Distribution by Scientific Domains

Terms modified by dsDNA

  • dsdna molecule

  • Selected Abstracts

    New Anthraquinone Derivatives as Electrochemical Redox Indicators for the Visualization of the DNA Hybridization Process

    ELECTROANALYSIS, Issue 1 2010
    Agata Kowalczyk
    Abstract Interactions of dsDNA and ssDNA, at the surface of gold and silver electrodes, with three novel anthraquinone derivatives: 3-(9,,10,-dioxo-9,,10,-dihydro-anthracen-1-yl)-7,11-di(carboxymethyl)-3,7,11-triazatridecanedioic acid, (AQ-1); 1-(9,,10,-dioxo-9,,10,-dihydro-anthracen-1yl)-9-carboxymethyl-5-methyl-1,5,9-triazaundecanoicacid, (AQ-2); and N -(2-(9,10-dioxo-9,10-dihydro-anthracen-1-ylamino)ethyl)-2-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)acetamide, (AQ-3) are studied. These derivatives are well soluble in water and phosphate buffer solutions. The square wave voltammetric behavior of these redox indicators is described and the parameters of interactions with DNA are reported. It is also pointed out that these compounds can be employed as the hybridization indicators. The difference in the binding ability of the particular redox indicator to single and double stranded DNA can be used for the detection of the complementary nucleic acids. [source]

    Self-Assembled Multilayers of Polyethylenimine and DNA: Spectrophotometric and Electrochemical Characterization and Application for the Determination of Acridine Orange Interaction

    ELECTROANALYSIS, Issue 15 2009
    F. Ferreyra
    Abstract This work deals with the study of the interaction between acridine orange (AO) and calf-thymus double stranded DNA (dsDNA) present in supramolecular architectures built on gold electrodes modified with mercapto-1-propanesulfonic acid (MPS) by self-assembling of polyethylenimine and dsDNA. The optimal conditions for building the supramolecular architecture were obtained from UV-vis spectrophotometric experiments. The electrochemical studies were performed by adsorptive transfer square wave voltammetry from the evaluation of the oxidation signal of AO accumulated within the multistructure. The effect of the number of PEI-dsDNA bilayers (Au/MPS/(PEI-dsDNA)n) on the accumulation and electrooxidation of AO is also discussed. [source]

    Square-Wave Voltammetry as a Tool for Investigation of Doxorubicin Interactions with DNA Isolated from Neuroblastoma Cells

    ELECTROANALYSIS, Issue 3-5 2009
    Dalibor Huska
    Abstract We investigated ethidium bromide intercalation into DNA molecule as a model system to test square-wave voltammetry (SWV) as a suitable method for this purpose We found that 0.13,,g EtBr intercalates into 1,,g dsDNA in average. Further, SWV was utilized for investigation of doxorubicin-DNA interactions. Intercalated doxorubicin reduced observed dsDNA cytosine and adenine (CA) signal, but also provided new signal called DOXO at ,0.35,V. This phenomenon was observed at both single and double stranded DNA standards. We also employed adsorptive transfer stripping technique coupled with SWV for study of doxorubicin-DNA interactions. Doxorubicin intercalation into dsDNA molecule adsorbed onto working electrode was fast, because we observed considerable changes in CA and DOXO signals after 360,s. Finally, we detected doxorubicin-DNA adducts formed in doxorubicin treated neuroblastoma cells. [source]

    Electrochemical Biosensor for the Detection of Interaction Between Arsenic Trioxide and DNA Based on Guanine Signal

    ELECTROANALYSIS, Issue 7 2003
    Mehmet Ozsoz
    Abstract The interaction of arsenic trioxide (As2O3) with calf thymus double-stranded DNA (dsDNA), calf thymus single-stranded DNA (ssDNA) and also 17-mer short oligonucleotide (Probe,A) was studied electrochemically by using differential pulse voltammetry (DPV) with carbon paste electrode (CPE) at the surface and also in solution. Potentiometric stripping analysis (PSA) was employed to monitor the interaction of As2O3 with dsDNA in solution phase by using a renewable pencil graphite electrode (PGE). The changes in the experimental parameters such as the concentration of As2O3, and the accumulation time of As2O3 were studied by using DPV; in addition, the reproducibility data for the interaction between DNA and As2O3 was determined by using both electrochemical techniques. After the interaction of As2O3 with dsDNA, the DPV signal of guanine was found to be decreasing when the accumulation time and the concentration of As2O3 were increased. Similar DPV results were also found with ssDNA and oligonucleotide. PSA results observed at a low DNA concentration such as 1,ppm and a different working electrode such as PGE showed that there could be damage to guanine bases. The partition coefficients of As2O3 after interaction with dsDNA and ssDNA in solution by using CPE were calculated. Similarly, the partition coefficients (PC) of As2O3 after interaction with dsDNA in solution was also calculated by PSA at PGE. The features of this proposed method for the detection of DNA damage by As2O3 are discussed and compared with those methods previously reported for the other type of DNA targeted agents in the literature. [source]

    Label-Free and Label Based Electrochemical Detection of Hybridization by Using Methylene Blue and Peptide Nucleic Acid Probes at Chitosan Modified Carbon Paste Electrodes

    ELECTROANALYSIS, Issue 24 2002
    Pinar Kara
    Abstract A chitosan modified carbon paste electrode (ChiCPE) based DNA biosensor for the recognition of calf thymus double stranded DNA (dsDNA), single stranded DNA (ssDNA) and hybridization detection between complementary DNA oligonucleotides is presented. DNA and oligonucleotides were electrostatically attached by using chitosan onto CPE. The amino groups of chitosan formed a strong complex with the phosphate backbone of DNA. The immobilized probe could selectively hybridize with the target DNA to form hybrid on the CPE surface. The detection of hybridization was observed by using the label-free and label based protocols. The oxidation signals of guanine and adenine greatly decreased when a hybrid was formed on the ChiCPE surface. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with target. The signals of MB were investigated at dsDNA modified ChiCPE and ssDNA modified ChiCPE and the increased peak currents were observed, in respect to the order of electrodes. The hybridization of peptide nucleic acid (PNA) probes with the DNA target sequences at ChiCPE was also investigated. Performance characteristics of the sensor were described, along with future prospects. [source]

    MCE-electrochemical detection for following interactions of ssDNA and dsDNA with methylene blue

    ELECTROPHORESIS, Issue 11 2009
    Mario Castańo-Įlvarez
    Abstract The interaction between the organic dye, methylene blue and DNA has been studied by MCE with electrochemical detection. Interaction produces two different signals, one corresponding to free methylene blue and other, for the complex methylene blue,DNA. The hybridization between a ssDNA and a complementary sequence, specific to the severe acute respiratory syndrome virus, has been performed and studied in a thermoplastic olefin polymer of amorphous structure CE-microchip with an end-channel gold wire detector. Moreover, studies with a longer dsDNA, an expression vector involved in the transitory or stable expression in mammals cells, pFLAG-CMV4, has also been performed. [source]

    Nanostructured copolymer gels for dsDNA separation by CE

    ELECTROPHORESIS, Issue 23 2008
    Fen Wan
    Abstract Pluronics are triblock copolymers of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) that are able to form many different ordered nanostructures at appropriate polymer concentrations and temperatures in selective solvents. These nanostructured "gels" showed desirable criteria when used as DNA separation media, especially in microchip electrophoresis, including dynamic coating and viscosity switching. A ternary system of F127 (E99P69E99)/TBE buffer/1-butanol was selected as a model system to test the sieving performance of different nanostructures in separating dsDNA by CE. The nanostructures and their lattice constants were determined by small-angle X-ray scattering. Viscosity measurements showed the sol,gel transition phenomena. In addition to the cubic structure, successful electrophoretic separation of dsDNA in 2-D hexagonally packed cylinders was achieved. Results showed that without further optimization, ,X174 DNA,Hae III digest was well separated within 15,min in a 7-cm separation channel, by using F127/TBE/1-butanol gel with a 2-D hexagonal structure. A mechanism for DNA separations by those gels with both hydrophilic and hydrophobic domains is discussed. [source]

    Multilayer poly(vinyl alcohol)-adsorbed coating on poly(dimethylsiloxane) microfluidic chips for biopolymer separation

    ELECTROPHORESIS, Issue 1 2005
    Dapeng Wu
    Abstract A poly(dimethylsiloxane) (PDMS) microfluidic chip surface was modified by multilayer-adsorbed and heat-immobilized poly(vinyl alcohol) (PVA) after oxygen plasma treatment. The reflection absorption infrared spectrum (RAIRS) showed that 88% hydrolyzed PVA adsorbed more strongly than 100% hydrolyzed one on the oxygen plasma-pretreated PDMS surface, and they all had little adsorption on original PDMS surface. Repeating the coating procedure three times was found to produce the most robust and effective coating. PVA coating converted the original PDMS surface from a hydrophobic one into a hydrophilic surface, and suppressed electroosmotic flow (EOF) in the range of pH 3,11. More than 1 000,000 plates/m and baseline resolution were obtained for separation of fluorescently labeled basic proteins (lysozyme, ribonuclease B). Fluorescently labeled acidic proteins (bovine serum albumin, ,-lactoglobulin) and fragments of dsDNA ,X174 RF/HaeIII were also separated satisfactorily in the three-layer 88% PVA-coated PDMS microchip. Good separation of basic proteins was obtained for about 70 consecutive runs. [source]

    DNA fragment analysis by an affordable multiple-channel capillary electrophoresis system

    ELECTROPHORESIS, Issue 1-2 2003
    Ming S. Liu
    Abstract We are demonstrating a cost-effective multichannel capillary electrophoresis system for a high-efficiency double-stranded DNA (dsDNA) fragments analysis. This bench-type high-performance DNA analysis (HDAŌ) system uses fluorescence-type detection with inexpensive solid-state light sources and nonmoving integrated emission collection micro-optics. DNA samples are analyzed simultaneously by using a multiple usage and disposable multicapillary cartridge, which contains integrated capillary channels, optical fibers and an integrated sieving gel reservoir. Using commercially available dsDNA size markers as indicators, the HDAŌ system provides high resolving power in 7 min separations. The system can hold a total of 192 samples in two 96-well polymerase chain reaction (PCR) plates, which can be automatically analyzed within 2.5 h. This affordable system can be used in laboratories to replace slab gel electrophoresis for routine and high-throughput dsDNA analysis. [source]

    Twisted Intercalating Nucleic Acids , Intercalator Influence on Parallel Triplex Stabilities

    Vyacheslav V. Filichev
    Abstract Phosphoramidites of several new twisted intercalating nucleic acid (TINA) monomers and the previously discovered (R)-1- O -[4-(1-pyrenylethynyl)phenylmethyl]glycerol (1) were synthesized and used in DNA synthesis. Stabilization of Hoogsteen-type triplexes was observed in cases of insertion of the novel (R)-1- O -[3-(naphthalen-1-ylethynyl)phenylmethyl]glycerol (2) as a bulge into homopyrimidine oligodeoxynucleotides (ONs), whereas phenylethynyl and 4-(biphenylylethynyl) derivatives of TINAs resulted in destabilization of parallel triplexes relative to the wild-type triplex. It was concluded that TINA monomers should possess at least two fused phenyl rings attached through the triple bond at the 4-position of bulged (R)-1- O -(phenylmethyl)glycerol in homopyrimidine ONs in order to stabilize parallel triplexes. Slight destabilization of DNA/DNA Watson,Crick type duplexes (,Tm = 1.0,4.5 °C) was detected for 2 inserted as a bulge, while RNA/DNA duplexes and duplexes with other TINA analogues were considerably destabilized (,Tm > 6.0 °C). In cases of double insertion of 1 opposite to base inversions in dsDNA, the thermal stabilities of the triplexes were higher than that of the wild-type triplex, which is a new solution to overcome the problem of targeting homopurine stretches with single base pair inversions. A DNA three-way junction was considerably stabilized (,Tm in a range of 10.0,15.5 °C) upon insertion of TINA monomers in the junction point as a bulge. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source]

    A new and efficient method for inhibition of RNA viruses by DNA interference

    FEBS JOURNAL, Issue 16 2009
    Monika Nowak
    We report here a new method for inhibition of RNA viruses induced by dsDNA. We demonstrated that both long dsDNA molecules and short interfering DNA with a sequence complementary to that of viral RNA inhibited tobacco mosaic virus expression and prevented virus spread. Also, the expression of the HIV-1 gp41 gene in HeLa cells was inhibited by complementary short interfering DNA. We showed that Dicer processed dsDNA, which suggests activation of the cellular machinery involved in silencing of RNA. For the silencing of viral RNA effected with dsDNA, we coined the term DNA interference technology. [source]

    Gradual phosphorylation regulates PC4 coactivator function

    FEBS JOURNAL, Issue 7 2006
    Hendrik R. A. Jonker
    The unstructured N-terminal domain of the transcriptional cofactor PC4 contains multiple phosphorylation sites that regulate activity. The phosphorylation status differentially influences the various biochemical functions performed by the structured core of PC4. Binding to ssDNA is slightly enhanced by phosphorylation of one serine residue, which is not augmented by further phosphorylation. The presence of at least two phosphoserines decreases DNA-unwinding activity and abrogates binding to the transcriptional activator VP16. Phosphorylation gradually decreases the binding affinity for dsDNA. These phosphorylation-dependent changes in PC4 activities correlate with the sequential functions PC4 fulfils throughout the transcription cycle. MS and NMR revealed that up to eight serines are progressively phosphorylated towards the N-terminus, resulting in gradual environmental changes in the C-terminal direction of the following lysine-rich region. Also within the structured core, primarily around the interaction surfaces, environmental changes are observed. We propose a model for co-ordinated changes in PC4 cofactor functions, mediated by phosphorylation status-dependent gradual masking of the lysine-rich region causing shielding or exposure of interaction surfaces. [source]

    Kinetics and thermodynamics of nick sealing by T4 DNA ligase

    FEBS JOURNAL, Issue 21 2003
    Alexey V. Cherepanov
    T4 DNA ligase is an Mg2+ -dependent and ATP-dependent enzyme that seals DNA nicks in three steps: it covalently binds AMP, transadenylates the nick phosphate, and catalyses formation of the phosphodiester bond releasing AMP. In this kinetic study, we further detail the reaction mechanism, showing that the overall ligation reaction is a superimposition of two parallel processes: a ,processive' ligation, in which the enzyme transadenylates and seals the nick without dissociating from dsDNA, and a ,nonprocessive' ligation, in which the enzyme takes part in the abortive adenylation cycle (covalent binding of AMP, transadenylation of the nick, and dissociation). At low concentrations of ATP (< 10 µm) and when the DNA nick is sealed with mismatching base pairs (e.g. five adjacent), this superimposition resolves into two kinetic phases, a burst ligation (, 0.2 min,1) and a subsequent slow ligation (, 2 × 10,3 min,1). The relative rate and extent of each phase depend on the concentrations of ATP and Mg2+. The activation energies of self-adenylation (16.2 kcal·mol,1), transadenylation of the nick (0.9 kcal·mol,1), and nick-sealing (16.3,18.8 kcal·mol,1) were determined for several DNA substrates. The low activation energy of transadenylation implies that the transfer of AMP to the terminal DNA phosphate is a spontaneous reaction, and that the T4 DNA ligase,AMP complex is a high-energy intermediate. To summarize current findings in the DNA ligation field, we delineate a kinetic mechanism of T4 DNA ligase catalysis. [source]

    Purification and characterization of the single-strand-specific and guanylic-acid-preferential deoxyribonuclease activity of the extracellular nuclease from Basidiobolus haptosporus

    FEBS JOURNAL, Issue 16 2000
    Neelam A. Desai
    An extracellular nuclease from Basidiobolus haptosporus (designated as nuclease Bh1) was purified to homogeneity by ammonium sulfate precipitation, heat treatment, negative adsorption on DEAE-cellulose, and chromatography on phenyl-Sepharose followed by FPLC on phenyl-Superose. The overall yield was 26%. The Mr of the purified enzyme, determined by gel filtration, was 41 000 whereas by SDS/PAGE (after deglycosylation) it was 30 000. It is a glycoprotein with a pI of 6.8. The optimum pH and temperature for DNA hydrolysis were 8.5 and 60 °C, respectively. Nuclease Bh1 is a metalloprotein but has no obligate requirement for metal ions to be active, nor is its activity stimulated in the presence of metal ions. The enzyme was inhibited by Zn2+, Ag2+, Hg2+, Fe3+ and Al3+, inorganic phosphate, pyrophosphate, dithiothreitol, 2-mercaptoethanol, NaCl and KCl. It was stable to high concentrations of organic solvents and urea but susceptible to low concentrations of SDS and guanidine hydrochloride. Nuclease Bh1 is a multifunctional enzyme and its substrate specificity is in the order of ssDNA , 3,AMP , RNA > dsDNA. Studies on its mode of action showed that it cleaved supercoiled pUC 18 DNA and phage M13 DNA, endonucleolytically, generating single base nicks. The enzyme hydrolyzed DNA with preferential liberation of 5,dGMP, suggesting it to be a guanylic acid preferential endoexonuclease. 5,dGMP, the end product of hydrolysis, was a competitive inhibitor of the enzyme. The absence of 5,dCMP as a hydrolytic product, coupled with the resistance of (dC)10 and deoxyribodinucleoside monophosphates having cytosine either at the 3, or the 5, end, indicates that C-linkages are resistant to cleavage by nuclease Bh1. [source]

    Visualization of the interaction between archaeal DNA polymerase and uracil-containing DNA by atomic force microscopy

    GENES TO CELLS, Issue 1 2006
    Yasuo Asami
    Deamination of cytosine to uracil is a hydrolytic reaction that is greatly accelerated at high temperatures. The resulting uracil pairs with adenine during DNA replication, thereby inducing G:C to A:T transitions in the progeny. Interestingly, B-family DNA polymerases from hyperthermophilic Archaea recognize the presence of uracil in DNA and stall DNA synthesis. To better understand the recognition mechanism, the binding modes of DNA polymerase B1 of Sulfolobus solfataricus (Pol B1) to uracil-containing DNA were examined by gel mobility shift assays and atomic force microscopy. Although PolB1 per se specifically binds to uracil-containing single-stranded DNA, the binding efficiency was substantially enhanced by the initiation of DNA synthesis. Analysis by the atomic force microscopy showed a number of double-stranded DNA (dsDNA) in the products of DNA synthesis. The generation of ds DNA was significantly inhibited, however, by the presence of template uracil, and intermediates where monomeric forms of Pol B1 appeared to bind to uracil-containing DNA were observed. These results suggest that Pol B1 more efficiently recognizes uracil in DNA during DNA synthesis rather than during random diffusion in solution, and that single molecules of Pol B1 bind to template uracil and stall DNA synthesis. [source]

    Intercalating Dye Harnessed Cationic Conjugated Polymer for Real-Time Naked-Eye Recognition of Double-Stranded DNA in Serum

    Kan-Yi Pu
    Abstract Thiazole orange (TO), an intercalating dye, is integrated into cationic poly(fluorene- alt -phenylene) (PFP) to develop a macromolecular multicolor probe (PFPTO) for double-stranded DNA (dsDNA) detection. This polymer design not only takes advantage of the high affinity between TO and dsDNA to realize dsDNA recognition in biological media, but also brings into play the light-harvesting feature of conjugated polymers to amplify the signal output of TO in situ. PFPTO differentiates dsDNA from single-stranded DNA (ssDNA) more effectively upon excitation of the conjugated backbone relative to that upon direct excitation of TO as a result of efficient fluorescence resonance energy transfer from the polymer backbone to the intercalated TO. In the presence of dsDNA, energy transfer within PFPTO is more efficient as compared to that for free TO/PFP system, which leads to better dsDNA discriminability for PFPTO in contrast to that for TO/PFP. The distinguishable fluorescent color for PFPTO solutions in the presence of dsDNA allows naked-eye detection of dsDNA with the assistance of a hand-held UV lamp. The significant advantage of this macromolecular fluorescent probe is that naked-eye detection of label-free dsDNA can be performed in biological media in real-time. [source]

    Cutaneous sarcoid-like granulomas with alveolar hemorrhage and c-ANCA PR-3

    Natividade Rocha MD
    A 28-year-old woman, employed as a leather factory worker, noted asymptomatic, well-delimited plaques on both knees, 6 years ago. The plaques were violaceous with a smooth surface. One appeared over a post-traumatic scar from childhood (Fig. 1). Two years later, she began to complain of symptoms suggestive of polyarthritis, first of the small joints of the hands (proximal interphalanges) and then of the larger joints (wrists, elbows, and knees). She was diagnosed with rheumatoid arthritis and began treatment with nonsteroidal anti-inflammatory drugs for 1 month without any change. Deflazacort, 12 mg/day, and hydroxychloroquine, 400 mg/day, were administered for 3 months, with improvement of her articular complaints, but not her skin lesions. Figure 1. Well-delimited, violaceous plaques with a smooth surface on the knees, one over an old post-traumatic scar One year later, she complained of dysphonia, which remitted spontaneously after some weeks. After one additional year, she noted papules, with similar characteristics to the plaques, on the elbows, and two well-delimited orange-to-brown plaques on the forehead (Fig. 2). Figure 2. Orange,brown plaques symmetrically placed on the forehead During the fifth year of the disease, she was referred for the first time to a dermatologist, who biopsied one of the knee lesions. The histologic result was compatible with "sarcoid granuloma." At that time, she presented with skin lesions as her only complaint. Sarcoidosis was suspected based on a chest X-ray, which revealed hilar lymphadenopathy and diffuse accentuation of the interstitium. In November 2000, she suddenly developed fever (40 °C), cough with hemoptysis, dysphonia, and subcutaneous nodules on the palmar surface of the fingers of both hands that were painless, well-delimited, 5 mm in diameter, and firm (Fig. 3). She reported a weight loss of 12 kg in the previous 3 months. Pulmonary condensation was found on auscultation, and she had palpable hepatomegaly. Peripheral lymphadenopathy was not present. Figure 3. Painless, well-delimited, firm subcutaneous nodules on the palmar surface of the fingers Laboratory investigations revealed normochromic, normocytic anemia (hemoglobin, 7.7 g/dL), iron deficit, a white blood cell count of 16,000/µL with neutrophilia, an erythrocyte sedimentation rate of 130 mm/h, elevation of liver enzymes, a slight increase in angiotensin-converting enzyme (ACE) level (72 U/L), hypergammaglobulinemia (IgG, 3350 mg/dL), antinuclear antibody (ANA) of 1 : 320, and a slight increase in CD4 and decrease in CD8 lymphocytes with normal cellular morphology in blood. Renal function, urine sediment, urine and serum calcium, complement (C4), dsDNA, antimitochondrial antibody, direct and indirect Coombs test, antineutrophil cytoplasmic antibody (ANCA), tuberculin skin tests, viral markers of hepatitis B, C, and human immunodeficiency virus (HIV), electrocardiogram (ECG), ophthalmic examinations, and culture for infectious agents in blood and sputum were all normal or negative. Computed tomography (CT) scan showed an infiltrate in the upper right pulmonary lobule with a central cavity and bilateral hilar lymphadenopathy (Fig. 4). Homogeneous hepatosplenomegaly was present. The bronchoalveolar lavage (BAL) showed a slight lymphocytic increase predominantly of CD8 cells and hemosiderosis. Stains for infectious agents, including acid-fast bacillus, fungi, Mycoplasma, and Legionella, were negative. Three biopsies from the forehead, elbows, and knees showed well-formed noncaseating epithelioid cell granulomas with giant cells of the Langhans type in the dermis, suggestive of sarcoidosis (Figs 5 and 6). A fourth biopsy from a finger nodule demonstrated inflammatory infiltration of the dermis and necrosis with cellular debris. Vasculitis was not seen (Fig. 7). Figure 4. Computed tomography scan showing an infiltrate in the upper right pulmonary lobule with a central cavity Figure 5. Beneath a flattened epidermis, several sarcoid granulomas composed of epithelioid histiocytes and several multinucleated giant cells of Langhans type can be seen (hematoxylin and eosin, ×10) Figure 6. Less well-formed sarcoid granulomas in a hyperkeratotic area, surrounded by a sparse rim of lymphocytes (hematoxylin and eosin, ×20) Figure 7. Foci of necrosis and fibrinoid degeneration with some neutrophil infiltration and nuclear dusting (hematoxylin and eosin, ×40) The patient was treated with a broad-spectrum empirical antimicrobial (levofloxacin, 500 mg daily intravenously) over 12 days, with prompt improvement in her symptoms and remission of the forehead and finger lesions. Nevertheless, on the first evaluation after hospitalization, the CT scan showed persistence of the pulmonary cavity (Fig. 8). A repeat ANCA determination was positive (cytoplasmic pattern, c-ANCA) at 1 : 640 by indirect immunofluorescence (IIF). Antiproteinase-3 antibody was demonstrated at 78 by enzyme-linked immunosorbent assay (ELISA). Figure 8. Computed tomography scan showing persistence of the pulmonary cavity She underwent an open lung biopsy which revealed intra-alveolar hemorrhage and scanty noncaseating epithelioid cell granulomas of the sarcoidosis type in the peripheral blood vessels without vasculitis. A diagnosis of Wegener's granulomatosis was made and she began prednisolone (1 mg/kg/day) and oral cyclophosphamide (2 mg/kg/day). One year later, she is asymptomatic, the skin lesions have completely remitted, c-ANCA is negative, and the CT scan shows partial regression of the pulmonary cavity. [source]

    Confocal imaging of chromatographic fouling under flow conditions

    Sun Chau Siu
    Abstract BACKGROUND: The fouling impact of selected fouling species was assessed by utilising confocal scanning laser microscopy (CSLM) to image a packed chromatographic bed during operation. A custom-made flow cell was packed with Q Sepharose FF and loaded with partially clarified E. coli homogenate. Selective, multicoloured fluorescent dyes were used to label a bovine serum albumin (BSA) test protein (Cy5.5), dsDNA (PicoGreen) and host cell proteins (HCPs) (Cy3). The fouling caused by the various fluorescently labelled components was visualised as a result of the fluorescence emitted by the PicoGreen-labelled dsDNA and the Cy3-labelled protein in the foulant stream, and by testing the adsorptive capacity of a test protein (BSA) onto the resin prior to and post-fouling as well as following the application of a common CIP procedure. RESULTS: Values for the effective diffusivity of BSA (De) were derived from the confocal images and the fouling impact was assessed by comparing De values obtained from different fouling scenarios. Under the most extreme conditions examined, fouling caused a 20% reduction in capacity compared to a fresh bed. BSA diffusivity did not appear to be affected by the fouling conditions studied. Sequential CIP using 15 CVs of 1 mol L,1 NaCl then 15 CVs of 1 mol L,1 NaOH was shown to be effective in removing nucleic acids and HCPs. Subsequent BSA adsorption showed that the CIP regime successfully restored the column capacity to its original value. In contrast, 15 CVs of 1 mol L,1 NaCl were ineffective in removing dsDNA but substantially removed HCPs. CONCLUSION: CSLM was demonstrated to be a useful tool for visualising fouling mechanisms. Comparing the results obtained by this technique using different modes of chromatographic operation provided insights into the fouling characteristics of finite baths versus packed beds. Copyright © 2007 Society of Chemical Industry [source]

    Anti-C1q antibodies: association with nephritis and disease activity in systemic lupus erythematosus

    Carlos Geraldo Moura
    Abstract Background: Anti-C1q antibodies have been described in systemic lupus erythematosus (SLE) as well as in other connective tissue diseases. They have been considered as a marker for disease activity and presence of nephritis. Objective: The aim of this study was to determine the prevalence of anti-C1q antibodies in Brazilian lupus patients as well as analyze their association with different clinical and serologic parameters. Methods: Sera from 81 SLE patients, based on the American College of Rheumatology (ACR) criteria, were collected from a lupus referral outpatient clinic in Salvador, Brazil. Antibodies to C1q were detected by an enzyme-linked immunoassay (ELISA) kit and antibodies to other cellular antigens identified by indirect immunofluorescence on HEp-2 cell substrate (ANA), or Crithidia luciliae (dsDNA), and to nucleosome by ELISA. A cutoff of 20,U wasestablished for anti-C1q and antinucleosome assays. Results: Anti-C1q antibodies were detected in 39.5% (32/81) of SLE sera. The presence of anti-C1q antibodies was associated with proteinuria (P=0.028) but not with other laboratory or clinical features, such as antinucleosome or anti-dsDNA antibodies, hematuria, urinary casts or renal failure, leukopenia, pericarditis, pleuritis, malar rash, seizures, and psychosis. There was a positive correlation between the titers of anti-C1q antibodies and the systemic lupuis erythematosus disease activity index (SLEDAI) score (r=0.370; P=0.001). Conclusion: This study in Brazilian SLE patients confirms previous findings of the association of anti-C1q antibodies with nephritis and disease activity. J. Clin. Lab. Anal. 23:19,23, 2009. © 2009 Wiley-Liss, Inc. [source]

    Evaluation of a new automated enzyme fluoroimmunoassay using recombinant plasmid dsDNA for the detection of anti-dsDNA antibodies in SLE

    D. Villalta
    Abstract ELISA methods to detect anti-double-stranded DNA (anti-dsDNA) antibodies are highly sensitive, but are less specific for the diagnosis of SLE than the immunofluorescence test on Crithidia luciliae (CLIFT) and the Farr assay because they also detect low-avidity antibodies. This study evaluated the specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) of a new automated fluoroimmunoassay (EliA dsDNA; Pharmacia, Freiburg, Germany). We compared the results with those obtained using a commercial CLIFT and an in-house anti-dsDNA IgG ELISA method, and verified its putative ability to detect only high-avidity anti-dsDNA antibodies. Sera from 100 SLE patients and 120 controls were studied. The control group included 20 healthy donors, 70 patients with other rheumatic diseases (32 systemic sclerosis (SSc); 18 primary Sjögren syndrome (pSS), 20 rheumatoid arthritis (RA)), and 30 patients with various infectious diseases (ID). Anti-dsDNA avidity was estimated using an ELISA method based upon the law of mass action, and a simplified Scatchard plot analysis for data elaboration; the apparent affinity constant (Kaa) was calculated and expressed as arbitrary units (L/U). Sensitivity, specificity, PPV, and NPV for SLE were 64%, 95.8%, 93.8% and 72.7%, respectively, for the EliA anti-dsDNA assay; 55%, 99.2%, 98.5%, and 68.8%, respectively, for the CLIFT; and 64%, 93.3%, 90.6%, and 72.3%, respectively, for the in-house ELISA. Although EliA anti-dsDNA was positive mainly in SLE patients with high- (Kaa>80 L/U) and intermediate- (Kaa 30,80 L/U) avidity antibodies (45.3% and 49.9%, respectively), it was also positive in five (7.8%) SLE patients with low-avidity anti-dsDNA antibodies, and five controls (three SSc, one pSS, and one ID) (mean Kaa = 16.4 ± 9.04 L/U). In conclusion, EliA anti-dsDNA assay showed a higher sensitivity than the CLIFT, and a good specificity and PPV for SLE. Its putative ability to detect only high-avidity anti-dsDNA antibodies remains questionable. J. Clin. Lab. Anal. 16:227,232, 2002. © 2002 Wiley-Liss, Inc. [source]

    RPA repair recognition of DNA containing pyrimidines bearing bulky adducts,

    Irina O. Petruseva
    Abstract Recognition of new DNA nucleotide excision repair (NER) substrate analogs, 48-mer ddsDNA (damaged double-stranded DNA), by human replication protein A (hRPA) has been analyzed using fluorescence spectroscopy and photoaffinity modification. The aim of the present work was to find quantitative characteristics of RPA,ddsDNA interaction and RPA subunits role in this process. The designed DNA structures bear bulky substituted pyrimidine nitrogen bases at the inner positions of duplex forming DNA chains. The photoreactive 4-azido-2,5-difluoro-3- pyridin-6-yl (FAP) and fluorescent antracenyl, pyrenyl (Antr, Pyr) groups were introduced via different linker fragments into exo-4N of deoxycytidine or 5C of deoxyuridine. J-dU-containing DNA was used as a photoactive model of undamaged DNA strands. The reporter group was a fluorescein residue, introduced into the 5,-phosphate end of one duplex-forming DNA strand. RPA,dsDNA association constants and the molar RPA/dsDNA ratio have been calculated based on fluorescence anisotropy measurements under conditions of a 1:1 RPA/dsDNA molar ratio in complexes. The evident preference for RPA binding to ddsDNA over undamaged dsDNA distinctly depends on the adduct type and varies in the following way: undamaged dsDNA,<,Antr-dC-ddsDNA,<,mmdsDNA,<,FAPdU-, Pyr-dU-ddsDNA,<,FAP-dC-ddsDNA (KD,=,68,±,1; 25,±,6; 13,±,1; 8,±,2, and 3.5,±,0.5,nM correspondingly) but weakly depends on the chain integrity. Interestingly the bulkier lesions not in all cases have a greater effect on RPA affinity to ddsDNA. The experiments on photoaffinity modification demonstrated only p70 of compactly arranged RPA directly interacting with dsDNA. The formation of RPA,ddsDNA covalent adducts was drastically reduced when both strands of DNA duplex contained virtually opposite located FAP-dC and Antr-dC. Thus RPA requires undamaged DNA strand presence for the effective interaction with dsDNA bearing bulky damages and demonstrates the early NER factors characteristic features underlying strand discrimination capacity and poor activity of the NER system toward double damaged DNA. Copyright © 2008 John Wiley & Sons, Ltd. [source]

    Red Blood Cell Templated Polyelectrolyte Capsules: A Novel Vehicle for the Stable Encapsulation of DNA and Proteins

    Oliver Kreft
    Abstract Summary: A novel method for the encapsulation of biomacromolecules, such as nucleic acids and proteins, into polyelectrolyte microcapsules is described. Fluorescence-labelled double-stranded DNA and human serum albumin (HSA) are used as model substances for encapsulation in hollow microcapsules templated on human erythrocytes. The encapsulation procedure involves an intermediate drying step. The accumulation of DNA and HSA in the capsules is observed by confocal laser scanning microscopy, UV spectroscopy, and fluorimetry. The mechanism of encapsulation is discussed. Confocal fluorescence microscopy images of encapsulated TRITC-HSA (left) and dsDNA (right). Inserts demonstrate fluorescence profiles for both compounds. [source]

    Acquisition of double-stranded DNA-binding ability in a hybrid protein between Escherichia coli CspA and the cold shock domain of human YB-1

    Nan Wang
    Escherichia coli CspA, a major cold shock protein, is dramatically induced upon temperature downshift. As it binds co-operatively to single-stranded DNA (ssDNA) and RNA without apparent sequence specificity, it has been proposed that CspA acts as an RNA chaperone to facilitate transcription and translation at low temperature. CspA consists of a five-stranded ,-barrel structure containing two RNA-binding motifs, RNP1 and RNP2. Eukaryotic Y-box proteins, such as human YB-1, are a family of nucleic acid-binding proteins that share a region of high homology with CspA (43% identity), termed the cold shock domain (CSD). Their cellular functions are very diverse and are associated with growth-related processes. Here, we replaced the six-residue loop region of CspA between the ,3 and ,4 strands with the corresponding region of the CSD of human YB-1 protein. The resulting hybrid protein became capable of binding to double-stranded DNA (dsDNA) in addition to ssDNA and RNA. The dsDNA-binding ability of an RNP1 point mutant (F20L) of the hybrid was almost unchanged. On the other hand, the dsDNA-binding ability of the hybrid protein was abolished in high salt concentrations in contrast to its ssDNA-binding ability. These results indicate that the loop region between the ,3 and ,4 strands of Y-box proteins, which is a little longer and more basic than that of CspA, plays an important role in their binding to dsDNA. [source]

    Effects of the Interaction Between , -Carboline-3-carboxylic acid N -Methylamide and Polynucleotides on Singlet Oxygen Quantum Yield and DNA Oxidative Damage

    Ińigo X. Garcķa-Zubiri
    The complexation of , -carboline-3-carboxylic acid N -methylamide (,CMAM) with the sodium salts of the nucleotides polyadenylic (Poly A), polycytidylic (Poly C), polyguanylic (Poly G), polythymidylic (Poly T) and polyuridylic (Poly U) acids, and with double stranded (dsDNA) and single stranded deoxyribonucleic acids (ssDNA) was studied at pH 4, 6 and 9. Predominant 1:1 complex formation is indicated from Job plots. Association constants were determined using the Benesi,Hildebrand equation. ,CMAM-sensitized singlet oxygen quantum yields were determined at pH 4, 6 and 9, and the effects on this of adding oligonucleotides, dsDNA and ssDNA were studied at the three pH values. With dsDNA, the effect on ,CMAM triplet state formation was also determined through triplet,triplet transient absorption spectra. To evaluate possible oxidative damage of DNA following singlet oxygen ,CMAM photosensitization, we used thiobarbituric acid-reactivity assays and electrophoretic separation of DNA assays. The results showed no oxidative damage at the level of DNA degradation or strand break. [source]

    Positive ion electrospray ionization mass spectrometry of double-stranded DNA/drug complexes

    Rajesh Gupta
    An Erratum has been published for this article in Rapid Communicatons in Mass Spectrometry 16(7) 2002,740,741. Positive ion electrospray ionization mass spectra of 16 base-pair double-stranded (ds)DNA have been obtained with essentially no ions from single-stranded DNA present. Single-stranded DNA was minimized by: (1) careful choice of DNA sequences; (2) the use of a relatively high salt concentration (0.1,M ammonium acetate, pH 8.5), and, (3) a low desolvation temperature (40,°C). Similarly, ESI-MS complexes of dsDNA with cisplatin, daunomycin and distamycin were obtained that contained only negligible amounts of single-stranded DNA. The complexes with daunomycin and distamycin were more stable to strand separation in the gas phase than dsDNA alone. This is in agreement with solution studies and with other recent gas phase results. These data contrast with many earlier ESI-MS studies of dsDNA and DNA/drug complexes in which ions from ssDNA are also normally observed. Copyright © 2001 John Wiley & Sons, Ltd. [source]

    A Graphene-Based Platform for the Assay of Duplex-DNA Unwinding by Helicase,

    ANGEWANDTE CHEMIE, Issue 33 2010
    Hongje Jang
    Mit bloßen Auge zu erkennen: Mithilfe von Graphenoxid (GO) kann die Helicase-abhängige Entwindung doppelsträngiger DNA (dsDNA) quantitativ und in Echtzeit verfolgt werden. GO bindet selektiv an entwundene fluoreszenzmarkierte einzelsträngige DNA und löscht die Fluoreszenz (siehe Bild). Die Helicaseaktivität wird über die Fluoreszenzänderung registriert. [source]

    A novel subpopulation of B-1 cells is enriched with autoreactivity in normal and lupus-prone mice

    ARTHRITIS & RHEUMATISM, Issue 12 2009
    Xuemei Zhong
    Objective B-1 cells have long been suggested to play an important role in lupus. However, reports to date have been controversial regarding their pathogenic or protective roles in different animal models. We undertook this study to investigate a novel subpopulation of B-1 cells and its roles in murine lupus. Methods Lymphocyte phenotypes were assessed by flow cytometry. Autoantibody secretion was analyzed by enzyme-linked immunosorbent assay, autoantigen proteome array, and antinuclear antibody assay. Cell proliferation was measured by thymidine incorporation and 5,6-carboxyfluorescein succinimidyl ester dilution. B cell Ig isotype switching was measured by enzyme-linked immunospot assay. Results Anti,double-stranded DNA (anti-dsDNA) autoantibodies were preferentially secreted by a subpopulation of CD5+ B-1 cells that expressed programmed death ligand 2 (termed L2pB1 cells). A substantial proportion of hybridoma clones generated from L2pB1 cells reacted to dsDNA. Moreover, these clones were highly cross-reactive with other lupus-related autoantigens. L2pB1 cells were potent antigen-presenting cells and promoted Th17 cell differentiation in vitro. A dramatic increase of circulating L2pB1 cells in lupus-prone BXSB mice was correlated with elevated serum titers of anti-dsDNA antibodies. A significant number of L2pB1 cells preferentially switched to IgG1 and IgG2b when stimulated with interleukin-21. Conclusion Our findings identify a novel subpopulation of B-1 cells that is enriched for autoreactive specificities, undergoes isotype switch, manifests enhanced antigen presentation, promotes Th17 cell differentiation, and is preferentially associated with the development of lupus in a murine model. Together, these findings suggest that L2pB1 cells have the potential to initiate autoimmunity through serologic and T cell,mediated mechanisms. [source]

    Natural killer T cells in families of patients with systemic lupus erythematosus: Their possible role in regulation of IGG production

    ARTHRITIS & RHEUMATISM, Issue 1 2007
    Matthew R. J. Green
    Objective To determine whether there is a link between the frequency of natural killer T (NKT) cells and high levels of IgG in patients with systemic lupus erythematosus (SLE) and their relatives. Methods Blood samples were obtained from patients with SLE, their first-degree relatives, patients with rheumatoid arthritis (RA), and healthy control subjects. The frequency of NKT cells (defined as CD56+ T cells) was expressed as a percentage of total blood lymphocytes. Plasma levels of total IgG and IgM, and IgG antibodies to double-stranded DNA (dsDNA) were determined. Results The frequency of NKT cells was lower in patients with SLE than in control subjects. No such decrease was observed in the relatives of patients with SLE or in patients with RA. High levels of IgG were observed in both patients with SLE and their relatives, while low levels of IgM were observed in these same groups. In relatives of patients with SLE, an inverse correlation between the frequency of NKT cells and IgG levels was observed. Moreover, raised levels of IgG in patients with SLE and their relatives and high levels of IgG anti-dsDNA in patients were associated with low frequencies of NKT cells. Conclusion These results suggest that NKT cells have an important role in the regulation of IgG production, although NKT cells with invariant T cell receptors may not necessarily be involved. NKT cells in the setting of SLE could lack the cytokine stimulus from NK or other cells that is needed to exert control on IgG production. Enhancement of NKT cell activity may provide a novel basis for therapy in SLE. [source]

    Annular erythema with eosinophilia: A subset of Wells' syndrome

    R. Howes
    We present a 52-year-old lady with a 5-year history of a persistent, widespread, annular erythema associated with lethargy, arthralgias, and an inflammatory synovitis. Skin biopsies have shown mild lichenoid change at the dermoepidermal junction; and oedema, mucin, and a diffuse lymphocytic and eosinophilic infiltrate without flame figures in the dermis. A full blood count including an eosinophil count; liver, renal and thyroid function; rheumatoid factor, ANA, ENA, dsDNA, complement studies, immunoglobulins, and serum protein elecrophoresis and immunoelectrophoresis; flow cytometry of peripheral blood for lymphocyte markers; stool examination for ova, cysts and parasites; and a CT scan of the chest and abdomen have shown no significant abnormality. Hydroxychloroquine has stabilised but not cleared her condition. Cases presenting clinically with annular erythema and histologically with eosinophilic cellulitis are difficult to classify. We discuss the classification of this case in the context of the literature. [source]

    Capturing of cell culture-derived modified Vaccinia Ankara virus by ion exchange and pseudo-affinity membrane adsorbers

    Michael W. Wolff
    Abstract Smallpox is an acute, highly infectious viral disease unique to humans, and responsible for an estimated 300,500 million deaths in the 20th century. Following successful vaccination campaigns through the 19th and 20th centuries, smallpox was declared eradicated by the World Health Organization in 1980. However, the threat of using smallpox as a biological weapon prompted efforts of some governments to produce smallpox vaccines for emergency preparedness. An additional aspect for the interest in smallpox virus is its potential use as a platform technology for vector vaccines. In particular, the latter requires a high safety level for routine applications. IMVAMUNE®, a third generation smallpox vaccine based on the attenuated Modified Vaccinia Ankara (MVA) virus, demonstrates superior safety compared to earlier generations and represents therefore an interesting choice as viral vector. Current downstream production processes of Vaccinia virus and MVA are mainly based on labor-intensive centrifugation and filtration methods, requiring expensive nuclease treatment in order to achieve sufficient low host-cell DNA levels for human vaccines. This study compares different ion exchange and pseudo-affinity membrane adsorbers (MA) to capture chicken embryo fibroblast cell-derived MVA-BN® after cell homogenization and clarification. In parallel, the overall performance of classical bead-based resin chromatography (Cellufine® sulfate and Toyopearl® AF-Heparin) was investigated. The two tested pseudo-affinity MA (i.e., sulfated cellulose and heparin) were superior over the applied ion exchange MA in terms of virus yield and contaminant depletion. Furthermore, studies confirmed an expected increase in productivity resulting from the increased volume throughput of MA compared to classical bead-based column chromatography methods. Overall virus recovery was ,60% for both pseudo-affinity MA and the Cellufine® sulfate resin. Depletion of total protein ranged between 86% and 102% for all tested matrices. Remaining dsDNA in the product fraction varied between 24% and 7% for the pseudo-affinity chromatography materials. Cellufine® sulfate and the reinforced sulfated cellulose MA achieved the lowest dsDNA product contamination. Finally, by a combination of pseudo-affinity with anion exchange MA a further reduction of host-cell DNA was achieved. Biotechnol. Bioeng. 2010. 105: 761,769. © 2009 Wiley Periodicals, Inc. [source]