Precision on duplicate analyses of a carbonate standard was ±4.7 μmol kg(-1) (n = 22). This prototype instrument can measure in situ AT hourly for one month, limited by consumption of reagent and standard solutions.

  13. Advanced inorganic separators for alkaline batteries

    NASA Technical Reports Server (NTRS)

    Sheibley, D. W. (Inventor)

    1982-01-01

    A flexible, porous battery separator comprising a coating applied to a porous, flexible substrate is described. The coating comprises: (1) a thermoplastic rubber-based resin which is insoluble and unreactive in the alkaline electrolyte; (2) a polar organic plasticizer which is reactive with the alkaline electrolyte to produce a reaction product which contains a hydroxyl group and/or a carboxylic acid group; and (3) a mixture of polar particulate filler materials which are unreactive with the electrolyte, the mixture comprising at least one first filler material having a surface area of greater than 25 meters sq/gram, at least one second filler material having a surface area of 10 to 25 sq meters/gram, wherein the volume of the mixture of filler materials is less than 45% of the total volume of the fillers and the binder, the filler surface area per gram of binder is about 20 to 60 sq meters/gram, and the amount of plasticizer is sufficient to coat each filler particle. A method of forming the battery separator is also described.

  14. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    SciTech Connect

    SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

    2004-03-24

    Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNA populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.

  15. Thermodynamic model for an alkaline fuel cell

    NASA Astrophysics Data System (ADS)

    Verhaert, Ivan; De Paepe, Michel; Mulder, Grietus

    Alkaline fuel cells are low temperature fuel cells for which stationary applications, e.g. cogeneration in buildings, are a promising market. In order to guarantee a long life, water and thermal management has to be done in a careful way. In order to better understand the water, alkali and thermal flows, a two-dimensional model for an Alkaline Fuel Cell is developed using a control volume approach. In each volume the electrochemical reactions together with the mass and energy balance are solved. The model is created in Aspen Custom Modeller, the development environment of Aspen Plus, where special attention is given to the physical flow of hydrogen, water and air in the system. In this way the developed component, the AFC-cell, can be built into stack configurations to understand its effect on the overall performance. The model is validated by experimental data from measured performance by VITO with their Cell Voltage Monitor at a test case, where the AFC-unit is used as a cogeneration unit.

  16. The effect of alkaline agents on retention of EOR chemicals

    SciTech Connect

    Lorenz, P.B.

    1991-07-01

    This report summarizes a literature survey on how alkaline agents reduce losses of surfactants and polymers in oil recovery by chemical injection. Data are reviewed for crude sulfonates, clean anionic surfactants, nonionic surfactants, and anionic and nonionic polymers. The role of mineral chemistry is briefly described. Specific effects of various alkaline anions are discussed. Investigations needed to improve the design of alkaline-surfactant-polymer floods are suggested. 62 refs., 28 figs., 6 tabs.

  17. The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum.

    PubMed Central

    Bailyes, E M; Seabrook, R N; Calvin, J; Maguire, G A; Price, C P; Siddle, K; Luzio, J P

    1987-01-01

    1. Liver and bone alkaline phosphatase isoenzymes were solubilized with the zwitterionic detergent sulphobetaine 14, and purified to homogeneity by using a monoclonal antibody previously raised against a partially-purified preparation of the liver isoenzyme. Both purified isoenzymes had a specific activity in the range 1100-1400 mumol/min per mg of protein with a subunit Mr of 80,000 determined by SDS/polyacrylamide gel electrophoresis. Butanol extraction instead of detergent solubilization, before immunoaffinity purification of the liver enzyme, resulted in the same specific activity and subunit Mr. The native Mr of the sulphobetaine 14-solubilized enzyme was consistent with the enzyme being a dimer of two identical subunits and was higher than that of the butanol-extracted enzyme, presumably due to the binding of the detergent micelle. 2. Pure bone and liver alkaline phosphatase were used to raise further antibodies to the two isoenzymes. Altogether, 27 antibody-producing cell lines were cloned from 12 mice. Several of these antibodies showed a greater than 2-fold preference for bone alkaline phosphatase in the binding assay used for screening. No antibodies showing a preference for liver alkaline phosphatase were successfully cloned. None of the antibodies showed significant cross-reaction with placental or intestinal alkaline phosphatase. Epitope analysis of the 27 antibodies using liver alkaline phosphatase as antigen gave rise to six groupings, with four antibodies unclassified. The six major epitope groups were also observed using bone alkaline phosphatase as antigen. 3. Serum from patients with cholestasis contains soluble and particulate forms of alkaline phosphatase. The soluble serum enzyme had the same size and charge as butanol-extracted liver enzyme on native polyacrylamide-gel electrophoresis. Cellulose acetate electrophoresis separated the soluble and particulate serum alkaline phosphatases as slow- and fast-moving forms respectively. In the

  18. Bluetongue virus VP6 protein binds ATP and exhibits an RNA-dependent ATPase function and a helicase activity that catalyze the unwinding of double-stranded RNA substrates.

    PubMed Central

    Stäuber, N; Martinez-Costas, J; Sutton, G; Monastyrskaya, K; Roy, P

    1997-01-01

    RNA-dependent ATPase and helicase activities have been identified associated with the purified VP6 protein of bluetongue virus, a member of the Orbivirus genus of double-stranded RNA (dsRNA; Reoviridae family) viruses. In addition, the protein has an ATP binding activity. RNA unwinding of duplexes occurred with both 3' and 5' overhang templates, as well as with blunt-ended dsRNA, an activity not previously identified in other viral helicases. Although little sequence similarity to other helicases was detected, certain similarities to motifs commonly attributed to such proteins were identified. PMID:9311795

  19. Homogeneous, bioluminescent proteasome assays.

    PubMed

    O'Brien, Martha A; Moravec, Richard A; Riss, Terry L; Bulleit, Robert F

    2015-01-01

    Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.

  20. SIGMA RECEPTOR BINDING ASSAYS

    PubMed Central

    CHU, UYEN B.; RUOHO, ARNOLD E.

    2016-01-01

    Sigma receptors belong to a class of small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated receptors, of which there are two subtypes: the Sigma-1 receptor (S1R) and the Sigma-2 receptor (S2R). Both S1R and S2R bind to a number of drugs including antipsychotic, haloperidol, and the opioid analgesic, (+)-pentazocine. Sigma receptors are implicated in multiple disease pathologies associated with the nervous system including diseases affecting motor control such as Amyotrophic Lateral Sclerosis (ALS) and Alzeimher's disease. This unit describes methods for the pharmacological characterization of S1R and S2R using radioligand-binding assays. In the first section, radioligand saturation binding assay to determine receptor densities and competitive inhibition assays to characterize affinities of novel compounds are presented for S1R using the selective S1R ligand, [3H]-(+)-pentazocine. The second section describes radioligand saturation binding assay and competitive inhibition assays for the S2R using a non-selective S1R and S2R ligand, [3H]-1,3-di(2-tolyl)guanidine ([3H]-DTG). PMID:26646191

  1. Enzymatic activity of alkaline phosphatase inside protein and polymer structures fabricated via multiphoton excitation.

    PubMed

    Basu, Swarna; Campagnola, Paul J

    2004-01-01

    We demonstrate micron scale control of bioactivity through the use of multiphoton excited photochemistry, where this technique has been used to cross-link three-dimensional matrixes of alkaline phosphatase, bovine serum albumin, and polyacrylamide and combinations therein. Using a fluorescence-based assay (ELF-97), the enzymatic activity has been studied using a Michaelis-Menten analysis, and we have measured the specificity constants kcat/KM for alkaline phosphatase in both the protein and polymer matrixes to be on the order of 10(5)-10(6) M(-1) s(-1)and are comparable to known literature values in other environments. It is found that the enzyme is simply entrapped in the polymer matrix, whereas it is completely covalently bound in the protein structures. The relative reaction rate of alkaline phosphatase bound to BSA with the ELF substrate was measured as a function of cross-link density and was found to decrease in the more tightly formed matrixes, indicating a decrease in the diffusion in the matrix.

  2. Enhanced coagulation with polyaluminum chlorides: role of pH/alkalinity and speciation.

    PubMed

    Yan, Mingquan; Wang, Dongsheng; Yu, Jianfeng; Ni, Jinren; Edwards, Marc; Qu, Jiuhui

    2008-04-01

    Enhanced coagulation is considered to be among the best available techniques (BAT) for disinfection by-product (DBP) precursor removal in water treatment. Improving existing understanding requires further consideration of nuances of chemical speciation relative to source water chemistry. In this paper, the effect of alkalinity/pH and speciation on inorganic polymer flocculants, polyaluminum chlorides (PACls) for enhanced particle and natural organic matter (NOM) removal was investigated. Three kinds of well-characterized typical source waters in China with low, moderate, and high alkalinity were selected. Performance of coagulants is controlled not only by preformed species but also by those formed in situ. At neutral and basic pH values, PACls with higher basicity (ratio of OH(-)/Al), which have more stable preformed Alb (the rapid reacted species as in ferron assay), are more efficient for turbidity and NOM removal. At slightly acidic pH, PACls with lower basicity are more efficient since more Alb can be formed in situ. Optimal NOM removal was achieved at pH 5.5-6.5 for all PACls. Basicity, speciation, and dosage of coagulant should be optimized based on raw water alkalinity to enhance the removal efficiency of NOM.

  3. Rover waste assay system

    SciTech Connect

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  4. Clonogenic Assay: Adherent Cells

    PubMed Central

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T.; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C.

    2011-01-01

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  5. Multiplex Flow Assays

    PubMed Central

    2016-01-01

    Lateral flow or dipstick assays (e.g., home pregnancy tests), where an analyte solution is drawn through a porous membrane and is detected by localization onto a capture probe residing at a specific site on the flow strip, are the most commonly and extensively used type of diagnostic assay. However, after over 30 years of use, these assays are constrained to measuring one or a few analytes at a time. Here, we describe a completely general method, in which any single-plex lateral flow assay is transformed into a multiplex assay capable of measuring an arbitrarily large number of analytes simultaneously. Instead of identifying the analyte by its localization onto a specific geometric location in the flow medium, the analyte-specific capture probe is identified by its association with a specific optically encoded region within the flow medium. The capture probes for nucleic acids, antigens, or antibodies are attached to highly porous agarose beads, which have been encoded using multiple lanthanide emitters to create a unique optical signature for each capture probe. The optically encoded capture probe-derivatized beads are placed in contact with the analyte-containing porous flow medium and the analytes are captured onto the encoded regions as the solution flows through the porous medium. To perform a multiplex diagnostic assay, a solution comprising multiple analytes is passed through the flow medium containing the capture probe-derivatized beads, and the captured analyte is treated with a suitable fluorescent reporter. We demonstrate this multiplex analysis technique by simultaneously measuring DNA samples, antigen–antibody pairs, and mixtures of multiple nucleic acids and antibodies. PMID:27819063

  6. Dolomite Dissolution in Alkaline Cementious Media

    NASA Astrophysics Data System (ADS)

    Mittermayr, Florian; Klammer, Dietmar; Köhler, Stephan; Dietzel, Martin

    2010-05-01

    Chemical alteration of concrete has gained much attention over the past years as many cases of deterioration due to sulphate attack, thaumasite formation (TSA) or alkali silica reactions (ASR) have been reported in various constructions (Schmidt et al, 2009). Much less is known about the so called alkali carbonate reaction (ACR). It is believed that dolomite aggregates can react with the alkalis from the cement, dissolve and form calcite and brucite (Katayama, 2004). Due to very low solubility of dolomite in alkaline solutions this reaction seems doubtful. In this study we are trying to gain new insides about the conditions that can lead to the dissolution of dolomite in concrete. Therefore we investigated concrete samples from Austrian tunnels that show partially dissolved dolomite aggregates. Petrological analysis such as microprobe, SEM and Raman spectroscopy as well as a hydrochemical analysis of interstitial solutions and ground water and modelling with PhreeqC (Parkhurst and Appelo, 1999) are carried out. In addition a series of batch experiments is set up. Modelling approaches by PhreeqC show a thermodynamically possibility in the alkaline range when additional Ca2+ in solution causes dolomite to become more and more undersaturated as calcite gets supersaturated. Interacting ground water is enriched in Ca2+and saturated with respect to gypsum as marine evaporites are found in situ rocks. Furthermore it is more likely that Portlandite (Ca(OH)2) plays a more important role than Na and K in the cement. Portlandite acts as an additional Ca2+ source and is much more abundant than the alkalies. Some interstitial solutions are dominated mainly by Na+ and SO42- and reach concentrations up to 30 g/l TDS. It is believed that solutions can even reach thenardite saturation as efflorescences are found on the tunnel walls. In consequence dolomite solubility increases with increasing ionic strength. pH > 11 further accelerate the process of dedolomitization by the removal

  7. Fluorometric assay for aflatoxins

    SciTech Connect

    Chakrabarti, A.G.

    1984-11-01

    The method that is now widely adopted by the government laboratories for the assay of individual aflatoxin components (B/sub 1/, B/sub 2/, G/sub 1/, and G/sub 2/) utilizes a TLC technique. The extraction and clean-up steps of this technique were further researched but the method is still time consuming. It is, therefore, very important to develop a rapid and accurate assay technique for aflatoxins. The current research proposes a technique which utilizes a Turner Fluorometer.

  8. CTL ELISPOT assay.

    PubMed

    Ranieri, Elena; Popescu, Iulia; Gigante, Margherita

    2014-01-01

    Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method

  9. Lateral flow strip assay

    SciTech Connect

    Miles, Robin R; Benett, William J; Coleman, Matthew A; Pearson, Francesca S; Nasarabadi, Shanavaz L

    2011-03-08

    A lateral flow strip assay apparatus comprising a housing; a lateral flow strip in the housing, the lateral flow strip having a receiving portion; a sample collection unit; and a reagent reservoir. Saliva and/or buccal cells are collected from an individual using the sample collection unit. The sample collection unit is immersed in the reagent reservoir. The tip of the lateral flow strip is immersed in the reservoir and the reagent/sample mixture wicks up into the lateral flow strip to perform the assay.

  10. Assays for calcitonin receptors

    SciTech Connect

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.

    1985-01-01

    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is /sup 125/I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed.

  11. Development of an alkaline fuel cell subsystem

    NASA Technical Reports Server (NTRS)

    1987-01-01

    A two task program was initiated to develop advanced fuel cell components which could be assembled into an alkaline power section for the Space Station Prototype (SSP) fuel cell subsystem. The first task was to establish a preliminary SSP power section design to be representative of the 200 cell Space Station power section. The second task was to conduct tooling and fabrication trials and fabrication of selected cell stack components. A lightweight, reliable cell stack design suitable for the SSP regenerative fuel cell power plant was completed. The design meets NASA's preliminary requirements for future multikilowatt Space Station missions. Cell stack component fabrication and tooling trials demonstrated cell components of the SSP stack design of the 1.0 sq ft area can be manufactured using techniques and methods previously evaluated and developed.

  12. Properties of cathode materials in alkaline cells

    NASA Astrophysics Data System (ADS)

    Salkind, A. J.; McBreen, J.; Freeman, R.; Parkhurst, W. A.

    1984-04-01

    Conventional and new cathode materials in primary and secondary alkaline cells were investigated for stability, structure, electrochemical reversibility and efficiency. Included were various forms of AgO for reserve type silver zinc batteries, a new material - AgNiO2 and several nickel electrodes for nickel cadmium and nickel hydrogen cells for aerospace applications. A comparative study was made of the stability of electroformed and chemically prepared AgO. Stability was correlated with impurities. After the first discharge AgNiO2 can be recharged to the monovalent level. The discharge product is predominantly silver. Plastic bonded nickel electrodes display a second plateau on discharge. Additions of Co(OH)2 largely eliminate this.

  13. Advanced-capability alkaline fuel cell powerplant

    NASA Astrophysics Data System (ADS)

    Deronck, Henry J.

    The alkaline fuel cell powerplant utilized in the Space Shuttle Orbiter has established an excellent performance and reliability record over the past decade. Recent AFC technology programs have demonstrated significant advances in cell durability and power density. These capabilities provide the basis for substantial improvement of the Orbiter powerplant, enabling new mission applications as well as enhancing performance in the Orbiter. Improved durability would extend the powerplant's time between overhaul fivefold, and permit longer-duration missions. The powerplant would also be a strong candidate for lunar/planetary surface power systems. Higher power capability would enable replacement of the Orbiter's auxiliary power units with electric motors, and benefits mass-critical applications such as the National AeroSpace Plane.

  14. Polyvinyl alcohol membranes as alkaline battery separators

    NASA Technical Reports Server (NTRS)

    Sheibley, D. W.; Gonzalez-Sanabria, O.; Manzo, M. A.

    1982-01-01

    Polyvinly alcohol (PVA) cross-linked with aldehyde reagents yields membranes that demonstrate properties that make them suitable for use as alkaline battery separators. Film properties can be controlled by the choice of cross-linker, cross-link density and the method of cross-linking. Three methods of cross-linking and their effects on film properties are discussed. Film properties can also be modified by using a copolymer of vinyl alcohol and acrylic acid as the base for the separator and cross-linking it similarly to the PVA. Fillers can be incorporated into the films to further modify film properties. Results of separator screening tests and cell tests for several variations of PBA films are discussed.

  15. Rechargeable alkaline manganese dioxide/zinc batteries

    NASA Astrophysics Data System (ADS)

    Kordesh, K.; Weissenbacher, M.

    The rechargeable alkaline manganese dioxide/zinc MnO 2/Zn) system, long established commercial as a primay battery, has reached a high level of performance as a secondary battery system. The operating principles are presented and the technological achievements are surveyed by referencing the recent publications and patent literature. A review is also given of the improvements obtained with newly formulated cathodes and anodes and specially designed batteries. Supported by modelling of the cathode and anode processes and by statistical evidence during cycling of parallel/series-connected modules, the envisioned performance of the next generation of these batteries is described. The possibility of extending the practical use of the improved rechargeable MnO 2/Zn system beyond the field of small electronics into the area of power tools, and even to kW-sized power sources, is demonstrated. Finally, the commercial development in comparison with other rechargeable battery systems is examined.

  16. Inhibition of renal alkaline phosphatase by cimetidine.

    PubMed

    Minai-Tehrani, Dariush; Khodai, Somayeh; Aminnaseri, Somayeh; Minoui, Saeed; Sobhani-Damavadifar, Zahra; Alavi, Sana; Osmani, Raheleh; Ahmadi, Shiva

    2011-08-01

    Alkaline phosphatase (ALP) belongs to hydrolase group of enzymes. It is responsible for removing phosphate groups from many types of molecules, including nucleotides and proteins. Cimetidine (trade name Tagamet) is an antagonist of histamine H2-receptor that inhibits the production of gastric acid. Cimetidine is used for the treatment of gastrointestinal diseases. In this study the inhibitory effect of cimetidine on mouse renal ALP activity was investigated. Our results showed that cimetidine can inhibit ALP by uncompetitive inhibition. In the absence of inhibitor the V(max) and K(m) of the enzyme were found to be 13.7 mmol/mg prot.min and 0.25 mM, respectively. Both the Vmax and Km of the enzyme decreased with increasing cimetidine concentrations (0- 1.2 mM). The Ki and IC(50) of cimetidine were determined to be about 0.5 mM and 0.52 mM, respectively.

  17. Oxygen electrodes for rechargeable alkaline fuel cells

    NASA Astrophysics Data System (ADS)

    Swette, Larry; Giner, Jose

    1987-09-01

    Electrocatalysts and supports for the positive electrode of moderate temperature single unit rechargeable alkaline fuel cells were investigated and developed. The electrocatalysts are defined as the material with a higher activity for the oxygen electrode reaction than the support. Advanced development will require that the materials be prepared in high surface area forms, and may also entail integration of various candidate materials. Eight candidate support materials and seven electrocatalysts were investigated. Of the 8 support, 3 materials meet the preliminary requirements in terms of electrical conductivity and stability. Emphasis is now on preparing in high surface area form and testing under more severe corrosion stress conditions. Of the 7 electrocatalysts prepared and evaluated, at least 5 materials remain as potential candidates. The major emphasis remains on preparation, physical characterization and electrochemical performance testing.

  18. Development of an alkaline fuel cell subsystem

    NASA Astrophysics Data System (ADS)

    1987-03-01

    A two task program was initiated to develop advanced fuel cell components which could be assembled into an alkaline power section for the Space Station Prototype (SSP) fuel cell subsystem. The first task was to establish a preliminary SSP power section design to be representative of the 200 cell Space Station power section. The second task was to conduct tooling and fabrication trials and fabrication of selected cell stack components. A lightweight, reliable cell stack design suitable for the SSP regenerative fuel cell power plant was completed. The design meets NASA's preliminary requirements for future multikilowatt Space Station missions. Cell stack component fabrication and tooling trials demonstrated cell components of the SSP stack design of the 1.0 sq ft area can be manufactured using techniques and methods previously evaluated and developed.

  19. Acylglucuronide in alkaline conditions: migration vs. hydrolysis.

    PubMed

    Di Meo, Florent; Steel, Michele; Nicolas, Picard; Marquet, Pierre; Duroux, Jean-Luc; Trouillas, Patrick

    2013-06-01

    This work rationalizes the glucuronidation process (one of the reactions of the phase II metabolism) for drugs having a carboxylic acid moiety. At this stage, acylglucuronides (AG) metabolites are produced, that have largely been reported in the literature for various drugs (e.g., mycophenolic acid (MPA), diclofenac, ibuprofen, phenylacetic acids). The competition between migration and hydrolysis is rationalized by adequate quantum calculations, combing MP2 and density functional theory (DFT) methods. At the molecular scale, the former process is a real rotation of the drug around the glucuconic acid. This chemical-engine provides four different metabolites with various toxicities. Migration definitely appears feasible under alkaline conditions, making proton release from the OH groups. The latter reaction (hydrolysis) releases the free drug, so the competition is of crucial importance to tackle drug action and elimination. From the theoretical data, both migration and hydrolysis appear kinetically and thermodynamically favored, respectively.

  20. The Alkaline Dissolution Rate of Calcite.

    PubMed

    Colombani, Jean

    2016-07-07

    Due to the widespread presence of calcium carbonate on Earth, several geochemical systems, among which is the global CO2 cycle, are controlled to a large extent by the dissolution and precipitation of this mineral. For this reason, the dissolution of calcite has been thoroughly investigated for decades. Despite this intense activity, a consensual value of the dissolution rate of calcite has not been found yet. We show here that the inconsistency between the reported values stems mainly from the variability of the chemical and hydrodynamic conditions of measurement. The spreading of the values, when compared in identical conditions, is much less than expected and is interpreted in terms of sample surface topography. This analysis leads us to propose benchmark values of the alkaline dissolution rate of calcite compatible with all the published values, and a method to use them in various chemical and hydrodynamic contexts.

  1. Alkaline oxide conversion coatings for aluminum alloys

    SciTech Connect

    Buchheit, R.G.

    1996-02-01

    Three related conversion coating methods are described that are based on film formation which occurs when aluminum alloys are exposed to alkaline Li salt solutions. Representative examples of the processing methods, resulting coating structure, composition and morphology are presented. The corrosion resistance of these coatings to aerated 0.5 M NaCl solution has been evaluated as a function of total processing time using electrochemical impedance spectroscopy (EIS). This evaluation shows that excellent corrosion resistance can be uniformly achieved using no more than 20 minutes of process time for 6061-T6. Using current methods a minimum of 80 minutes of process time is required to get marginally acceptable corrosion resistance for 2024-T3. Longer processing times are required to achieve uniformly good corrosion resistance.

  2. Oxygen electrodes for rechargeable alkaline fuel cells

    NASA Technical Reports Server (NTRS)

    Swette, Larry; Giner, Jose

    1987-01-01

    Electrocatalysts and supports for the positive electrode of moderate temperature single unit rechargeable alkaline fuel cells were investigated and developed. The electrocatalysts are defined as the material with a higher activity for the oxygen electrode reaction than the support. Advanced development will require that the materials be prepared in high surface area forms, and may also entail integration of various candidate materials. Eight candidate support materials and seven electrocatalysts were investigated. Of the 8 support, 3 materials meet the preliminary requirements in terms of electrical conductivity and stability. Emphasis is now on preparing in high surface area form and testing under more severe corrosion stress conditions. Of the 7 electrocatalysts prepared and evaluated, at least 5 materials remain as potential candidates. The major emphasis remains on preparation, physical characterization and electrochemical performance testing.

  3. Alkaline pulping of some eucalypts from Sudan.

    PubMed

    Khristova, P; Kordsachia, O; Patt, R; Dafaalla, S

    2006-03-01

    Four eucalypts (Eucalyptus camaldulensis, Eucalyptus microtheca, Eucalyptus tereticornis and Eucalyptus citriodora) grown in Sudan were examined for their suitability for pulping and papermaking with different alkaline methods. Their physical, morphological and chemical characteristics are reported. The pulping trials with E. citriodora and E. tereticornis were carried out using the kraft-AQ, soda-AQ, modified AS/AQ (ASA), ASAM and kraft methods. For the other two species, only the ASAM and the kraft process were applied. ASAM pulping gave the best results in terms of yield, degree of delignification, mechanical and optical pulp properties. The best pulps, obtained in kraft and ASAM cooking of E. citriodora, were bleached to 88% ISO brightness in a totally chlorine free bleaching sequence (OQ1O/PQ2P). The bleached pulps, especially the ASAM pulp, showed good papermaking properties and would be suitable for manufacture of writing and printing grades of paper.

  4. The Nickel(111)/Alkaline Electrolyte Interface

    NASA Technical Reports Server (NTRS)

    Wang, Kuilong; Chottiner, G. S.; Scherson, D. A.; Reid, Margaret A.

    1991-01-01

    The electrochemical properties of Ni (111) prepared and characterized in ultra high vacuum, UHV, by surface analytical techniques have been examined in alkaline media by cyclic voltammetry using an UHV-electrochemical cell transfer system designed and built in this laboratory. Prior to the transfer, the Ni(111) surfaces were exposed to saturation coverages of CO in UHV in an attempt to protect the surface from possible contamination with other gases during the transfer. Temperature Programmed Desorption, TPD, of CO-dosed Ni (111) surfaces displaying sharp c(4x2), LEED patterns, subsequently exposed to water-saturated Ar at atmospheric pressure in an auxiliary UHV compatible chamber and finally transferred back to the main UHV chamber, yielded CO2 and water as the only detectable products. This indicates that the CO-dosed surfaces react with water and/or bicarbonate and hydroxide as the most likely products. Based on the integration of the TPD peaks, the combined amounts of H2O and CO2 were found to be on the order of a single monolayer. The reacted c(4x2)CO/Ni(111) layer seems to protect the surface from undergoing spontaneous oxidation in strongly alkaline solutions. This was evidenced by the fact that the open circuit potential observed immediately after contact with deaerated 0.1 M KOH was about 0.38 V vs. DHE, drifting slightly towards more negative values prior to initiating the voltametric scans. The average ratio of the integrated charge obtained in the first positive linear scan in the range of 0.35 to 1.5 V vs. DHE (initiated at the open circuit potential) and the first (and subsequent) linear negative scans in the same solution yielded for various independent runs a value of 3.5 +/- 0.3. Coulometric analysis of the cyclic voltammetry curves indicate that the electrochemically formed oxyhydroxide layer involves a charge equivalent to 3.2 +/- 0.4 layers of Ni metal.

  5. Sigma Receptor Binding Assays.

    PubMed

    Chu, Uyen B; Ruoho, Arnold E

    2015-12-08

    Sigma receptors, both Sigma-1(S1R) and Sigma-2 (S2R), are small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated sites. A number of drugs bind to sigma receptors, including the antipsychotic haloperidol and (+)-pentazocine, an opioid analgesic. Sigma receptors are implicated in many central nervous system disorders, in particular Alzheimer's disease and conditions associated with motor control, such as Amyotrophic Lateral Sclerosis (ALS). Described in this unit are radioligand binding assays used for the pharmacological characterization of S1R and S2R. Methods detailed include a radioligand saturation binding assay for defining receptor densities and a competitive inhibition binding assay employing [³H]-(+)-pentazocine for identifying and characterizing novel ligands that interact with S1R. Procedures using [³H]-1,3-di(2-tolyl)guanidine ([³H]-DTG), a nonselective sigma receptor ligand, are described for conducting a saturation binding and competitive inhibition assays for the S2R site. These protocols are of value in drug discovery in identifying new sigma ligands and in the characterization of these receptors.

  6. New oligosaccharyltransferase assay method.

    PubMed

    Kohda, Daisuke; Yamada, Masaki; Igura, Mayumi; Kamishikiryo, Jun; Maenaka, Katsumi

    2007-11-01

    We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.

  7. Kinetic tetrazolium microtiter assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L. (Inventor); Stowe, Raymond P. (Inventor); Koeing, David W. (Inventor)

    1992-01-01

    A method for conducting an in vitro cell assay using a tetrazolium indicator is disclosed. The indicator includes a nonionic detergent which solubilizes a tetrazolium reduction product in vitro and has low toxicity for the cells. The incubation of test cells in the presence of zolium bromide and octoxynol (TRITON X-100) permits kinetics of the cell metabolism to be determined.

  8. Instrument for assaying radiation

    DOEpatents

    Coleman, Jody Rustyn; Farfan, Eduardo B.

    2016-03-22

    An instrument for assaying radiation includes a flat panel detector having a first side opposed to a second side. A collimated aperture covers at least a portion of the first side of the flat panel detector. At least one of a display screen or a radiation shield may cover at least a portion of the second side of the flat panel detector.

  9. Detection of salmonellas by DNA hybridization with a fluorescent alkaline phosphatase substrate.

    PubMed

    Cano, R J; Torres, M J; Klem, R E; Palomares, J C; Casadesus, J

    1992-05-01

    This study evaluates a DNA hybridization assay for salmonella with AttoPhos (JBL Scientific, San Luis Obispo, CA), a fluorescent substrate for alkaline phosphatase. The probe used (50 ng/ml) was a biotinylated 600 bp fragment consisting of a tandem repeat of an insertion sequence (IS200) found in most Salmonella spp. evaluated. The hybridization was carried out at 65 degrees C for 2 h without prior prehybridization and hybrids were detected by the addition of a streptavidin-alkaline phosphatase conjugate. Circles (5 mm) were cut from the membrane and placed in a cuvette containing 1 ml of 1 mmol/l AttoPhos. The reaction was evaluated after 30 min at 37 degrees C with a fluorometer with an excitation wavelength of 440 nm and an emission wavelength of 550 nm. The sensitivity of the probe was estimated to be 10,000 copies of target DNA or 5 x 10(-20) mol of DNA. All 74 salmonella strains tested reacted with the probe but none of the 98 heterologous species tested gave positive results. The results of this study indicate that our assay method, which employs a biotinylated tandem repeat of IS200 and AttoPhos, is a specific and highly sensitive quantitative method for the detection of salmonellas.

  10. Alkaline cleaner replacement for printed wiring board fabrication

    SciTech Connect

    Goldammer, S.E.; Pemberton, S.E.; Tucker, D.R.

    1997-04-01

    A replacement alkaline cleaning chemistry was qualified for the copper cleaning process used to support printed wiring board fabrication. The copper cleaning process was used to prepare copper surfaces for enhancing the adhesion of dry film photopolymers (photoresists and solder masks) and acrylic adhesives. The alkaline chemistry was used to remove organic contaminates such as fingerprints.

  11. Exploring Alkaline Stable Organic Cations for Polymer Hydroxide Exchange Membranes

    DTIC Science & Technology

    2015-04-29

    Organic Cations for Polymer Hydroxide Exchange Membranes Hydroxide exchange membranes (HEMs) are important polymer electrolytes for electrochemical...Exploring Alkaline Stable Organic Cations for Polymer Hydroxide Exchange Membranes Report Title Hydroxide exchange membranes (HEMs) are important polymer ...constructing HEMs. EXPLORING ALKALINE STABLE ORGANIC CATIONS FOR POLYMER HYDROXIDE EXCHANGE MEMBRANES by Bingzi Zhang

  12. The Chemistry of Paper Preservation Part 4. Alkaline Paper.

    ERIC Educational Resources Information Center

    Carter, Henry A.

    1997-01-01

    Discusses the problem of the inherent instability of paper due to the presence of acids that catalyze the hydrolytic degradation of cellulose. Focuses on the chemistry involved in the sizing of both acid and alkaline papers and the types of fillers used. Discusses advantages and problems of alkaline papermaking. Contains 48 references. (JRH)

  13. Removal of plutonium and americium from alkaline waste solutions

    DOEpatents

    Schulz, Wallace W.

    1979-01-01

    High salt content, alkaline waste solutions containing plutonium and americium are contacted with a sodium titanate compound to effect removal of the plutonium and americium from the alkaline waste solution onto the sodium titanate and provide an effluent having a radiation level of less than 10 nCi per gram alpha emitters.

  14. Sensitive, coupled assay for plasminogen activator using a thiol ester substrate for plasmin

    SciTech Connect

    Coleman, P L; Green, G D.J.

    1980-01-01

    Several assays for plasminogen activator employ a direct assay method. These are remarkably sensitive methods, yet they suffer in comparison to the sensitivity of coupled methods. Coupling the assay with plasminogen not only amplifies the sensitivity by the multiplicative effect of plasmin, but insures that only those proteases specific for plasminogen are assayed. The choice of substrate for plasmin is critical. A thiol ester substrate, thiobenzyl benzyloxy-carbonyl-L-lysinate (Z-Lys-SBzl), has been synthesized which combines high k/sub cat/ with alkaline stability. In an effort to characterize the plasminogen activator from hepatoma tissue culture (HTC) and its hormonally-controlled inhibitor, Z-Lys-SBzl was used in a coupled approach providing an assay which is superior to the /sup 125/I-fibrinolytic assay. It is also extremely sensitive to plasminogen activator and can be used for routine analysis of purification as well as kinetic and binding studies. (ERB)

  15. Alkaline stability of quaternary ammonium cations for alkaline fuel cell membranes and ionic liquids.

    PubMed

    Marino, M G; Kreuer, K D

    2015-02-01

    The alkaline stability of 26 different quaternary ammonium groups (QA) is investigated for temperatures up to 160 °C and NaOH concentrations up to 10 mol L(-1) with the aim to provide a basis for the selection of functional groups for hydroxide exchange membranes in alkaline fuel cells and of ionic-liquid cations stable in basic conditions. Most QAs exhibit unexpectedly high alkaline stability with the exception of aromatic cations. β-Protons are found to be far less susceptible to nucleophilic attack than previously suggested, whereas the presence of benzyl groups, nearby hetero-atoms, or other electron-withdrawing species promote degradation reactions significantly. Cyclic QAs proved to be exceptionally stable, with the piperidine-based 6-azonia-spiro[5.5]undecane featuring the highest half-life at the chosen conditions. Absolute and relative stabilities presented herein stand in contrast to literature data, the differences being ascribed to solvent effects on degradation.

  16. Salt- and alkaline-tolerance are linked in Acacia.

    PubMed

    Bui, Elisabeth N; Thornhill, Andrew; Miller, Joseph T

    2014-07-01

    Saline or alkaline soils present a strong stress on plants that together may be even more deleterious than alone. Australia's soils are old and contain large, sometimes overlapping, areas of high salt and alkalinity. Acacia and other Australian plant lineages have evolved in this stressful soil environment and present an opportunity to understand the evolution of salt and alkalinity tolerance. We investigate this evolution by predicting the average soil salinity and pH for 503 Acacia species and mapping the response onto a maximum-likelihood phylogeny. We find that salinity and alkalinity tolerance have evolved repeatedly and often together over 25 Ma of the Acacia radiation in Australia. Geographically restricted species are often tolerant of extreme conditions. Distantly related species are sympatric in the most extreme soil environments, suggesting lack of niche saturation. There is strong evidence that many Acacia have distributions affected by salinity and alkalinity and that preference is lineage specific.

  17. Salt- and alkaline-tolerance are linked in Acacia

    PubMed Central

    Bui, Elisabeth N.; Thornhill, Andrew; Miller, Joseph T.

    2014-01-01

    Saline or alkaline soils present a strong stress on plants that together may be even more deleterious than alone. Australia's soils are old and contain large, sometimes overlapping, areas of high salt and alkalinity. Acacia and other Australian plant lineages have evolved in this stressful soil environment and present an opportunity to understand the evolution of salt and alkalinity tolerance. We investigate this evolution by predicting the average soil salinity and pH for 503 Acacia species and mapping the response onto a maximum-likelihood phylogeny. We find that salinity and alkalinity tolerance have evolved repeatedly and often together over 25 Ma of the Acacia radiation in Australia. Geographically restricted species are often tolerant of extreme conditions. Distantly related species are sympatric in the most extreme soil environments, suggesting lack of niche saturation. There is strong evidence that many Acacia have distributions affected by salinity and alkalinity and that preference is lineage specific. PMID:25079493

  18. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    SciTech Connect

    Jemmerson, R.; Low, M.G.

    1987-09-08

    Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either /sup 3/H-fatty acids or (/sup 3/H)ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the /sup 3/H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of (/sup 3/H)ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from /sup 3/H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the /sup 3/H-fatty acid and the (/sup 3/H)ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the (/sup 3/H)ethanolamine label from the purified alkaline phosphatase. The /sup 3/H radioactivity in alkaline phosphatase purified from (/sup 3/H)ethanolamine-labeled cells comigrated with authentic (/sup 3/H)ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the /sup 3/H-fatty acid and (/sup 3/H)ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.

  19. DNA conformation of Chinese hamster V79 cells and sensitivity to ionizing radiation

    SciTech Connect

    Olive, P.L.; Hilton, J.; Durand, R.E.

    1986-07-01

    Chinese hamster V79 cells grown for 20 h in suspension culture form small clusters of cells (spheroids) which are more resistant to killing by ionizing radiation than V79 cells grown as monolayers. This resistance appears to be due to the greater capacity of cells grown in contact to repair radiation damage. Attempts to relate this ''contact effect'' to differences in DNA susceptibility or DNA repair capacity have provided conflicting results. Two techniques, alkaline sucrose gradient sedimentation and alkaline elution, show no difference in the amounts of radiation-induced DNA single-strand breakage or its repair between suspension or monolayer cells. However, using the alkali-unwinding assay, the rate of DNA unwinding is much slower for suspension cells than for monolayer cells. Interestingly, a decrease in salt concentration or in pH of the unwinding solution eliminates these differences in DNA unwinding kinetics. A fourth assay, sedimentation of nucleoids on neutral sucrose gradients, also shows a significant decrease in radiation damage produced in suspension compared to monolayer cultures. It is believed that this assay measures differences in DNA conformation (supercoiling) as well as differences in DNA strand breakage. We conclude from these four assays that the same number of DNA strand breaks/Gy is produced in monolayer and spheroid cells. However, changes in DNA conformation or packaging occur when cells are grown as spheroids, and these changes are responsible for reducing DNA damage by ionizing radiation.

  20. The corneal pocket assay.

    PubMed

    Ziche, Marina; Morbidelli, Lucia

    2015-01-01

    The cornea in most species is physiologically avascular, and thus this assay allows the measurement of newly formed vessels. The continuous monitoring of neovascular growth in the same animal allows the evaluation of drugs acting as suppressors or stimulators of angiogenesis. Under anesthesia a micropocket is produced in the cornea thickness and the angiogenesis stimulus (tumor tissue, cell suspension, growth factor) is placed into the pocket in order to induce vascular outgrowth from the limbal capillaries. Neovascular development and progression can be modified by the presence of locally released or applied inhibitory factors or by systemic treatments. In this chapter the experimental details of the avascular cornea assay, the technical challenges, and advantages and disadvantages in different species are discussed. Protocols for local drug treatment and tissue sampling for histology and pharmacokinetic profile are reported.

  1. Kinetic Tetrazolium Microtiter Assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L.; Stowe, Raymond; Koenig, David

    1993-01-01

    Kinetic tetrazolium microtiter assay (KTMA) involves use of tetrazolium salts and Triton X-100 (or equivalent), nontoxic, in vitro color developer solubilizing colored metabolite formazan without injuring or killing metabolizing cells. Provides for continuous measurement of metabolism and makes possible to determine rate of action of antimicrobial agent in real time as well as determines effective inhibitory concentrations. Used to monitor growth after addition of stimulatory compounds. Provides for kinetic determination of efficacy of biocide, greatly increasing reliability and precision of results. Also used to determine relative effectiveness of antimicrobial agent as function of time. Capability of generating results on day of test extremely important in treatment of water and waste, disinfection of hospital rooms, and in pharmaceutical, agricultural, and food-processing industries. Assay also used in many aspects of cell biology.

  2. Batteries: from alkaline to zinc-air.

    PubMed

    Dondelinger, Robert M

    2004-01-01

    There is no perfect disposable battery--one that will sit on the shelf for 20 years, then continually provide unlimited current, at a completely constant voltage until exhausted, without producing heat. There is no perfect rechargeable battery--one with all of the above characteristics and will also withstand an infinite overcharge while providing an equally infinite cycle life. There are only compromises. Every battery selection is a compromise between the ideally required characteristics, the advantages, and the limitations of each battery type. General selection of a battery type to power a medical device is largely outside the purview of the biomed. Initially, these are engineering decisions made at the time of medical equipment design and are intended to be followed in perpetuity. However, since newer cell types evolve and the manufacturer's literature is fixed at the time of printing, some intelligent substitutions may be made as long as the biomed understands the characteristics of both the recommended cell and the replacement cell. For example, when the manufacturer recommends alkaline, it is usually because of the almost constant voltage it produces under the devices' design load. Over time, other battery types may be developed that will meet the intent of the manufacturer, at a lower cost, providing longer operational life, at a lower environmental cost, or with a combination of these advantages. In the Obstetrical Doppler cited at the beginning of this article, the user had put in carbon-zinc cells, and the biomed had unknowingly replaced them with carbonzinc cells. If the alkaline cells recommended by the manufacturer had been used, there would have been the proper output voltage at the battery terminals when the [table: see text] cells were at their half-life. Instead, the device refused to operate since the battery voltage was below presumed design voltage. While battery-type substitutions may be easily and relatively successfully made in disposable

  3. B cell helper assays.

    PubMed

    Abrignani, Sergio; Tonti, Elena; Casorati, Giulia; Dellabona, Paolo

    2009-01-01

    Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC-peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC-peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called "B cell helper assays" that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell responses. We show here examples of B cell helper assays in vitro, which can be reproduced with any subset of T lymphocytes that displays the appropriate helper signals.

  4. Microbial Thiocyanate Utilization under Highly Alkaline Conditions

    PubMed Central

    Sorokin, Dimitry Y.; Tourova, Tatyana P.; Lysenko, Anatoly M.; Kuenen, J. Gijs

    2001-01-01

    Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS−) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division Proteobacteria (the Halomonas group for the heterotrophs and the genus Thioalkalivibrio for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus Thioalkalivibrio. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genus Thioalkalivibrio according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase

  5. Net alkalinity and net acidity 2: Practical considerations

    USGS Publications Warehouse

    Kirby, C.S.; Cravotta, C.A.

    2005-01-01

    The pH, alkalinity, and acidity of mine drainage and associated waters can be misinterpreted because of the chemical instability of samples and possible misunderstandings of standard analytical method results. Synthetic and field samples of mine drainage having various initial pH values and concentrations of dissolved metals and alkalinity were titrated by several methods, and the results were compared to alkalinity and acidity calculated based on dissolved solutes. The pH, alkalinity, and acidity were compared between fresh, unoxidized and aged, oxidized samples. Data for Pennsylvania coal mine drainage indicates that the pH of fresh samples was predominantly acidic (pH 2.5-4) or near neutral (pH 6-7); ??? 25% of the samples had pH values between 5 and 6. Following oxidation, no samples had pH values between 5 and 6. The Standard Method Alkalinity titration is constrained to yield values >0. Most calculated and measured alkalinities for samples with positive alkalinities were in close agreement. However, for low-pH samples, the calculated alkalinity can be negative due to negative contributions by dissolved metals that may oxidize and hydrolyze. The Standard Method hot peroxide treatment titration for acidity determination (Hot Acidity) accurately indicates the potential for pH to decrease to acidic values after complete degassing of CO2 and oxidation of Fe and Mn, and it indicates either the excess alkalinity or that required for neutralization of the sample. The Hot Acidity directly measures net acidity (= -net alkalinity). Samples that had near-neutral pH after oxidation had negative Hot Acidity; samples that had pH < 6.3 after oxidation had positive Hot Acidity. Samples with similar pH values before oxidation had dissimilar Hot Acidities due to variations in their alkalinities and dissolved Fe, Mn, and Al concentrations. Hot Acidity was approximately equal to net acidity calculated based on initial pH and dissolved concentrations of Fe, Mn, and Al minus the

  6. Low pH alkaline chemical formulations

    SciTech Connect

    French, T.R.; Peru, D.A.; Thornton, S.D.

    1989-01-01

    This report describes the development of a surfactant-enhanced alkaline flooding system that is applicable to specific reservoir conditions in Wilmington (California) field. The cost of the chemicals for an ASP (alkali/surfactant/polymer) flood is calculated to be $3.90/bbl of oil produced, with 78% of that cost attributable to polymer. This research included phase behavior tests, oil displacement tests, mineral dissolution tests, and adsorption measurements. It was discovered that consumption of low pH alkalis is low enough in the Wilmington field to be acceptable. In addition, alkali dramatically reduced surfactant adsorption and precipitation. A mixture of NaHCO3 and Na2CO3 was recommended for use as a preflush and in the ASP formulation. Research was also conducted on the synergistic effect that occurs when a mixture of alkali and synthetic surfactant contacts crude oil. It appears that very low IFT is predominantly a result of the activation of the natural surfactants present in the Wilmington oil, and the sustained low IFT is primarily the result of the synthetic surfactant. It also appears that removal of acids from the crude oil by the alkali renders the oil more interfacially reactive to synthetic surfactant. These phenomena help to explain the synergism that results from combining alkali and synthetic surfactant into a single oil recovery formulation. 19 refs., 24 figs., 10 tabs.

  7. Process for treating alkaline wastes for vitrification

    SciTech Connect

    Hsu, Chia-lin W.

    1994-01-01

    According to its major aspects and broadly stated, the present invention is a process for treating alkaline waste materials, including high level radioactive wastes, for vitrification. The process involves adjusting the pH of the wastes with nitric acid, adding formic acid (or a process stream containing formic acid) to reduce mercury compounds to elemental mercury and MnO{sub 2} to the Mn(II) ion, and mixing with class formers to produce a melter feed. The process minimizes production of hydrogen due to noble metal-catalyzed formic acid decomposition during, treatment, while producing a redox-balanced feed for effective melter operation and a quality glass product. An important feature of the present invention is the use of different acidifying and reducing, agents to treat the wastes. The nitric acid acidifies the wastes to improve yield stress and supplies acid for various reactions; then the formic acid reduces mercury compounds to elemental mercury and MnO{sub 2}) to the Mn(II) ion. When the pH of the waste is lower, reduction of mercury compounds and MnO{sub 2}) is faster and less formic acid is needed, and the production of hydrogen caused by catalytically-active noble metals is decreased.

  8. Extracellular alkaline proteinase of Colletotrichum gloeosporioides.

    PubMed

    Dunaevsky, Ya E; Matveeva, A R; Beliakova, G A; Domash, V I; Belozersky, M A

    2007-03-01

    The main proteinase of the filamentous fungus Colletotrichum gloeosporioides causing anthracnoses and serious problems for production and storage of agricultural products has molecular mass of 57 kD and was purified more than 200-fold to homogeneity with the yield of 5%. Maximal activity of the proteinase is at pH 9.0-10.0, and the enzyme is stable at pH 6.0-11.5 (residual activity not less than 70%). The studied enzyme completely kept its activity to 55 degrees C, with a temperature optimum of 45 degrees C. The purified C. gloeosporioides proteinase is stable at alkaline pH values, but rapidly loses its activity at pH values lower than 5.0. Addition of bovine serum albumin stabilizes the enzyme under acidic conditions. Data on inhibitor analysis and substrate specificity of the enzyme allow its classification as a serine proteinase of subtilisin family. It is demonstrated that the extracellular proteinase of C. gloeosporioides specifically effects plant cell wall proteins. It is proposed that the studied proteinase--via hydrolysis of cell wall--provides for penetration of the fungus into the tissues of the host plant.

  9. Production of alkaline protease from Cellulosimicrobium cellulans

    PubMed Central

    Ferracini-Santos, Luciana; Sato, Hélia H

    2009-01-01

    Cellulosimicrobium cellulans is one of the microorganisms that produces a wide variety of yeast cell wall-degrading enzymes, β-1,3-glucanase, protease and chitinase. Dried cells of Saccharomyces cerevisiae were used as carbon and nitrogen source for cell growth and protease production. The medium components KH2PO4, KOH and dried yeast cells showed a significant effect (p<0.05) on the factorial fractional design. A second design was prepared using two factors: pH and percentage of dried yeast cells. The results showed that the culture medium for the maximum production of protease was 0.2 g/l of MgSO4.7H2O, 2.0 g/l of (NH4)2SO4 and 8% of dried yeast cells in 0.15M phosphate buffer at pH 8.0. The maximum alkaline protease production was 7.0 ± 0.27 U/ml over the center point. Crude protease showed best activity at 50ºC and pH 7.0-8.0, and was stable at 50ºC. PMID:24031317

  10. Solubility of pllutonium in alkaline salt solutions

    SciTech Connect

    Hobbs, D.T.; Edwards, T.B.

    1993-02-26

    Plutonium solubility data from several studies have been evaluated. For each data set, a predictive model has been developed where appropriate. In addition, a statistical model and corresponding prediction intervals for plutonium solubility as a quadratic function of the hydroxide concentration have been developed. Because of the wide range of solution compositions, the solubility of plutonium can vary by as much as three orders of magnitude for any given hydroxide concentration and still remain within the prediction interval. Any nuclear safety assessments that depend on the maximum amount of plutonium dissolved in alkaline salt solutions should use concentrations at least as great as the upper prediction limits developed in this study. To increase the confidence in the prediction model, it is recommended that additional solubility tests be conducted at low hydroxide concentrations and with all of the other solution components involved. To validate the model for application to actual waste solutions, it is recommended that the plutonium solubilities in actual waste solutions be determined and compared to the values predicted by the quadratic model.

  11. Process for treating alkaline wastes for vitrification

    DOEpatents

    Hsu, Chia-lin W.

    1995-01-01

    A process for treating alkaline wastes for vitrification. The process involves acidifying the wastes with an oxidizing agent such as nitric acid, then adding formic acid as a reducing agent, and then mixing with glass formers to produce a melter feed. The nitric acid contributes nitrates that act as an oxidant to balance the redox of the melter feed, prevent reduction of certain species to produce conducting metals, and lower the pH of the wastes to a suitable level for melter operation. The formic acid reduces mercury compounds to elemental mercury for removal by steam stripping, and MnO.sub.2 to the Mn(II) ion to prevent foaming of the glass melt. The optimum amounts of nitric acid and formic acid are determined in relation to the composition of the wastes, including the concentrations of mercury (II) and MnO.sub.2, noble metal compounds, nitrates, formates and so forth. The process minimizes the amount of hydrogen generated during treatment, while producing a redox-balanced feed for effective melter operation and a quality glass product.

  12. Process for treating alkaline wastes for vitrification

    DOEpatents

    Hsu, C.L.W.

    1995-07-25

    A process is described for treating alkaline wastes for vitrification. The process involves acidifying the wastes with an oxidizing agent such as nitric acid, then adding formic acid as a reducing agent, and then mixing with glass formers to produce a melter feed. The nitric acid contributes nitrates that act as an oxidant to balance the redox of the melter feed, prevent reduction of certain species to produce conducting metals, and lower the pH of the wastes to a suitable level for melter operation. The formic acid reduces mercury compounds to elemental mercury for removal by steam stripping, and MnO{sub 2} to the Mn(II) ion to prevent foaming of the glass melt. The optimum amounts of nitric acid and formic acid are determined in relation to the composition of the wastes, including the concentrations of mercury (II) and MnO{sub 2}, noble metal compounds, nitrates, formates and so forth. The process minimizes the amount of hydrogen generated during treatment, while producing a redox-balanced feed for effective melter operation and a quality glass product. 4 figs.

  13. Engineering challenges of ocean alkalinity enhancement

    NASA Astrophysics Data System (ADS)

    Kruger, T.; Renforth, P.

    2012-04-01

    The addition of calcium oxide (CaO) to the ocean as a means of enhancing the capacity of the ocean as a carbon sink was first proposed by Haroon Kheshgi in 1995. Calcium oxide is created by heating high purity limestone in a kiln to temperatures of approximately 1000°C. Addition of this material to the ocean draws carbon dioxide out of the atmosphere (approximately 1 tonne of CaO could sequester 1.3 tonnes of CO2). Abiotic carbonate precipitation is inhibited in the surface ocean. This is a carbon and energy expensive process, where approximately 0.8 tonnes of CO2 are produced at a point source for every tonne sequestered. The feasibility of ocean alkalinity enhancement requires capture and storage of the point source of CO2. We present details of a feasibility study of the engineering challenges of Kheshgi's method focusing on the potential scalability and costs of the proposed process. To draw down a PgC per year would require the extraction and processing of ~6Pg of limestone per year, which is similar in scale to the current coal industry. Costs are estimated at ~USD30-40 per tonne of CO2 sequestered through the process, which is favourable to comparative processes. Kheshgi, H. (1995) Energy 20 (9) 915-922

  14. Response of Desulfovibrio vulgaris to Alkaline Stress

    SciTech Connect

    Stolyar, S.; He, Q.; He, Z.; Yang, Z.; Borglin, S.E.; Joyner, D.; Huang, K.; Alm, E.; Hazen, T.C.; Zhou, J.; Wall, J.D.; Arkin, A.P.; Stahl, D.A.

    2007-11-30

    The response of exponentially growing Desulfovibrio vulgarisHildenborough to pH 10 stress was studied using oligonucleotidemicroarrays and a study set of mutants with genes suggested by microarraydata to be involved in the alkaline stress response deleted. The datashowed that the response of D. vulgaris to increased pH is generallysimilar to that of Escherichia coli but is apparently controlled byunique regulatory circuits since the alternative sigma factors (sigma Sand sigma E) contributing to this stress response in E. coli appear to beabsent in D. vulgaris. Genes previously reported to be up-regulated in E.coli were up-regulated in D. vulgaris; these genes included three ATPasegenes and a tryptophan synthase gene. Transcription of chaperone andprotease genes (encoding ATP-dependent Clp and La proteases and DnaK) wasalso elevated in D. vulgaris. As in E. coli, genes involved in flagellumsynthesis were down-regulated. The transcriptional data also identifiedregulators, distinct from sigma S and sigma E, that are likely part of aD. vulgaris Hildenborough-specific stress response system.Characterization of a study set of mutants with genes implicated inalkaline stress response deleted confirmed that there was protectiveinvolvement of the sodium/proton antiporter NhaC-2, tryptophanase A, andtwo putative regulators/histidine kinases (DVU0331 andDVU2580).

  15. Baseline values of micronuclei and comet assay in the lizard Tupinambis merianae (Teiidae, Squamata).

    PubMed

    Schaumburg, Laura G; Poletta, Gisela L; Siroski, Pablo A; Mudry, Marta D

    2012-10-01

    The Micronucleus test (MN) and Comet assay (CA) are currently the most widely used methods that allow the characterization of DNA damage induced by physical and chemical agents in wild species. The continuous expansion of the cultivated areas in Argentina, since the introduction of transgenic crops, mainly soy, in association with the increased use of pesticides, transformed deeply the natural environments where the lizard Tupinambis merianae (tegu lizard) occurs. Despite the fact that reptiles have shown to be excellent bioindicators of environmental contaminants, there is no record of genotoxicity studies in T. merianae. The aim of the present study was to adjust the MN test and CA protocols to be applied in erythrocytes of T. merianae, and determine the baseline values of DNA damage in this species. We used 20 adult lizards (10 males: 10 females) from Estación Zoológica Experimental "Granja La Esmeralda" (Santa Fe, Argentina). Peripheral blood samples were collected from all animals and the MN test and CA applied according to the protocols established for other reptilian species. We test critical parameters of CA protocol (cell density, unwinding and electrophoresis times) using increasing concentrations of H2O2 (10, 25 and 50 μM) as a known genotoxic agent to induce DNA damage. Based on this, we determined the most suitable conditions for the CA in this species: a cell density of 4×10(3) erythrocytes per slide, 10 min of unwinding and 15 min of electrophoresis at 0.90 V/cm approximately. The baseline frequency of micronuclei (BFMN=MN/1000 erythrocytes counted) determined for this species was 0.95±0.27 and the basal damage index (BDI: calculated from 100 comet images classified in arbitrary units)=103.85±0.97. No differences were observed between sexes in the BFMN or BDI (p>0.05), and no relation was found between baseline values and length or weight of the analyzed animals (p>0.05). These results demonstrated the sensitivity of both biomarkers of

  16. Copper release in low and high alkaline water.

    PubMed

    D'Antonio, L; Fabbricino, M; Nasso, M; Trifuoggi, M

    2008-04-01

    Copper release in drinking water, caused by electrochemical corrosion of household distribution systems, was investigated. Experiments were developed testing both low and high alkaline water in stagnant conditions. The effect of varying stagnation time was investigated also. Both soluble and insoluble copper compounds, produced by corrosion processes are quantified, using appropriate experimental procedures. On the basis of obtained results, copper concentration in stagnant water is defined as a function of water alkalinity, while total metal release is defined as a function of stagnation length, and is not dependent on water alkalinity.

  17. Method of increasing the sulfation capacity of alkaline earth sorbents

    DOEpatents

    Shearer, J.A.; Turner, C.B.; Johnson, I.

    1980-03-13

    A system and method for increasing the sulfation capacity of alkaline earth carbonates to scrub sulfur dioxide produced during the fluidized bed combustion of coal in which partially sulfated alkaline earth carbonates are hydrated in a fluidized bed to crack the sulfate coating and convert the alkaline earth oxide to the hydroxide. Subsequent dehydration of the sulfate-hydroxide to a sulfate-oxide particle produces particles having larger pore size, increased porosity, decreased grain size and additional sulfation capacity. A continuous process is disclosed.

  18. Method of increasing the sulfation capacity of alkaline earth sorbents

    DOEpatents

    Shearer, John A.; Turner, Clarence B.; Johnson, Irving

    1982-01-01

    A system and method for increasing the sulfation capacity of alkaline earth carbonates to scrub sulfur dioxide produced during the fluidized bed combustion of coal in which partially sulfated alkaline earth carbonates are hydrated in a fluidized bed to crack the sulfate coating and convert the alkaline earth oxide to the hydroxide. Subsequent dehydration of the sulfate-hydroxide to a sulfate-oxide particle produces particles having larger pore size, increased porosity, decreased grain size and additional sulfation capacity. A continuous process is disclosed.

  19. Polyvinyl alcohol battery separator containing inert filler. [alkaline batteries

    NASA Technical Reports Server (NTRS)

    Sheibley, D. W.; Hsu, L. C.; Manzo, M. A. (Inventor)

    1981-01-01

    A cross-linked polyvinyl alcohol battery separator is disclosed. A particulate filler, inert to alkaline electrolyte of an alkaline battery, is incorporated in the separator in an amount of 1-20% by weight, based on the weight of the polyvinyl alcohol, and is dispersed throughout the product. Incorporation of the filler enhances performance and increases cycle life of alkaline batteries when compared with batteries containing a similar separator not containing filler. Suitable fillers include titanates, silicates, zirconates, aluminates, wood floor, lignin, and titania. Particle size is not greater than about 50 microns.

  20. Radon assay for SNO+

    SciTech Connect

    Rumleskie, Janet

    2015-12-31

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  1. Growth cone collapse assay.

    PubMed

    Cook, Geoffrey M W; Jareonsettasin, Prem; Keynes, Roger J

    2014-01-01

    The growth cone collapse assay has proved invaluable in detecting and purifying axonal repellents. Glycoproteins/proteins present in detergent extracts of biological tissues are incorporated into liposomes, added to growth cones in culture and changes in morphology are then assessed. Alternatively purified or recombinant molecules in aqueous solution may be added directly to the cultures. In both cases after a defined period of time (up to 1 h), the cultures are fixed and then assessed by inverted phase contrast microscopy for the percentage of growth cones showing a collapsed profile with loss of flattened morphology, filopodia, and lamellipodia.

  2. Radon assay for SNO+

    NASA Astrophysics Data System (ADS)

    Rumleskie, Janet

    2015-12-01

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  3. Zebrafish Assays of Ciliopathies

    PubMed Central

    Zaghloul, Norann A.; Katsanis, Nicholas

    2013-01-01

    In light of the growing list of human disorders associated with their dysfunction, primary cilia have recently come to attention as being important regulators of developmental signaling pathways and downstream processes. These organelles, present on nearly every vertebrate cell type, are highly conserved structures allowing for study across a range of species. Zebrafish, in particular, have emerged as useful organisms in which to explore the consequences of ciliary dysfunction and to model human ciliopathies. Here, we present a range of useful techniques that allow for investigation of various aspects of ciliary function. The described assays capitalize on the hallmark gastrulation defects associated with ciliary defects as well as relative ease of visualization of cilia in whole-mount embryos. Further, we describe our recently developed assay for querying functionality of human gene variants in live developing embryos. Finally, a current catalog of known zebrafish ciliary mutant lines is included. The techniques presented here provide a basic toolkit for in vivo investigation of both the biological and genetic mechanisms underlying a growing class of human diseases. PMID:21951534

  4. RAS - Screens & Assays - Drug Discovery

    Cancer.gov

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  5. Biosensors: Viruses for ultrasensitive assays

    NASA Astrophysics Data System (ADS)

    Donath, Edwin

    2009-04-01

    A three-dimensional assay based on genetically engineered viral nanoparticles and nickel nanohairs can detect much lower levels of protein markers associated with heart attacks than conventional assays.

  6. The Martian ocean: First acid, then alkaline

    NASA Technical Reports Server (NTRS)

    Schaefer, M. W.

    1992-01-01

    In Mars' distant past, carbon dioxide and water may have been plentiful. Values of total outgassed CO2 from several to about 10 bar are consistent with present knowledge, and this amount of CO2 implies an amount of water outgassed equal to an equivalent depth of 500 to 1000 m. It is quite reasonable, therefore, to envision an early Mars in which there was a body or bodies of liquid water, perhaps in the northern plains, and a dense carbon dioxide atmosphere. Under such conditions, the pH of the water will be low, due to the dissolution of carbon dioxide in the water to form carbonic acid. This acidic water is capable of weathering the available rock quite intensely, particularly because this rock is likely to be heavily fractured (from meteorite bombardment) or even consist of fine particles (such as pyroclastic deposits). As time goes on, however, the carbon dioxide atmosphere will rapidly pass through the ocean to form carbonate deposits. As the density of the atmosphere decreases, so will the flux of carbonic acid into the ocean. Without this input of carbonic acid, the effect of the dissolved weathering products will be to increase the pH of the water. The ocean will then become alkaline. To study this process, I have developed a geochemical cycle model for the atmosphere-hydrosphere-regolith system of Mars. The treatment of geochemical cycles as complex kinetic chemical reactions has been undertaken for terrestrial systems in recent years with much success. This method is capable of elegantly handling the interactions between the simultaneous chemical reactions needed to understand such a system.

  7. Spectroscopic studies of alkaline activated slag geopolymers

    NASA Astrophysics Data System (ADS)

    Mozgawa, W.; Deja, J.

    2009-04-01

    In the work, results of structural studies of different geopolymers, obtained using a granulated blast furnace slag, are presented. The slag was subjected to an alkaline activation process. As activators, NaOH, Na 2CO 3 and liquid glass were applied. IR and NMR spectroscopy were the main experimental methods used, the results obtained were compared with XRD phase analysis and SEM observations. In the IR spectra of raw slag as well as in the spectra of products of paste hydration, the bands due to the characteristic vibrations of bonds observed in both types of oxygen bridges: Si-O-Si and Si-O-Al, were assigned. These bridges constitute basic structural units, forming tetrahedral geopolymer chains. It was found that the slag composition, mainly SiO 2/Al 2O 3 ratio and modification in oxides concentration, influences the presence of the bands connected with the phases (mainly C-S-H) formed during the hydration in the IR spectra. Additionally, significant effect of amorphous phases share on the spectra shape was established. 29Si and 27Al MAS-NMR spectra of initial slag geopolymers and pastes provided information concerning coordination of both atoms in the structures. It was revealed that the kind of slag geopolymers and the conditions of paste hydration influence connectedness of silicooxygen tetrahedra and coordination number of aluminium atoms. Based on IR spectra, it was also possible to determine the influence of the activator type, activation time and hydration conditions on the products formed. Significant changes were observed for the bands assigned to vibrations of carbonate and hydroxide groups. The changes were also noticed in the case of bands due to vibrations of silicate and aluminosilicate bonds.

  8. Alkaline decomposition of synthetic jarosite with arsenic

    PubMed Central

    2013-01-01

    The widespread use of jarosite-type compounds to eliminate impurities in the hydrometallurgical industry is due to their capability to incorporate several elements into their structures. Some of these elements are of environmental importance (Pb2+, Cr6+, As5+, Cd2+, Hg2+). For the present paper, AsO43- was incorporated into the lattice of synthetic jarosite in order to carry out a reactivity study. Alkaline decomposition is characterized by removal of sulfate and potassium ions from the lattice and formation of a gel consisting of iron hydroxides with absorbed arsenate. Decomposition curves show an induction period followed by a conversion period. The induction period is independent of particle size and exponentially decreases with temperature. The conversion period is characterized by formation of a hydroxide halo that surrounds an unreacted jarosite core. During the conversion period in NaOH media for [OH-] > 8 × 10-3 mol L-1, the process showed a reaction order of 1.86, and an apparent activation energy of 60.3 kJ mol-1 was obtained. On the other hand, during the conversion period in Ca(OH)2 media for [OH-] > 1.90 × 10-2 mol L-1, the reaction order was 1.15, and an apparent activation energy of 74.4 kJ mol-1 was obtained. The results are consistent with the spherical particle model with decreasing core and chemical control. PMID:23566061

  9. Alkaline decomposition of synthetic jarosite with arsenic.

    PubMed

    Patiño, Francisco; Flores, Mizraim U; Reyes, Iván A; Reyes, Martín; Hernández, Juan; Rivera, Isauro; Juárez, Julio C

    2013-01-01

    The widespread use of jarosite-type compounds to eliminate impurities in the hydrometallurgical industry is due to their capability to incorporate several elements into their structures. Some of these elements are of environmental importance (Pb(2+), Cr(6+), As(5+), Cd(2+), Hg(2+)). For the present paper, AsO4 (3-) was incorporated into the lattice of synthetic jarosite in order to carry out a reactivity study. Alkaline decomposition is characterized by removal of sulfate and potassium ions from the lattice and formation of a gel consisting of iron hydroxides with absorbed arsenate. Decomposition curves show an induction period followed by a conversion period. The induction period is independent of particle size and exponentially decreases with temperature. The conversion period is characterized by formation of a hydroxide halo that surrounds an unreacted jarosite core. During the conversion period in NaOH media for [OH(-)] > 8 × 10(-3) mol L(-1), the process showed a reaction order of 1.86, and an apparent activation energy of 60.3 kJ mol(-1) was obtained. On the other hand, during the conversion period in Ca(OH)2 media for [OH(-)] > 1.90 × 10(-2) mol L(-1), the reaction order was 1.15, and an apparent activation energy of 74.4 kJ mol(-1) was obtained. The results are consistent with the spherical particle model with decreasing core and chemical control.

  10. Improving the alkaline stability of imidazolium cations by substitution.

    PubMed

    Dong, Huilong; Gu, Fenglou; Li, Min; Lin, Bencai; Si, Zhihong; Hou, Tingjun; Yan, Feng; Lee, Shuit-Tong; Li, Youyong

    2014-10-06

    Imidazolium cations are promising candidates for preparing anion-exchange membranes because of their good alkaline stability. Substitution of imidazolium cations is an efficient way to improve their alkaline stability. By combining density functional theory calculations with experimental results, it is found that the LUMO energy correlates with the alkaline stability of imidazolium cations. The results indicate that alkyl groups are the most suitable substituents for the N3 position of imidazolium cations, and the LUMO energies of alkyl-substituted imidazolium cations depend on the electron-donating effect and the hyperconjugation effect. Comparing 1,2-dimethylimidazolium cations (1,2-DMIm+) and 1,3-dimethylimidazolium cations (1,3-DMIm+) with the same substituents reveals that the hyperconjugation effect is more significant in influencing the LUMO energy of 1,3-DMIms. This investigation reveals that LUMO energy is a helpful aid in predicting the alkaline stability of imidazolium cations.

  11. Processes affecting the oceanic distributions of dissolved calcium and alkalinity

    SciTech Connect

    Shiller, A.M.; Gieskes, J.M.

    1980-05-20

    Recent studies of the CO/sub 2/ system have suggested that chemical processes in addition to the dissolution and precipitation of calcium carbonate affect the oceanic calcium and alkalinity distributions. Calcium and alkalinity data from the North Pacific have been examined both by using the simple physical-chemical model of previous workers and by a study involving the broader oceanographic context of these data. The simple model is shown to be an inadequate basis for these studies. Although a proton flux associated with organic decomposition may affect the alkalinity, previously reported deviations of calcium-alkalinity correlations from expected trends appear to be related to boundary processes that have been neglected rather than to this proton flux. The distribution of calcium in the surface waters of the Pacific Ocean is examined.

  12. ESTIMATION OF PHOSPHATE ESTER HYDROLYSIS RATE CONSTANTS - ALKALINE HYDROLYSIS

    EPA Science Inventory

    SPARC (SPARC Performs Automated Reasoning in Chemistry) chemical reactivity models were extended to allow the calculation of alkaline hydrolysis rate constants of phosphate esters in water. The rate is calculated from the energy difference between the initial and transition state...

  13. ESTIMATION OF PHOSPHATE ESTER HYDROLYSIS RATE CONSTANTS. I. ALKALINE HYDROLYSIS

    EPA Science Inventory

    SPARC (SPARC Performs Automated Reasoning in Chemistry) chemical reactivity models were extended to allow the calculation of alkaline hydrolysis rate constants of phosphate esters in water. The rate is calculated from the energy difference between the initial and transition state...

  14. Regulation of alkaline phosphatase expression in human choriocarcinoma cell lines.

    PubMed Central

    Hamilton, T A; Tin, A W; Sussman, H H

    1979-01-01

    The coincident expression of two structurally distinct isoenzymes of human alkaline phosphatase was demonstrated in two independently derived gestational choriocarcinoma cell lines. These proteins were shown to have enzymatic, antigenic, and physical-chemical properties resembling those of isoenzymes from term placenta and adult liver. The regulation of these isoenzymes has been studied during the exposure of both cell lines to 5-bromodeoxyuridine and dibutyryl cyclic AMP. The responses of the alkaline phosphatase isoenzymes to these agents have also been compared with the response of another protein phenotypic to placenta, the alpha subunit of chorionic gonadotropin. The results show that (i) the separate structural genes coding for placental and liver alkaline phosphatases are regulated in a noncoordinate fashion; (ii) both alkaline phosphatase genes respond independently of the alpha subunit; and (iii) the induction of the placental type isoenzyme occurs via at least two independent pathways. Images PMID:218197

  15. Net alkalinity and net acidity 1: Theoretical considerations

    USGS Publications Warehouse

    Kirby, C.S.; Cravotta, C.A.

    2005-01-01

    Net acidity and net alkalinity are widely used, poorly defined, and commonly misunderstood parameters for the characterization of mine drainage. The authors explain theoretical expressions of 3 types of alkalinity (caustic, phenolphthalein, and total) and acidity (mineral, CO2, and total). Except for rarely-invoked negative alkalinity, theoretically defined total alkalinity is closely analogous to measured alkalinity and presents few practical interpretation problems. Theoretically defined "CO 2-acidity" is closely related to most standard titration methods with an endpoint pH of 8.3 used for determining acidity in mine drainage, but it is unfortunately named because CO2 is intentionally driven off during titration of mine-drainage samples. Using the proton condition/mass- action approach and employing graphs to illustrate speciation with changes in pH, the authors explore the concept of principal components and how to assign acidity contributions to aqueous species commonly present in mine drainage. Acidity is defined in mine drainage based on aqueous speciation at the sample pH and on the capacity of these species to undergo hydrolysis to pH 8.3. Application of this definition shows that the computed acidity in mg L -1 as CaCO3 (based on pH and analytical concentrations of dissolved FeII, FeIII, Mn, and Al in mg L -1):aciditycalculated=50{1000(10-pH)+[2(FeII)+3(FeIII)]/56+2(Mn)/ 55+3(Al)/27}underestimates contributions from HSO4- and H+, but overestimates the acidity due to Fe3+ and Al3+. However, these errors tend to approximately cancel each other. It is demonstrated that "net alkalinity" is a valid mathematical construction based on theoretical definitions of alkalinity and acidity. Further, it is shown that, for most mine-drainage solutions, a useful net alkalinity value can be derived from: (1) alkalinity and acidity values based on aqueous speciation, (2) measured alkalinity minus calculated acidity, or (3) taking the negative of the value obtained in a

  16. Establishment of hybridomas secreting monoclonal antibodies to placental alkaline phosphatase and development of an enzyme immunoassay for its determination.

    PubMed

    Kinoshita, Y; Okamoto, T; Mano, H; Furuhashi, Y; Goto, S; Tomoda, Y

    1990-06-01

    We established seven hybridomas secreting murine IgG monoclonal antibodies (MoAbs) to placental alkaline phosphatase (PLAP). The seven hybridomas were designated (1) 7C6, (2) 6G10, (3) 5B9, (4) 6D5, (5) 6B5, (6) 11G6 and (7) 3E10, respectively. The characteristics of these hybridomas were evaluated by radioimmunoassay (RIA) with 125I-PLAP. Their reactivity with the intestinal alkaline phosphatase, one of the alkaline phosphatase isozymes, was (1) 0.04, (2) 0.2, (3) 1.4, (4) 1.8, (5) 0, (6) 4.0 and (7) 6.2(%), respectively. None of them showed signs of cross-reactivity with the liver-type alkaline phosphatase, also one of the alkaline phosphatase isozymes, within a PLAP concentration of 2,000 IU/l. The subtype of 5B9 was IgG1, and that of the others was IgG2a. We then used 7C6, to develop a sensitive, specific and convenient enzyme immunoassay (EIA) for the determination of PLAP, and assayed sera from patients with various gynecologic diseases. The incidence of increased PLAP was 6.4% in patients with benign diseases, 21.5% in cervical cancer, 36.4% in endometrial carcinoma, and 39.5% in malignant ovarian tumors. The specificity for malignant diseases seemed to be higher than that of CA125. Among endometrial carcinomas, well-differentiated adenocarcinoma had the highest incidence of an increased concentration. Among malignant ovarian tumors, serous cystadenocarcinoma, endometrioid carcinoma, dysgerminoma and Krukenberg's tumor showed a higher incidence than the other types.

  17. Dual-color ELISPOT assay for analyzing cytokine balance.

    PubMed

    Okamoto, Yoshihiro; Nishida, Mikio

    2005-01-01

    A dual-color enzyme-linked immunospot (ELISPOT) assay enabled us to analyze three kinds of cytokine-secreting cells simultaneously. T helper (Th) cells can be subdivided into at least two distinct functional subsets based on their cytokine secretion profiles. The first type of clones (Th1) produces interleukin (IL)-2 and interferon (IFN)-gamma but not IL-4 or IL-5. The second type of clones (Th2) produces IL-4 and IL-5 but not IL-2 or IFN-gamma. Furthermore, the presence of the third type (Th0) cell, which is a precursor of Th1 or Th2 cells, has been demonstrated to produce both Th1- and Th2-type cytokines. The dual-color ELISPOT assay is developed to differentiate these three subtypes of Th cells in an identical well. In the system, the red spots corresponding to IL-2-secreting cells (Th1) were developed with horseradish peroxidase and amino-ethyl-carbazole/H2O2. The light blue spots corresponding to IL-4-secreting cells (Th2) were developed with alkaline phosphatase and Vector blue (chromogenic substrate for alkaline phosphatase). The mixed colored (indigo) spots corresponding to both kinds of cytokine-secreting cells (Th0 cells) were developed with both chromogenic substrates. With this system, we could detect the IL-2- and/or IL-4-secreting cells simultaneously in a murine spleen cell or human peripheral mononuclear cell preparation.

  18. Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

    PubMed Central

    Valencia, Laura Carolina; García, Adriana; Ramírez-Pinilla, Martha Patricia; Fuentes, Jorge Luis

    2011-01-01

    The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies. PMID:22215974

  19. Cation exchange properties of zeolites in hyper alkaline aqueous media.

    PubMed

    Van Tendeloo, Leen; de Blochouse, Benny; Dom, Dirk; Vancluysen, Jacqueline; Snellings, Ruben; Martens, Johan A; Kirschhock, Christine E A; Maes, André; Breynaert, Eric

    2015-02-03

    Construction of multibarrier concrete based waste disposal sites and management of alkaline mine drainage water requires cation exchangers combining excellent sorption properties with a high stability and predictable performance in hyper alkaline media. Though highly selective organic cation exchange resins have been developed for most pollutants, they can serve as a growth medium for bacterial proliferation, impairing their long-term stability and introducing unpredictable parameters into the evolution of the system. Zeolites represent a family of inorganic cation exchangers, which naturally occur in hyper alkaline conditions and cannot serve as an electron donor or carbon source for microbial proliferation. Despite their successful application as industrial cation exchangers under near neutral conditions, their performance in hyper alkaline, saline water remains highly undocumented. Using Cs(+) as a benchmark element, this study aims to assess the long-term cation exchange performance of zeolites in concrete derived aqueous solutions. Comparison of their exchange properties in alkaline media with data obtained in near neutral solutions demonstrated that the cation exchange selectivity remains unaffected by the increased hydroxyl concentration; the cation exchange capacity did however show an unexpected increase in hyper alkaline media.

  20. Acid transformation of bauxite residue: Conversion of its alkaline characteristics.

    PubMed

    Kong, Xiangfeng; Li, Meng; Xue, Shengguo; Hartley, William; Chen, Chengrong; Wu, Chuan; Li, Xiaofei; Li, Yiwei

    2017-02-15

    Bauxite residue (BR) is a highly alkaline solid hazardous waste produced from bauxite processing for alumina production. Alkaline transformation appears to reduce the environmental risk of bauxite residue disposal areas (BRDAs) whilst potentially providing opportunities for the sustainable reuse and on-going management of BR. Mineral acids, a novel citric acid and a hybrid combination of acid-gypsum treatments were investigated for their potential to reduce residue pH and total alkalinity and transform the alkaline mineral phase. XRD results revealed that with the exception of andradite, the primary alkaline solid phases of cancrinite, grossular and calcite were transformed into discriminative products based on the transformation used. Supernatants separated from BR and transformed bauxite residue (TBR) displayed distinct changes in soluble Na, Ca and Al, and a reduction in pH and total alkalinity. SEM images suggest that mineral acid transformations promote macro-aggregate formation, and the positive promotion of citric acid, confirming the removal or reduction in soluble and exchangeable Na. NEXAFS analysis of Na K-edge revealed that the chemical speciation of Na in TBRs was consistent with BR. Three acid treatments and gypsum combination had no effect on Na speciation, which affects the distribution of Na revealed by sodium STXM imaging.